WO2006138316A2

WO2006138316A2 - Methods for delivering molecules to the central nervous system

Info

Publication number: WO2006138316A2
Application number: PCT/US2006/023001
Authority: WO
Inventors: John M. Lincecum; Steven N. Perrin
Original assignee: Biogen Idec Ma Inc.
Priority date: 2005-06-14
Filing date: 2006-06-13
Publication date: 2006-12-28
Also published as: WO2006138316A3

Abstract

The invention, in part, provides methods and compositions for delivering molecules to sensory and motor neurons in a subject. Molecules delivered using the methods of the invention may be “administered” or “injected” into innervated tissues of a subject and may be diagnostic and/or therapeutic molecules. The invention also provides methods and compositions for delivering molecules to neurons of the spinal cord and other neurons of the CNS.

Description

METHODS FOR DELIVERING MOLECULES TO THE CENTRAL NERVOUS SYSTEM

Related Applications This application claims priority under 35 U.S. C. §119(6) from U.S. provisional application serial number 60/690,583, filed June 14, 2005, the entire content of which is incorporated by reference herein.

Field of the Invention The invention relates in some aspects to methods, compositions, and kits for delivering molecules to motor neurons and neurons of the central nervous system (CNS).

Background of the Invention

Difficulties in delivering molecules such as polynucleotides and polypeptides products to the CNS prevent the effective diagnosis and/or treatment of many neurological conditions that may be responsive to exogenous molecules.

Summary of the Invention

The invention relates to methods for delivering a molecule to a cell (e.g., a nerve, etc.) in a subject and to diagnostic methods and treatment methods for disorders such as neurological disorders. In some embodiments, the cell to which the molecule is delivered is a cell in the CNS.

According to one aspect of the invention, methods for delivering a molecule to a neuron in a subject are provided. The methods include administering an immunomodulatory agent to a subject, and injecting a molecule into an innervated tissue of the subject in an amount sufficient to be transported into a neuron of the subject. In some embodiments, the innervated tissue is a muscle. In some embodiments, the injection is an intramuscular injection. Ia certain embodiments, the subject has or is predisposed to or is suspected of having a neurological disorder. In some embodiments, the neurological disorder is amyotrophic lateral sclerosis ("ALS")- In certain embodiments, the molecule is a therapeutic, non-therapeutic, or diagnostic molecule. In some embodiments, the immunomodulatory agent is an inhibitor of the co-stimulatory pathway. In some embodiments, the immunomodulatory agent is an antibody that binds to CD40, CD40 ligand (CD154), CD80, CD86, or CD28. In certain embodiments, the antibody is MRl or the anti- human CD40L (hu5C8). Ih some embodiments, two or more immunomodulatory agents are administered. In some embodiments, the immunomodulatory agent is administered prior to injection of the molecule. In certain embodiments, the molecule is hIGF-1. In some embodiments, the therapeutic molecule is a polynucleotide encoding hIGF-1, or a fragment thereof, or an hIGF-1 polypeptide or fragment thereof. In some embodiments, the neuron lies fully within the CNS.

According to another aspect of the invention, methods of treating a neurological disorder are provided. The methods include administering one or more immunomodulatory agents to a subject with a neurological disorder, and injecting one or more therapeutic molecules into an innervated tissue of the subject in an amount effective to treat the neurological disorder. In certain embodiments, the innervated tissue is muscle tissue. In some embodiments, the injection is an intramuscular injection. In certain embodiments, the subject has or is predisposed to or is suspected of having a neurological disorder. In some embodiments, the neurological disorder is a neurological disease or condition, hi some embodiments, the neurological disease is a neurodegenerative disease. In certain embodiments, the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury, spinal cord injury, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis ("ALS"), epilepsy, peripheral neuropathy, or peripheral nerve injury. In some embodiments, the neurological disease is amyotrophic lateral sclerosis ("ALS"). In some embodiments, the therapeutic molecule is a polynucleotide encoding hIGF-1, or a fragment thereof, or an hIGF-1 polypeptide or fragment thereof.

According to yet another aspect of the invention, methods of monitoring the onset, progression, and/or regression of a neurological disease or condition in a subject are provided. The methods include administering an immunomodulatory agent to the subject; injecting a diagnostic molecule to an innervated tissue in an amount sufficient to be transported into a neuron of the subject, and detecting the diagnostic molecule in the subject as a means of monitoring onset, progression, and/or regression of a neurological disease or condition in the subject. In some embodiments, the innervated tissue is muscle tissue. In some embodiments, the injection is an intramuscular injection. In certain embodiments, the subject has or is predisposed to or is suspected of having a neurological disorder. In certain embodiments, the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury, spinal cord injury, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis ("ALS"), epilepsy, peripheral neuropathy, or peripheral nerve injury. In some embodiments, the subject has the neurological disease or condition. In some embodiments, the subject is a model for the neurological disease or condition.

According to another aspect of the invention, methods of diagnosing a neurological disease or condition in a subject are provided. The methods include administering an immunomodulatory agent to the subject; injecting a diagnostic molecule into an innervated tissue in the subject in an amount sufficient to be delivered into a neuron of the subject, and detecting the delivery of the diagnostic molecule in a neuron of the subject, wherein the delivery, pattern of delivery, or amount of delivery of the diagnostic molecule to a neuron of the subject is diagnostic for a neurological disease or condition in the subject, hi certain embodiments, the innervated tissue is muscle tissue. In some embodiments, the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury, spinal cord injury, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis ("ALS"). epilepsy, peripheral neuropathy, or peripheral nerve injury. In some embodiments, the subject is suspected of having the neurological disease or condition.

According to yet another aspect of the invention, methods are provided for delivering one or more detectable molecules to a neuron by administering an immunomodulatory agent to a subject and injecting a detectable molecule to the subject in an amount effective to detect the presence or absence of a neuron and/or the presence or absence of a phenotype in a neuron (e.g., ability to process/modify the detectable molecule), hi some embodiments, the injection is an intramuscular injection, hi certain embodiments, the subject has or is predisposed to or is suspected of having a neurological disorder, hi some embodiments, the neurological disorder is a neurological disease, condition, or injury, hi some embodiments, the neurological disease is a neurodegenerative disease, hi certain embodiments, the neurological disease is Amyotropic Lateral Sclerosis ("ALS"). In certain embodiments, the therapeutic molecule is a polynucleotide encoding hIGF-1 or a fragment thereof, or an hIGF-1 polypeptide or fragment thereof.

Detailed Description Aspects of the invention provide methods and compositions for delivering molecules to one or more sensory neurons, motor neurons, and/or neurons of the CNS and/or CNS in general, or other tissues/cells associated with the CNS. According to methods of the invention, immunomodulation maybe used to enhance the delivery of molecule(s) to a neuron when the molecule(s) are injected into an innervated tissue (e.g., a muscle or organ) in a subject. Aspects of the invention may enhance retrograde transport of a molecule from an injection site by modulating the subject's immune response to the injected molecule (e.g., by inhibiting one or more steps in the co-stimulatory pathway leading to T-cell activation). In one embodiment, a molecule may be taken up by a neuron (e.g., a sensory neuron, a motor neuron, etc.) that innervates the site of injection. In certain embodiments, a molecule may be transported to one or more neurons of the CNS (e.g., the spinal cord, the cortex, etc.) that are separated from the site of injection by at least one neuron (e.g., a sensory neuron, a motor neuron, etc.). In some embodiments of the invention, a molecule may be delivered from a neuron that lies at least in part outside the CNS to a neuron that lies fully within CNS tissue. Thus, methods of the invention can be used to transport a molecule from outside the CNS into CNS tissue that is protected by the blood brain barrier. Accordingly, aspects of the invention provide methods for delivering one or more molecules across the blood brain barrier.

