WO2008094583A2 - Methods and devices for mri-based measurement of axonal transport in vivo and delivery of therapeutic substances to the cns - Google Patents

Abstract

Aspects of the invention relate to tetanus toxin C fragment conjugated to a MRI contrast agent, iron nanoparticle, or biodegradable nanoparticle. Aspects of the invention relate to methods of determining neurodegeneration in a subject by administering a tetanus toxin C fragment conjugated to a MRI contrast agent, iron nanoparticle, or biodegradable nanoparticle and measuring axonal transport.

Description

METHODS AND DEVICES FOR MRI-BASED MEASUREMENT OF AXONAL

TRANSPORT IN VIVO AND DELIVERY OF THERAPEUTIC

SUBSTANCES TO THE CNS

Field of the Invention

Aspects of the invention relate to tetanus toxin C fragment conjugates comprising MRI contrast agents, iron core nanoparticles or biodegradable nanoparticles, directly conjugated to tetanus toxin C fragment. Aspects of the invention relate to methods for determining neurodegeneration in a subject comprising administering a tetanus toxin C fragment conjugate that is retrogradely transported to the central nervous system.

Background of the Invention

ALS remains a lethal and untreatable neurodegenerative disorder. Diagnosis of ALS in a subject is delayed. No ALS biomarkers have been discovered to aid in diagnosis. Multiple factors impeded the delivery of therapeutics to the central nervous system. A need exists for improved methods of delivering therapeutics to the central nervous system to treat neurodegenerative disorders such as ALS. Methods for determining axonal transport of compositions to the central nervous system will provide valuable information to aid in the delivery of therapeutics and the treatment of neurodegenerative disorders.

Summary of the Invention

Aspects of the invention relate to tetanus toxin C fragment conjugates capable of retrograde axonal transport and methods for determining retrograde axonal transport. According to aspects of the invention, a conjugate comprising tetanus toxin C fragment (TTC) directly conjugated to a magnetic resonance imaging (MRI) contrast agent, iron core nanoparticle, or biodegradable nanoparticle, is provided. In certain embodiments, the MRI contrast agent-TTC conjugate, iron core nanoparticle-TTC conjugate or biodegradable nanoparticle-TTC conjugate binds to neuron cells. In some embodiments, the MRI contrast agent-TTC conjugate, iron core nanoparticle-TTC conjugate or biodegradable nanoparticle-TTC conjugate binds to neuron cells with high affinity. In certain embodiments, the MRI contrast agent-TTC conjugate, iron core nanoparticle-TTC conjugate or biodegradable nanoparticle-TTC conjugate is retrogradely transported from the neuronal periphery to the central nervous system. In some embodiments, the conjugate retains similar retrograde transport properties as the tetanus toxin C fragment alone. In certain embodiments, the MRI contrast agent, iron core nanoparticle or biodegradable nanoparticle is directly conjugated to the tetanus toxin C fragment by amine reactive chemistry. In other embodiments, the MRI contrast agent of any of the conjugates is gadolinium. In certain embodiments, a therapeutic molecule is attached to or incorporated into the

MRI contrast agent-TTC conjugate, the iron core nanoparticle-TTC conjugate, or the biodegradable nanoparticle-TTC conjugate. In some embodiments, the biodegradable nanoparticle encapsulates the therapeutic molecule. In some embodiments, the biodegradable nanoparticle is formulated to release the therapeutic molecule over a predetermined time. According to aspects of the invention, compositions comprising the conjugates contemplated by the invention are provided. In some embodiments, the composition further includes a therapeutic molecule. In certain embodiments, the composition further includes a pharmaceutically acceptable carrier. In some embodiments, the composition comprising the conjugate includes any conjugate of the invention. In certain embodiments, the composition of the invention further includes a pharmaceutically acceptable carrier.