Aspects of the invention maybe useful for delivering one or more molecules to a target neuron or target neuronal network or region. Non-limiting examples of molecules that can be delivered include therapeutic and non-therapeutic molecules (e.g., therapeutic and non- therapeutic peptides; therapeutic and non-therapeutic polynucleotides; genes encoding therapeutic or non-therapeutic peptides or polynucleotides; etc.) and diagnostic molecules (e.g., imaging molecules, polynucleotides that encode detectable molecules, detectable molecules, molecules that are attached to detectable labels, etc.). In some embodiments, therapeutic molecule(s) may be administered to a subject to treat a neurological disorder such as a neurological disease, condition, or injury. In certain embodiments, diagnostic molecule(s) may be administered to a subject to detect neuronal connectivity; determine cell or tissue condition; detect the presence or absence of cells, ascertain the stage of a disease or condition; determine the onset, progression, or regression of a disease or condition, etc.

Aspects of the invention are useful for delivering doses of the same molecule(s) to a subject at a single time or at two or more points in time. For example, a subject may be administered a molecule using methods of the invention repeatedly at regular time intervals (e.g., weekly, monthly, annually, etc.) or whenever the subject needs further administration after an initial administration, or wherever administration is recommended for the subject. A subject may be administered a diagnostic molecule one, two, or more times using methods of the invention, which may allow an assessment of a subject's condition and/or a determination of changes in a subject's disease or condition. For example, the onset of a disease or condition may be determined by comparing the results of administering a diagnostic compound at two or more time points and detecting a change between the amount or location of delivery of a diagnostic molecule in the subject resulting from a first administration and the amount or location of delivery resulting from a subsequent administration of a diagnostic molecule to the subject.

Immunomodulation

Aspects of the invention involve immunomodulating a subject that has been, is being, and/or will be injected with a molecule to be delivered to one or more neurons. hi one aspect, immunomodulation involves inhibiting (partially or completely) one or more branches of the co-stimulatory pathway that leads to T-cell activation. The inhibition may be transient and may involve administering one or more doses of an immunomodulating agent such as an antibody that binds to and inhibits a moiety that is involved in the co- stimulatory pathway. Accordingly, any one or more of the following molecules may be targeted for inhibition: CD40, CD40 ligand (CD154), CD80 (B7.1), CD86 (Bl JZ), CD28, and any other molecule involved in the co-stimulatory pathway. In certain embodiments, one or more of the following immunomodulatory agents maybe used, anti-murine CD40L (MRl), anti-human CD40L (hu5C8), CTLA4-Fc (or other CTLA4-Ig), anti-CD86 antibodies, and anti-CD80 antibodies, hi some embodiments, an immunomodulating agent may be a ligand, or other inhibitory molecule such as a polypeptide or antibody, hi certain embodiments, inhibition may involve administering one or more immunomodulating agents that inhibit transcription and/or translation (e.g., an siRNA) of a moiety involved in the co-stimulatory pathway. An inhibitor that is useful in methods of the invention may be a polynucleotide, a polypeptide, an antibody or antigen-binding fragment thereof, a small molecule, or any other molecule or compound that inhibits the co-stimulatory pathway. One of ordinary skill in the art will understand how to select and administer an inhibitor for use in methods of the invention. An immunomodulatory agent may be administered before, and/or with (e.g., at the same time as), and/or after the administration of a molecule to be taken up by a neuron. In some embodiments, an immunomodulatory agent is administered at least about 168, 167, 166, 165, 164, 163, 162, 161, 160, 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124,123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 1081, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 hours (including all times in between, including, for example, any recited hourly time point minus 59 minutes, 58 minutes, 57 minutes, 56 minutes, 55 minutes, 54 minutes, 53 minutes, 52 minutes, 51 minutes, 50 minutes, 49 minutes, 48 minutes, 47 minutes, 46 minutes, 45 minutes, 44 minutes, 43 minutes, 42 minutes, 41 minutes, 40 minutes, 39 minutes, 38 minutes, 37 minutes, 36 minutes, 35 minutes, 34 minutes, 33 minutes, 32 minutes, 31 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes, 21 minutes, 20 minutes, 19 minutes, 18 minutes, 17 minutes, 16 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes orl minute) before administration of a molecule to be taken up by a neuron. In certain embodiments, an immunomodulatory agent is administered at least about 168, 144, 120, 96, 72, 48, 24, 18, 12, 6, or 0 hours (including all times in between, minus any number of minutes, as recited above) before administration of a molecule to be taken up by a neuron. In certain embodiments, an immunomodulatory agent may also be administered following the administration of a molecule to be taken up by a neuron. In some embodiments, the agent administration following the administration of the molecule maybe at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168 hours (including all times in between, minus any number of minutes, as recited above) after the administration of the molecule. It will be understood that in some embodiments there may be a single administration of an immunomodulatory agent prior to and/or after administration of a molecule to be taken up by a neuron, and in certain embodiments an immunomodulatory agent maybe administered 2, 3, 4, 5 or more times before and/or after administration of a molecule to be taken up by a neuron. It will also be understood that in some embodiments of the invention, two or more different immunomodulatory agents maybe administered, one or more times to a subject. In some embodiments, an immunomodulatory agent is administered before or simultaneously with administration of a molecule to be taken up by a neuron and in certain embodiments, an immunomodulatory agent is administered before and/or simultaneously with a molecule to be taken up by an neuron and the immunomodulatory agent is also administered after administration of the molecule to be taken up by a neuron.

In certain embodiments, an immunomodulatory ligand or inhibitor molecule may be an antibody. In certain embodiments, an immunomodulatory antibody maybe a single-chain antibody, a single-domain antibody, or a Nanobody™. Characteristics of each of these antibody types and methods for their use are well known in the art. Nanobodies™ are the smallest functional fragments of antibodies and are derived from naturally occurring single- chain antibodies (see Ablynx, Technologiepark 4, 9052 Ghent, Belgium; http/www.ablynx.com). Nanobody™ technology was developed following the discovery that camelidae (camels and llamas) possess a unique repertoire of fully functional antibodies that lack light chains (see Ablynx, Technologiepark 4, 9052 Ghent, Belgium; http/www.ablynx.com). Nanobody™ structure consists of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a stable polypeptide harboring the full antigen-binding capacity of the original heavy chain. Nanobodies™ combine the features of conventional antibodies with features of small molecule drugs. Nanobodies™ show high target specificity and low inherent toxicity. Additionally, Nanobodies™ are very stable, can be administered by means other than injection, and are easy to manufacture. In certain embodiments, an immunomodulatory antibody may be a humanized Nanobody™. One of ordinary skill in the art will realize that conservative amino acid substitutions may be made in immunomodulatory antibodies or in other immunomodulatory polypeptide ligands or polypeptide inhibitors of the invention, to provide functionally equivalent variants of the antibodies, ligands, or inhibitor molecules that retain their functional capabilities. A conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering a polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g., Molecular Cloning: A Labomtoiγ Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Functionally equivalent variants of antibodies, ligands, or inhibitor molecules may include conservative amino acid substitutions of in the amino acid sequences. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In certain embodiments, the antibodies can be modified, e.g., humanized. In certain embodiments, the invention provides for F(ab')₂, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; chimeric F(ab')₂ fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; and/or chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.