In some embodiments of the foregoing conjugates or compositions, the therapeutic molecule is a RNAi molecule. Preferably the RNAi molecule is a siRNA molecule or a vector that expresses a RNAi molecule. More preferably, the RNAi molecule is targeted to mutant SODl . According to aspects of the invention, methods of determining neurodegeneration of a subject comprising, administering to a subject a conjugate comprising tetanus toxin C fragment (TTC) directly conjugated to a MRI contrast agent, iron core nanoparticle, or biodegradable nanoparticle, wherein said conjugate is retrogradely transported to the central nervous system, and measuring retrograde axonal transport of the conjugate in the subject to determine neurodegeneration are provided. In certain embodiments, retrograde axonal transport is measured quantitatively. In other embodiments, the conjugate is administered intramuscularly, intraperitoneally, systemically, intravascularly, or via infusion into the cerebrospinal fluid. In some embodiments, the conjugate binds neuron cells. In certain embodiments, the conjugate binds neuron cells with high affinity. In some embodiments, the conjugate is retrogradely transported from the neuronal periphery to the central nervous system. In other embodiments, the MRI contrast agent is gadolinium. In some embodiments, the neurodegeneration is a result of amyotrophic lateral sclerosis. In certain embodiments, the conjugate further comprises a therapeutic molecule. In other embodiments, the conjugate delivers the therapeutic molecule to motor neurons. In certain embodiments, the therapeutic molecule is attached to or incorporated into the MRI contrast agent-TTC conjugate, the iron core nanoparticle-TTC conjugate, or the biodegradable nanoparticle-TTC conjugate. In some embodiments, the biodegradable nanoparticle encapsulates the therapeutic molecule. In other embodiments, the biodegradable nanoparticle is formulated to release the therapeutic molecule over a predetermined time.

In some embodiments of the foregoing methods, the therapeutic molecule is a RNAi molecule. Preferably the RNAi molecule is a siRNA molecule or a vector that expresses a RNAi molecule. More preferably the RNAi molecule is targeted to mutant SOD l . According to aspects of the invention, methods for treating neurodegeneration in a subject are provided. The methods include administering to a subject the foregoing conjugate or composition, which contains a therapeutic molecule, wherein the conjugate or composition is administered in an amount effective to treat neurodegeneration, and wherein said therapeutic molecule is retrogradely transported to the central nervous system. In some embodiments, the neurodegeneration is a result of amyotrophic lateral sclerosis.

According to aspects of the invention, any of the foregoing conjugates prepared by the process of amine reactive chemistry are provided. Also provided are compositions containing such conjugates.

Brief Description of the Drawings

Figure 1 : Diagrammatic representation of a neuronal cell with mutant SOD l .

Figure 2: Diagrammatic representation of tetanus toxin C fragment conjugated to A) gadolinium, B) an iron core nanoparticle, and C) a biodegradable nanoparticle.

Figure 3: Diagrammatic representation of amine-reactive chemistry conjugation of gadolinium to tetanus toxin C fragment.

Figure 4: SDS protein gel showing tetanus toxin C fragment conjugated to gadolinium, tetanus toxin C fragment alone, gadolinium conjugated to BSA, and BSA alone.

Figure 5: Graphs showing flow cytometry analysis of fluorescein conjugated to TTC or BSA. Figure 6: In vitro MRI data showing detection and quantification of tetanus toxin C fragment conjugated to gadolinium.

Figure 7A-D: MRI results of in vivo administration of tetanus toxin C fragment alone and conjugated.

Figure 8A-E: MRI results of in vivo administration of tetanus toxin C fragment alone and conjugated. Sections of mouse brain are shown.

Detailed Description of the Invention

Amyotrophic lateral sclerosis (ALS) is an orphan disease with an incidence of 1 - 2/100,000. ALS is uniformly fatal within 4-5 years. The hallmark symptom of ALS is the death of motor neurons leading to weakness, muscle denervation and atrophy. Motor neuron death begins focally and then spreads throughout the body. Damage to a neuron results in the neuron being unable to control the muscle as a normal undamaged neuron does. Cytoskeletal pathology in affected motor neurons is also associated with ALS. Ubiquitinated protein aggregates form in the motor neurons. ALS reflects a confluence of three main factors: aging, the environment, and genetic predisposition. Environmental factors such as pesticides, infection with atypical retroviruses or lyme-like agents, and toxins have been associated with ALS. ALS has also been associated with genes such as SODl , ALSIN, senataxin, VAPB and dynactin. 3% of ALS cases have been associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SODl). The wildtype SOD 1 gene converts superoxide anion to hydrogen peroxide through the reduction and oxidation of copper. Mutations in the SOD l gene cause toxicity in the neuronal cell (Figure 1).