Molecules and delivery vehicles Aspects of the invention may be used to promote the delivery of any molecule (e.g., a therapeutic molecule, a diagnostic molecule, etc) or any combination of such molecules to a neuron. A molecule may be a protein, a polynucleotide (e.g., a transgene), a carbohydrate, etc. A molecule may be delivered in a delivery vehicle such as a micelle or a liposome or any other natural or synthetic delivery vehicle. A molecule also may be a vector such as a viral, plasmid, or any other suitable vector. The vector may be provided in the form of a viral particle. The vector may include a gene that expresses a polynucleotide (e.g., an inhibitory RNA such as a siRNA) and/or a protein to be delivered to a neuron. The vector may include one or more neuron-specific promoter and/or regulatory sequences that promote or enhance expression in neurons. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to one of ordinary skill in the art. Aspects of the invention may be particularly useful when the molecule(s) being delivered is immunogenic or if the subject is allergic and/or sensitized to the molecule(s).

Aspects of the invention also comprise molecules that are chemically modified. One example of a chemically modified molecule is one that comprises a polyalkyl oxide moiety. PEG is one suitable polyalkyl oxide. As an illustration, a molecule of the invention can be modified with PEG, a process known as "PEGylation." PEGylation of molecule of the invention can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol 68:1 (1998)). For example, PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule. In an alternative approach, conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657). PEGylation by acylation typically requires reacting an active ester derivative of PEG with a molecule of the invention. An example of an activated PEG ester is PEG esterified to N-hydroxysucciiimide. As used herein, the term "acylation" includes the following types of linkages between a molecule of the invention and a water soluble polymer: amide, carbamate, urethane, and the like. Methods for preparing PEGylated molecules of the invention by acylation will typically comprise the steps of (a) reacting a polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to a molecule of the invention, and (b) obtaining the reaction product(s). Generally, the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG:molecule of the invention, the greater the percentage of polyPEGylated product.

The product of PEGylation by acylation is typically a polyPEGylated product, wherein the lysine amino groups are PEGylated via an acyl linking group. An example of a connecting linkage is an amide. Typically, the resulting molecule of the invention will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions. PEGylated species can be separated from unconjugated polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like. PEGylation by allcylation generally involves reacting a terminal aldehyde derivative of PEG with a molecule of the invention in the presence of a reducing agent. PEG groups are preferably attached to the polypeptide via a -CH₂ --NH group.

Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization. Typically, the reaction is performed at a pH that allows one to take advantage of the pKa differences between the .epsilon. -amino groups of the lysine residues and the .alpha.-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water-soluble polymer that contains a reactive group such as an aldehyde, to a protein is controlled. The conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups. The present invention provides a substantially homogenous preparation of monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population of monopolymer conjugate molecules can comprise the steps of: (a) reacting polypeptide molecule of the invention with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the alpha-amino group at the amino terminus of the molecule of the invention, and (b) obtaining the reaction products). The reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive allylation. Preferred reducing agents include sodium borohydride, sodium cyanoborohydri.de, dimethylamine borane, trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer conjugates, the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terminus. Such reaction conditions generally provide for pKa differences between the lysine amino groups and the α-amino group at the N-terminus. The pH also affects the ratio of polymer to protein to be used, hi general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal a- group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer:molecule of the invention need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3-9, or 3-6.

Another factor to consider is the molecular weight of the water-soluble polymer. Generally, the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. For PEGylation reactions, the typical molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of water-soluble polymer to a molecule of the invention will generally be in the range of 1 : 1 to 100 : 1. Typically, the molar ratio of water- soluble polymer to a molecule of the invention will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising interferon and water-soluble polymer moieties are known in the art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat. No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem. 247:434 (1997).

Aspects of the invention may be particularly useful for molecules that are efficiently taken up by neurons and/or efficiently transported by retrograde transport.

In certain embodiments, methods of the invention maybe used to deliver a transgene to neuron(s) of a subject. In some embodiments, the transgene is delivered via an adenoviral vector. Additional viral vectors may also be used for delivery. In certain embodiments, a molecule to be delivered (e.g., a therapeutic molecule, a non-therapeutic molecule, a diagnostic molecule, etc.) may be a peptide. A peptide may be delivered to neuron(s) of a subject using methods of the invention. A peptide or other molecule for delivery may be fused to, attached to, or delivered in conjunction with a portion of a neurotoxin that is useful for rapid uptake by neurons. Neurotoxins or functional portions thereof (e.g., a portion of a neurotoxin that is useful for neuronal delivery) can include, but are not limited to, one or more of the following: cholera toxin, rabies toxin, Agatoxin (Funnel Web Spider), Agitoxin (Scorpion), alpha-bungarotoxin (Krait snake), Anatoxin (Algae), Apamin (Honey bee), Atracotoxin (Blue Mountains Funnel Web Spider), Batrachotoxin (Poison Arrow Frog), beta-bungarotoxin (Krait snake), Botulinum toxin (Clostridium botulinum), Brevetoxin (Red Tide Dinoflagellate), Capsaicin (Cayenne Pepper), Charybdotoxin (Scorpion), Ciguatoxin (Dinoflagellate), Cobrotoxin (Cobra), Conotoxin (Marine Snail), Crotoxin (South American Rattlesnake), Dendrotoxin (Green Mamba), Domoic acid (Blue mussel), Erabutoxin (Sea Snake), Grammotoxin SIA

(South American Rose Tarantula), Gonyautoxin (Dinoflagellate), Holocyclotoxin (Australian paralysis tick), Homobatrachotoxin (Pitohui bird), HWTX-I (Chinese bird spider), Iberiotoxin (Scorpion), Joro spider toxin (Joro spider), Kaliotoxin (Scorpion), Kurtoxin (South African Scorpion), Latrotoxin (Black Widow Spider), Maculotoxin (Blue-Ringed Octopus), Margatoxin (Scorpion), Noxiustoxin (Scorpion), Palytoxin (Soft coral), Philanthotoxin (Predaceous Wasp), Phoneutriatoxin (Banana spider), Phrixotoxin (Chilean fire tarantula), Robustotoxin (Funnel web spider), Saxitoxin (Dinoflagellate), SNX-482 (African Tarantula), Stichodactyla Toxin (Sea Anemone), Tetanus toxin {Clostridium tetani), Taicatoxin (Australian Taipan snake), Tetrodotoxin (TTX) (Pufferfish), Textilotoxin (Australian common brown snake), Tityustoxin-K (Brazilian Scorpion), and Versutoxin (Funnel web spider). In some embodiments, a toxin may be attached to a detectable label and used as a diagnostic molecule in the methods of the invention.

Therapeutic and non-therapeutic molecules useful in the invention, may be antibodies, ligands, aptmers, polynucleotides, small molecules, polypeptides, etc.

Aspects of the invention maybe used to deliver one or more therapeutic molecules to treat a neurological disorder. A therapeutic molecule may be a transgene. A transgene may be any gene encoding a polynucleotide (e.g., siRNA) or protein that can treat a neurological disorder such as a neurodegenerative disease. For example, IGF-I is a neurotrophic factor essential for normal development of the nervous system and shows protection of motor neurons in animal models and cell culture systems. It is thought to block cell death pathways and promote muscle re-innervation and axonal growth and regeneration. Efficient delivery of polynucleotides encoding IGF-I, or fragments thereof, or IGF-I polypeptides or fragments thereof, by a method of the invention may alleviate some of the symptoms of ALS or slow the progression of the disease. In another embodiment, siRNA targeted to the mutated gene variant in hereditary forms of neurodegenerative disease maybe delivered to affected motor neurons to downregulate the mutant gene product thereby alleviating symptoms and reversing a pathophysiological course, hi other embodiments, agents that are known to be useful in the treatment of a β -amyloid-associated diseases such as Alzheimer's maybe delivered to neurons. For example, acetylcholinesterase inhibitors maybe delivered according to methods of the invention.

A wide variety of therapeutic molecules may be administered using methods of the invention. Examples, though not intended to be limiting may include, growth factors, nerve specific growth factors, neurotransmitters, membrane components, lysosomal enzymes, post- translational modification enzymes, etc., and polynucleotides encoding them, including, but not limited to, for example, PDGF, EGF₅ TGF-alpha, TGF-beta, FGF, NGF, erythropoietin, IFG-II, IL-lalpha, IL-lbeta, IL-2, EL-3, IL-4, IL-5, IL-6, JL-I, EL-8, IL-9, IL-IO, IL-11, EL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IFN-alpha, IFN-beta, IFN-gamma, CSF, GCSF, MCSF, GMCSF₃ and the like. Aspects of the invention may be used to deliver one or more diagnostic molecules to assess or detect a neurological disorder. Diagnostic molecules useful in the invention, may be antibodies, ligands, aptmers, polynucleotides, small molecules, polypeptides, etc. Diagnostic molecules may target and/or bind to molecules found preferentially (e.g., specifically) in or on neurons, components of neurons, growth factors, nerve specific growth factors, neurotransmitters, neuromodulators, membrane components, lysosomal enzymes, post- translational modification enzymes, etc., and polynucleotides encoding them. Diagnostic molecules may bind to specific neuronal cells, cell types, neuronal epitopes, etc. Diagnostic molecules useful in the invention may include molecules that include a detectable label or that result in a detectable product after administration. For example, a diagnostic molecule may include or be attached to a detectable label or may encode a detectable expression product. A wide variety of detectable labels are available for use in methods of the invention, such as those that provide direct detection (e.g., fluorescence, colorimetric, or optical, etc.) or indirect detection (e.g., enzyme-generated luminescence, epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, labeled antibody, etc.). A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc., or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for using and detecting labels are well known by those of ordinary skill in the art. Methods of the invention may be used for in vivo, in vitro, and/or ex vivo imaging, including but not limited to real-time imaging. The presence of a diagnostic molecule in a subject can be detected by in vivo, ex vivo, or in vitro imaging using standard methods. Examples of detection methods include, but are not limited to, MRI, functional MRI, X-Ray detection, PET, CT imaging, immunohistochemistry, Western blot of tissues or cells, or by any other suitable detection method. The term "diagnostic molecule" as used here means a molecule preferably selected from, but not limited to, the group consisting of fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, and bioluminescent molecules. As used herein, the molecule may include a colorimetric label, e.g., a chromophore molecule. In some aspects of the invention, a label may be a combination of the foregoing molecule types.

Radioactive or isotopic labels may be, for example, ¹⁴C, ³H, ³⁵S, ¹²⁵I, and ³²P.

Fluorescent labels may be any compound that emits an electromagnetic radiation, preferably visible light, resulting from the absorption of incident radiation and persisting as long as the stimulating radiation is continued.

Examples of fluorescent labels that maybe used in the methods of the invention include but are not limited to 2,4-dinitrophenyl, acridine, cascade blue, rhodamine, 4- benzoylphenyl, 7-nitrobenz-2-oxa-l ,3-diazole, 4,4-difluoro-4-bora-3a,4a-diaza-3-indacene and fluorescamine. Absorbance-based labels may be molecules that are detectable by the level of absorption of various electromagnetic radiation. Such molecules may be, for example, the fluorescent labels indicated above.

Chemiluminescent labels in this invention refer to compounds that emit light as a result of a non-enzymatic chemical reaction. Methods of the invention may also include the use of a luminescent detectable diagnostic molecule such as enhanced green fluorescent protein (EGFP), luciferase {Luc), or another detectable expression product.

Enzymatic methods for detection may be used including the use of alkaline phosphatase and peroxidase. Additional or alternative enzymes may also be used for detection in methods and kits of the invention. As used herein, fluorophores include, but are not limited to amine-reactive fluorophores that cover the entire visible and near-infrared spectrum. Examples of such fluorophores include, but are not limited to, 4-methylumbelliferyl phosphate, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate ("TRITC"), BODIPY dyes;

Oregon Green, rhodamine green dyes; the red-fluorescent Rhodamine Red-X, Texas Red dyes; and the UV light-excitable Cascade Blue, Cascade Yellow, Marina Blue, Pacific Blue and AMCA-X fluorophores. Fluorophores may also include non-fluorescent dyes used in fluorescence resonance energy transfer ("FRET").

The diagnostic molecules of the invention can be prepared from standard moieties known in the art. As is recognized by one of ordinary skill in the art, the labeling process for preparing a diagnostic molecule may vary according to the molecular structure of the molecule and the detectable label. Methods of labeling molecules with one or more types of detectable labels are routinely used and are well understood by those of ordinary skill in the art.

In some aspects of the invention, a diagnostic molecule may be a molecule that causes a change or modification in a cell to which it is delivered and it is that change or modification that is detectable. For example, a diagnostic molecule or its product may not be directly detectable, the presence of the diagnostic molecule in a neuronal cell or tissue may result in a detectable alteration in the cell or tissue that can be assessed as a determination of delivery of the diagnostic molecule and/or status of the cell or tissue or of a neurological disease or condition, hi some embodiments of the invention, one or more diagnostic molecules and one or more therapeutic molecules may be delivered to a subject. In some embodiments, such deliveries may be simultaneous or sequential deliveries.

Injection site

Molecules (e.g., diagnostic molecules, therapeutic molecules, etc.) maybe injected at any site that is suitable for uptake by one or more neurons. Molecules may be injected into innervated tissues for delivery to a neuron. Innervated tissues include, but are not limited to muscle, organs, sensory organs, etc. Molecules may be injected intramuscularly, intrathecally, intracranially, intraocularly, into the tongue, or other innervated tissue. Additional suitable sites of injection may be used, hi certain embodiments, molecules may be injected into highly innervated sites that are characterized by an elevated concentration of nerve endings (e.g., motor end-plates). In certain embodiments, injection of a diagnostic molecule and/or a therapeutic molecule may be made at more than one injection site. One of ordinary skill in the art would be well aware of specific anatomical injection sites that could be used to target therapeutic, diagnostic, and/or other molecules of the invention to specific areas of the brain or spinal cord. For example, many maps correlating innervation of the dorsal root ganglia ("DRG") of a large number of animals have been widely available for some time. For example, the thirty-one pairs of human DRG are numbered by the corresponding vertebral foramen thorough which the root enters the spinal cord. There are seven cervical (C), 12 thoracic (T), five lumbar (L), and five sacral (S) DRG, which are numbered rostrally to caudally for each division of the vertebral column, and that the human facial skin is innervated by the three branches of the trigeminal nerve (i.e., the ophthalmic, maxillary, and mandibular branches) (see, e.g., Principals of Neural Science, Fourth Ed., Kandel, Eric R., et al., eds. 2000). Therapeutic and Diagnostic Applications

Methods of the invention can be used to diagnose, assess, prevent, or treat a neurological disorder such as a neurodegenerative disorder in a subject. As used herein, treating may include preventing, reducing, and/or delaying the onset of symptoms, and/or prolonging the life of a subject with a neurological disorder. Diagnosing maybe the detection and/or confirmation of the presence or absence of a disease or condition in a subject. A subject may be a vertebrate, for example a mammal (e.g., a human, mouse, rat, horse, dog, cat, etc.). In some embodiments, a therapeutic molecule may also function as a diagnostic molecule and a diagnostic molecule may also function as a therapeutic molecule. Thus a therapeutic molecule may include a detectable label and a detectable molecule may provide a therapeutic effect upon administration.

Methods of the invention also maybe used to diagnose and/or treat neurological disorders or conditions of the CNS, including but not limited to neuromuscular disorders. In some embodiments, methods of the invention may be used to treat one or more of the following neurological and/or neurodegenerative disorders or conditions: Motor Neuron disease, Multiple Sclerosis, epilepsy, Alzheimer's disease, Parkinson's disease, Multiple System Atrophy and other synucleopathies, Segawa syndrome, Friedreich Ataxia 1, Holgcuin Ataxia, Spastic Ataxia, Sensory Ataxia, Spinocerebellar Ataxia 1, Spinocerebellar Ataxia 2, Spinocerebellar Ataxia 3 (Machado- Joseph disease), Spinocerebellar Ataxia 4,

Spinocerebellar Ataxia 5, Spinocerebellar Ataxia 6, Spinocerebellar Ataxia 7, Spinocerebellar Ataxia 8, Spinocerebellar Ataxia 9, Spinocerebellar Ataxia 10, Spinocerebellar Ataxia 11 , Spinocerebellar Ataxia 12, Spinocerebellar Ataxia 13, Spinocerebellar Ataxia 14, Spinocerebellar Ataxia 15, Spinocerebellar Ataxia 16, Spinocerebellar Ataxia 17 (Huntington disease-like 4), Cerebellar Ataxia types I, II, and DI, Ataxia-Oculomotor Apraxia 1 and 2, Ataxia-Talangiectasia, Pallidopontonigral Degeneration, ischemic stroke, spinal cord injury, brain injury; Benign Focal Amyotrophy, Infantile Spinal Muscular Atrophy, Duchenne Muscular Dystrophy, Becher Muscular Dystrophy; Schinzel-Giedon syndrome, Spinal Bulbar Muscular Atrophy (Kennedy's disease), Tay-Sachs disease, Sandhoff disease, Dentatorubral- Pallidoluysian Atrophy, Haw River Syndrome, Frontotemporal Dementia, Myotonic

Dystrophy, Leigh syndrome, Brain Iron Accumulation (Hallervorden-Spatz disease), NARP syndrome, Retinal Pigmentosa, Glaucoma, various tauopathies including Frontotemporal Dementia, Pick disease, and Steele-Richardson-Olszewski syndrome, Amyotrophic Lateral Sclerosis (Lou Gehrig disease), early-onset Alzheimer disease, Huntington disease, Huntington disease-like 1, Huntington disease-like 3, Pontocerebellar hypoplasia, Gertstmann-Straussler disease, Fatal Familial Insomnia, Creutzfeldt- Jakob disease (CJD), variant Creutzfeldt- Jakob disease (vCJD), Iatrogenic CJD, Kuru, Jansky-Bielschowsky disease, Ceroid Lipofuscinosis Neuronal 1 (Santavuori disease), Ceroid Lipofuscinosis Neuronal 2 (Vogt-Spielmeyer disease), Ceroid Lipofuscinosis Neuronal Ceroid Lipofuscinosis Neuronal 4 (Kufs disease), Ceroid Lipofuscinosis Neuronal 5, Ceroid Lipofuscinosis Neuronal 6, normal aging, abnormal development, tumor (e.g. brain tumor), cancer (e.g., in the brain or CNS), or neoplasm (either benign or malignant), and Juvenile Primary Lateral Sclerosis. In certain embodiments, methods of the invention may be used to treat a neurodegenerative disease in which motor neurons are affected. Jn certain embodiments, the neurodegenerative disease may be Amyotrophic Lateral Sclerosis ("ALS"). It will be clear to those of ordinary skill in the art, that not all neurological and/or neurodegenerative conditions are abnormal or are indicative of illness. Some neurological and/or neurodegenerative conditions represent a normal state of a cell or tissue in development, growth, aging, and day-to-day cellular operations. In other embodiments, a neurological and/or neurodegenerative disease or condition may be an illness, injury, or other abnormal indication in a cell, tissue, or animal. In some aspects, the invention involves, in part, administration of an immunomodulatory agent to a subject and the administration of an additional molecule that prevents and/or treats diseases or conditions, including, but not limited to neurological and/or neurodegenerative diseases or conditions. Thus, methods of the invention may be useful to prevent or treat a neurological and/or neurodegenerative disease or condition in a subject. Administration of immunomodulatory agents and therapeutic molecules and delivery of the therapeutic molecules to neurons using methods of the invention may restore a population of neuronal cells and/or may protect an existing population from death, abnormal activity, and/or malfunction, m some embodiments, a therapeutic molecule may be a toxin or other compound that impairs function of or kills a target neuronal cell or tissue. For example, a therapeutic molecule may be a toxin or other molecule that either eliminates a cell or tissue (e.g., tumor tissue) and/or reduces an activity or function of a cell or tissue that is abnormal or undesirable. Methods of the invention may be used to reduce or eliminate a neurological and/or neurodegenerative disease or condition. It will be understood by one of ordinary skill in the art that the reduction of a neurological or neurodegenerative disease or condition need not always be the elimination of the disease or condition. Aspects of the invention involve, in part, the administration of an effective amount of an immunomodulatory agent and an effective amount of a therapeutic molecule to prevent and/or treat a neurological and/or neurodegenerative disease or condition. An "effective amount" is also referred to herein as an "amount sufficient." Typically an effective amount of an immunomodulatory agent or therapeutic molecule delivered using a method of the invention will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study. In some embodiments, an effective amount will be that amount that diminishes or eliminates a negative effect (e.g., symptom and/or physiological effect and/or clinical effect) of a neurological or neurodegenerative disease or condition in a subject. Thus, in some embodiments, an effective amount may be the amount that when administered prevents or treats a neurological or neurodegenerative disease or condition relative to what would occur in the subject without the administration of the immunomodulatory agent or therapeutic molecule using the methods of the invention. In certain embodiments, an effective amount of an immunomodulatory agent or therapeutic molecule delivered using a method of the invention, may each be an amount of the agent or molecule, respectively, that reduces symptoms and/or clinical manifestations from the level of symptoms or clinical manifestations that would occur in the subject or tissue without the administration of the immunomodulatory agent or therapeutic molecule using methods of the invention.

Aspects of the invention also involve, in part, administration of an immunomodulatory agent and therapeutic molecule using methods of the invention, in an amount that that reduces symptoms and/or clinical manifestations of a disease or condition in a subject as compared to the symptoms and/or clinical manifestations in a control subject. The invention, in part, relates to the administration of immunological agents and therapeutic molecules for the prevention and/or treatment of neurological and/or neurodegenerative diseases or conditions. As used herein the term "reduce" or "inhibit" a neurological or neurodegenerative disease or condition means to lower or decrease the likelihood that the neurological or neurodegenerative disease will be present or will be as severe as in an equivalent, untreated subject. As used herein, to "prevent" or "treat" a neurological or neurodegenerative disease or condition in a subject may include lowering the likelihood that a subject will have a neurological or neurodegenerative disease or condition; decreasing the severity of the disease or condition in the subject; delaying the onset or progression of the disease or condition; and/or increasing the likelihood of survival of a subject with the disease or condition. Thus, the administration of an immunomodulatory agent and therapeutic molecule will reduce symptoms and/or clinical manifestations in a subject may be an amount that is statistically significant versus a level or type of symptoms or clinical manifestations of the neurological and neurodegenerative disease or condition in a control subject or subjects. In certain embodiments, prevention or treatment may include an increase in cell number, cell or tissue function and/or activity in a subject or may be a reduction in loss of cells (e.g., a reduction in the amount of cell death) in the subject. Thus, the invention may involve, in part, the administration of an immunomodulatory agent and a therapeutic molecule using methods of the invention in amounts that that increase cell function, activity, and/or cell number thus reducing symptoms or clinical manifestations of a disease or condition in the subject. The reduction of symptoms or clinical manifestation in a subject administered an immunomodulatory agent and therapeutic molecule may be compared to the symptoms or clinical manifestations in a control subject not administered the immunomodulatory agent and therapeutic molecule. As used herein the term "increase" cell function, number, or activity means to raise the amount of function, number, or activity of cells, which may result in reduced symptoms or clinical manifestations of a disease or condition. It will be understood that an "increase" in cell number may be the result of a decrease in loss of cells — not necessarily an increase in overall number of cells. Thus, for example, a therapeutic molecule may act to reduce cell death in a neurodegenerative disease or condition, which would result in an increase number of cells as compared to (e.g., relative to) an untreated control with the neurodegenerative disease or condition. In some embodiments, a therapeutic molecule may kill, prevent proliferation, and/or prevent undesirable functioning of unwanted cells, e.g., cancer cells or diseased cells in certain neurological conditions. The invention relates in part to the administration of an amount of a therapeutic molecule using the methods of the invention, in an amount effective to treat, inhibit, or prevent a neurological or neurodegenerative disease or condition. In some embodiments, a therapeutic molecule that may be administered to prevent or treat a neurological or neurodegenerative disease or condition is a molecule that reduces or inhibits the disease or condition. In some embodiments, the disease or condition may be not be detectable in a subject - e.g., may be clinically asymptomatic. In some aspects, the invention involves, in part, administration of an immunomodulatory agent to a subject and the administration of a molecule that is detectable, or has a detectable product, and can be used to diagnose and/or detect the presence of a disease or condition, including, but not limited to neurological and/or neurodegenerative diseases or conditions. Thus, methods of the invention may be useful to diagnose or assess a neurological and/or neurodegenerative disease or condition in a subject. Administration of immunomodulatory agents and diagnostic molecules and delivery of the diagnostic molecules to neurons using methods of the invention may be used to detect the presence (or absence) of a target neuron or neurons; the size or expanse of a target neuron or neurons; and/or to determine connectivity between tissues, such as muscles, organs, and neurons, including, but not limited to: neuron-neuron connectivity, etc. A non-limiting example of a use of a diagnostic molecule administered using methods of the invention may be for the detection of a tumor (e.g., brain tumor) or for detection of a location and/or size/extent of a tumor in a subject.

The invention involves, in part, the administration of an effective amount of an immunomodulatory agent and an effective amount of a diagnostic molecule to permit detection of a target neuron or neuronal region in a subject. In some embodiment, a subject will have a neurological disorder or condition. Typically an effective amount of an immunomodulatory agent or diagnostic molecule delivered using a method of the invention will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study. In some embodiments, an effective amount will be that amount that permits sufficient detection of the desired neurons or region in a subject, ha some embodiments, an effective amount may be the amount that when administered permits detection of the presence or absence of neurons and/or neuronal regions relative to what could be detected in the subject without the administration of the administration of the immunomodulatory agent or therapeutic molecule using the methods of the invention. In certain embodiments, an effective amount of an immunomodulatory agent or diagnostic molecule delivered using a method of the invention, may each be an amount of the agent or molecule, respectively, that permits detection of a target neuron or neurons suitable for diagnosis and/or assessment of a neurological disorder or condition. The invention also involves, in part, administration of an immunomodulatory agent and diagnostic molecule using methods of the invention, in an amount that permits detection of neurons or target regions in a subject as compared to the detection possible in a control subject.

The invention also includes methods to assess and/or diagnose a disease or condition and methods to monitor the onset, progression, or regression of a disease or condition (e.g., a neurological disease or condition) in a subject. These methods may include, determining the delivery of a diagnostic molecule in a subject at sequential times and assaying such samples for the presence or absence of a diagnostic molecule administered according to a method of the invention. The characteristics of the delivery detected can be used as a marker of the disease or condition. Delivery characteristics include, but are not limited to: distance the molecule travels, amount of molecule delivered, expanse of the labeled cell(s) or regions, presence or absence of delivery, etc. A subject may be suspected of having a disease or condition or may be believed not to have a disease or condition and in the latter case, an initial determination of diagnostic molecule delivery may serve as a normal baseline level for comparison with subsequent determinations.

Methods of the invention maybe used to determine the onset of a disease or condition in a subject. Onset of a disease or condition is the initiation of changes associated with the disease or condition in a subject. Such changes may be evidenced by physiological symptoms, or may be clinically asymptomatic. For example, the onset of ALS may be followed by a period during which there may be ALS-associated pathogenic changes in the subject, even though clinical symptoms may not be evident at that time. The progression of a condition follows onset and is the advancement of the pathogenic (e.g., physiological) elements of the condition, which may or may not be marked by an increase in clinical symptoms. In contrast, the regression of a condition is a decrease in physiological characteristics of the condition, perhaps with a parallel reduction in symptoms, and may result from a treatment or may be a natural reversal in the condition.

A marker (e.g., a diagnostic sign) for a neurological disease or condition maybe the absence of, or an abnormal number of, neurons, or may be a sign of abnormal proliferation (e.g., a tumor in the CNS) that can be detected using the methods of the invention. Another example of a marker may be a change in connectivity of one or more neurons in a subject. Thus, a spinal injury may be assessed/diagnosed using the methods of the invention to detect whether normal connections between a tissue and its innervating neurons is damaged or intact. Methods of the invention can be used to compare neurons and/or connectivity in a control subject to that of a test subject. In a subject free of a condition (e.g., a control subject) administration of a diagnostic molecule may result in delivery to and labeling of a neuron or region of the CNS or may result in an innervation or connectivity pattern that can be detected and the administration of a diagnostic molecule to a subject with a neurological disease or condition may result in different delivery to or labeling of a neuron or region of the CNS and/or a different result in an innervation or connectivity pattern. In some embodiments, there may be a decrease or change in location of labeling of neuron(s) or brain regions in a subject with a neurological disease or condition compared to a control. In certain embodiments, there may be an increase in the amount or a change in the location of labeling of neuron(s) or brain regions in a subject with a neurological disease or condition compared to a control. In some embodiments, administration of a diagnostic molecule using methods of the invention may be repeated two or more times (e.g., monthly, annually, biannually etc.) allowing monitoring of onset, progression, and/or regression of a disease or condition in a subject.

Onset of a disease or condition may be indicated by the appearance of such a change in the labeling with a diagnostic molecule using a method of the invention. A change in the amount or location of a diagnostic label in a subj ect as compared to a previous determination for that subject may indicate the onset of a disease or condition in that subject. For example, if a "normal" (e.g., disease-free) pattern of delivery of a diagnostic molecule is present in a first determination in a subject, and a different pattern of delivery of the diagnostic molecule is determined to be present in a second or subsequent determination in the subject, it may indicate the onset of a neurological disease or condition in the subject.

Methods of the invention may also be used to determine the progression or regression of a disease or condition in a subject. Progression and regression of a neurological disease or condition may be generally indicated by changes in the pattern or delivery of a diagnostic molecule of the invention. Depending on the disease or disorder, a change maybe an increase or a decrease in delivery and/or a pattern change of the delivered diagnostic molecule. One of ordinary skill in the art can ascertain a specific change in delivery of a diagnostic molecule that maybe associated with progress or regression of a specific disease or condition based on the physiological characteristics of the disorder. For example, if a neurodegenerative disease is characterized by the loss of neurons in a specific region, then one of ordinary skill in the art would be able to use the methods of the invention to assess changes in delivery in the region to determine the onset, progression, or regression of the disease in a subject. Two or more determinations made a different time points can be used to assess changes in a subject's condition over time. Thus, methods of the invention may be used to monitor a subject who has been treated for a disease or condition and may be used to determine an effect of a therapeutic treatment.

Controls

In some embodiments of the invention, a "control" subject may be a reference subject who has a neurological or neurodegenerative disease or condition or may be a subject who is neurological or neurodegenerative-disease free. Identification of a subject as a control subject will depend upon the particular population selected. For example, an apparently healthy population of subjects can be used as control subjects in some embodiments, and a population of subjects that is known to have a disease or condition (e.g., a neurological or neurodegenerative disease or condition) may be used in some embodiments as control subjects. In certain embodiments, effects of administration of a molecule of the invention can be evaluated using animal models, e.g., mouse or rat models, etc. Accordingly, the methods of assessing a response to an immunomodulatory agent and a therapeutic molecule administered using methods of the invention may take into account the category in which an individual subject falls. Appropriate controls can be readily selected by those of ordinary skill in the art. In some embodiments, a control will be based on apparently healthy normal subject in an appropriate age bracket. hi some embodiments a control (e.g., comparative group) maybe a group of subjects with one or more specific neurological or neurodegenerative conditions or diseases. It will be understood that disease-free subjects may be used as comparative groups for subjects that have a neurological or neurodegenerative disease or condition.

Kits

The invention includes kits that include a container containing an immunomodulatory agent and a container containing a molecule (e.g., therapeutic molecule or diagnostic molecule) for administration using methods of the invention. In some embodiments, a container may contain a combination of a more than one molecule, and/or may contain a combination of an immunomodulatory agent and a molecule (e.g., therapeutic or diagnostic molecule). A kit of the invention may include one or more molecules (e.g., diagnostic and/or therapeutic molecules) and/or immunomodulatory agents. Kits may include also materials for use in standard detection methods.

In some embodiments, a kit may include components for labeling a molecule for use as a diagnostic molecule in the methods of the invention. A kit may also include solutions, tubes, vials, detectable-molecule readers, etc. A kit may include one or more therapeutic and/or diagnostic molecules, which may be antibodies, polypeptides, polynucleotides, etc. along with components useful for use of the molecules in the methods of the invention. The foregoing kits can include instructions or other printed material on how to use the various components of the kits for diagnostic and/or therapeutic purposes.

The following examples are illustrative only and are not intended to limit the scope of the invention in any way.

Examples

Example 1 : Materials and Methods Adenovirus vector: The Ad5 vector is a first generation El-, E3-deleted adenoviral type 5 vector. The modified Ad5 vector, optimized for neuronal uptake, was constructed with a fragment of the CMV early promoter/enhancer fused to a fragment of the chick beta-actin promoter, followed by a chick beta-actin intron fused to a rabbit beta-globin intron, followed by the transgene (including appropriate Kozak sequences/ ATG start site, coding sequence, and a termination codon), followed by a WPRE sequence (the Woodchuck post-translational regulatory element), followed by SV40 pA (SV40 polyadenylation sequences).

Transgene delivery:

Viral vector (1.3 x 10¹⁰ viral particles/injection site) was administered by intramuscular injection into motor end plates of the gastrocnemius and triceps brachii muscles. Mouse model ofAtnyotropic Lateral Sclerosis:

The murine model used for some of these studies was a transgenic model overexpressing the human mutant variant of superoxide dismutase (G93A).

Immunomodulation of the costimulatory pathways affecting T-cell activation:

Monoclonal antibodies to block the costimulatory pathways affecting T-cell activation, were injected systemically into mice. CD40-CD154 interaction was blocked with an anti- CD 154 monoclonal antibody (MRl, Biogen Idee, Cambridge MA).

Histology:

For histochemistry, mice were sacrificed and perfused with paraformaldehyde. Spinal cords and muscle tissue were embedded in OCT and processed for cryosectioning. GFP expression was monitored by direct epifluorescense. IGF-I expression was detected by colorimetric immunohistochemistry.

Example 2: Neuronal GFP expression

Ten C57B165 mice were dosed with 250 μg each of hamster anti-CD40 ligand monoclonal antibody (MRl) 48 hours prior to injection with an Ad5 adenoviral vector expressing the GFP marker protein as the transgene (pDC315 MCS-WRE/eGFP). In addition, the mice were dosed with MRl at the time of viral injection and again 48 hours after viral injection. The MRl antibody was injected intra-peritoneally in a 100 μl volume of sterile saline (the MRl solution was prepared by adding 1 ml of sterile saline solution to 300 μl of9.6 mg/ml MRl).

The adenoviral vector was injected bilaterally near the motor end plates of gastrocnemius muscles at a concentration of 1.31x10¹⁰ viral particles per injection site. A stock preparation of viral vector was diluted into sterile saline immediately prior to injection (80 μl into 2.5 ml saline). A volume of 25 μl was used per injection site.

Ten control mice were treated similarly except that they did not receive MRl or any other immunomodulatory agent prior to viral delivery. The presence of marker protein in the motor neurons and other neurons was analyzed in two experimental mice and two control mice at each of several time points after viral injection (14 days, 30 days, 60 days, 90 days, and 120 days). The marker protein was retrograde transported by motor neurons innervating the gastrocnemius muscle and GFP uptake was seen in the mouse neurons. There was widespread expression of GFP in neurons and processes of the lumbar spinal cord at 30 days post injection. Under both low and high power microscopy, cross sections from the mice showed the extensive expression of the cytosolic marker protein. Expression of the GFP protein continued in some cases through day 150, indicating long-term expression of the transgene. Expression of GFP also was observed at 60 days in non-motor neurons (e.g., tongue etc.) indicating that the transgene had been transported from the motor neurons to hypoglossal neurons.

Example 3: Neuronal IGF-I expression

To extend the work described in Example 2, a viral vector ( Ad5. CB A-hIGF-1- WPRE) encoding human insulin like growth factor 1 (hIGF-1) was delivered to neurons innervating the gastrocnemius and triceps brachii muscles after immunomodulation as previously described. Expression of hIGF-1 was demonstrated by colorimetric immunohistochemistry in motor neurons of the lumbar spinal cord. Results indicated the presence of hIGF-1 expression in the cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. These data demonstrate that, not only was the transgene efficiently taken up by motor neurons, but that it traveled through the central nervous system. Accordingly, these results show that immunomodulation combined with high dosage viral administration produces high efficiency uptake and retrograde transport of the virus to sensory and motor neurons innervating the injected muscle. The fact that a transgene can travel from peripheral motor neurons to the spinal cord has important implications for the treatment of a broad range of neurological conditions including neurodegenerative diseases. Methods of the invention can be used to efficiently deliver transgenes to neurons. Methods of the invention can be used (e.g., in gene therapies) for the treatment of a range of neurological disorders including neurodegenerative diseases.

Example 4: Mouse model of ALS

Prior to the onset of symptoms at 9 weeks of age, ALS (SOD1-G93A mutant) mice and control littermates were injected into four limbs (gastrocnemius and triceps brachii muscles) with one of three viral vectors encoding a non-toxic marker protein (eGFP). Seven weeks after injection, the mice were sacrificed and transgene expression was analyzed in the injected muscles and spinal lumbar motor neurons to determine if the marker protein was expressed and transported. The efficiency of the three vector systems, adenovirus, adeno- associated virus, or lenti virus was determined by comparing the relative levels of marker protein expression. Based on results from the marker protein experiments, the adenoviral vector system was selected and coupled with immunomodulation to express the known neuroprotective protein, IGF, in the ALS mice. Mutant mice and control littermates were injected with the IGF vector into four limbs as described above. The animals were behaviorally tested using a standard rotorod apparatus to determine whether the IGF treatment delays the onset of disease, improves motor function, and impacts the survival of the ALS mice. Upon completion of the behavior testing the mice were sacrificed and the expression of the exogenous IGF analyzed to confirm expression in the spinal motor neurons and skeletal muscle.

All of the references, patents and patent publications identified or cited herein are incorporated, in their entirety, by reference.

Although this invention has been described with respect to specific embodiments, the details of these embodiments are not to be construed as limitations. Various equivalents, changes and modifications can be made without departing from the spirit and scope of this invention, and it is understood that such equivalent embodiments are part of this invention. We claim:

Claims

1. A method for delivering a molecule to a neuron in a subject, the method comprising: administering an immunomodulatory agent to a subject; and injecting a molecule into an innervated tissue of the subject in an amount sufficient to be transported into a neuron of the subject.

2. The method of claim 1, wherein the innervated tissue is a muscle.

3. The method of claim 1, wherein the molecule is a therapeutic or diagnostic molecule.

4. The method of claim 1, wherein the immunomodulatory agent is an inhibitor of the co-stimulatory pathway.

5. The method of claim 2, wherein the immunomodulatory agent is an antibody that binds to CD40, CD40 ligand (CD154), CD80, CD86, or CD28.

6. The method of claim 5, wherein the antibody is MRl or the anti-human CD40L (hu5C8).

7. The method of claim 1 , wherein two or more immunomodulatory agents are administered.

8. The method of claim 1 , wherein the immunomodulatory agent is administered prior to inj ection of the molecule.

9. The method of claim 1 , wherein the molecule is hIGF- 1.

10. The method of claim 1 , wherein the neuron lies fully within the CNS.

11. A method of treating a neurological disorder, the method comprising: administering an immunomodulatory agent to a subject with a neurological disorder, and injecting a therapeutic molecule into an innervated tissue of the subject in an amount effective to treat the neurological disorder.

12. The method of claim 11, wherein the innervated tissue is muscle tissue.

13. The method of claim 11, wherein the neurological disorder is a neurological disease or condition.

14. The method of claim 13, wherein the neurological disease is a neurodegenerative disease.

15. The method of claim 13 , wherein the neurological disease or condition is : Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury, spinal cord injury, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, or peripheral nerve injury.

16. The method of claim 13 , wherein the neurological disease is Amyotropic Lateral Sclerosis.

17. The method of claim 11 , wherein the therapeutic molecule is hIGF- 1.

18. A method of monitoring the onset, progression, and/or regression of a neurological disease or condition in a subject, the method comprising; administering an immunomodulatory agent to the subject, injecting a diagnostic molecule to an innervated tissue in an amount sufficient to be transported into a neuron of the subject, and detecting the diagnostic molecule in the subject as a means of monitoring onset, progression, and/or regression of a neurological disease or condition in the subject.

19. The method of claim 18, wherein the innervated tissue is muscle tissue.

20. The method of claim 18, wherein the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury, spinal cord injury, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, or peripheral nerve injury.

21. The method of claim 18 , wherein the subj ect has the neurological disease or condition.

22. The method of claim 18, wherein the subject is a model for the neurological disease or condition.

23. A method of diagnosing a neurological disease or condition in a subject, the method comprising; administering an immunomodulatory agent to the subject, injecting a diagnostic molecule into an innervated tissue in the subject in an amount sufficient to be delivered into a neuron of the subject, and detecting the delivery of the diagnostic molecule in a neuron of the subject, wherein the delivery, pattern of delivery, or amount of delivery of the diagnostic molecule to a neuron of the subject is diagnostic for a neurological disease or condition in the subject.

24. The method of claim 23, wherein the innervated tissue is muscle tissue.

25. The method of claim 23, wherein the neurological disease or condition is: Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury, spinal cord injury, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), epilepsy, peripheral neuropathy, or peripheral nerve injury.

26. The method of claim 23, wherein the subject is suspected of having the neurological disease or condition.