ALS is a neurodegenerative disorder causing the death of motor neurons leading to the breakdown of muscle function. Axonal transport of compositions provides a method for determining delivery of compositions and therapeutic substances to the central nervous system. Compositions capable of binding to neuron cells and being retrogradely transported to the central nervous system provide a means for quantitatively measuring axonal transport.

Aspects of the invention relate to tetanus toxin C fragment conjugates. The tetanus toxin C fragment may be conjugated directly to MRI contrast agents, iron core nanoparticles or biodegradable nanoparticles. A tetanus toxin C (TTC) fragment conjugate encompasses a TTC-MRI contrast agent conjugate, a TTC-iron core nanoparticle conjugate, and a TTC- biodegradable nanoparticle conjugate. Other TTC-conjugates are also contemplated, such as TTC-conjugated to proteins, peptides, RNA, DNA, therapeutic molecules, or antibodies.

According to aspects of the invention, tetanus toxin C fragment may be directly conjugated to MRI contrast agents, iron core nanoparticles or biodegradable nanoparticles using amine-reactive chemistry.

A tetanus toxin C fragment conjugate is one that retains similar binding and retrograde transport properties as the tetanus toxin C fragment alone. A tetanus toxin C fragment conjugate may bind neuron cells. As shown by the in vitro and in vivo studies described in the Examples below, a tetanus toxin C fragment conjugate binds neuron cells with high affinity.

According to aspects of the invention, a tetanus toxin C fragment conjugate may be retrogradely transported to the central nervous system. Retrograde transport involves the transport of the conjugate from the neuronal periphery to the central nervous system. In aspects of the invention, a MRI contrast agent is any agent capable of producing a signal that can be detected using magnetic resonance imaging or other similar imaging method. Contrast agents are chemical substances that may be introduced to the anatomical or functional region being imaged, to increase the differences between different tissues or between normal and abnormal tissue, by altering the relaxation times. MRI contrast agents are classified by the different changes in relaxation times after their injection. Positive contrast agents cause a reduction in the Tl relaxation time (increased signal intensity on Tl weighted images). They (appearing bright on MRI) are generally small molecular weight compounds containing as their active element Gadolinium, Manganese, or Iron. All of these elements have unpaired electron spins in their outer shells and long relaxivities. Some typical contrast agents as gadopentetate dimeglumine, gadoteridol, and gadoterate meglumine may be utilized for the central nervous system and the complete body. Contrast agent mangafodipir trisodium is generally used for lesions of the liver and gadodiamide for the central nervous system. Negative contrast agents (appearing predominantly dark on MRI) may be small particulate aggregates, also known as superparamagnetic iron oxide (SPIO). These agents produce predominantly spin-spin relaxation effects (local field inhomogeneities), which results in shorter Tl and T2 relaxation times. SPIO's and ultrasmall superparamagnetic iron oxides (USPIO) generally include a crystalline iron oxide core containing thousands of iron atoms and a shell of polymer, dextran, polyethyleneglycol, and produce very high T2 relaxivities. USPIOs smaller than 300 nm cause a substantial T 1 relaxation. T2 weighted effects may be predominant. Another group of negative contrast agents (appearing dark on MRI) are perfluorocarbons (perfluorochemicals). Such contrast agents appear dark on MRI because their presence excludes the hydrogen atoms responsible for the signal in MR imaging. A MRI contrast agent may be nonionic or iodinated. In certain embodiments, a MRI contrast agent is gadolinium or gadolinium based.

According to aspects of the invention, a tetanus toxin C fragment conjugate may be formulated as a composition. Such formulation may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.

According to aspects of the invention, a tetanus toxin C fragment conjugate may further include one or more therapeutic molecules. Therapeutic molecules may be attached to or incorporated into the MRI contrast agent-TTC conjugate, the iron core nanoparticle-TTC conjugate, or the biodegradable nanoparticle-TTC conjugate. The biodegradable nanoparticle-TTC conjugate may encapsulate the therapeutic molecule.

Therapeutic molecules may be included in an effective amount. Effective amounts or amounts effective are well known to those of ordinary skill in the art and are described in the literature. A therapeutically effective amount will be determined by the parameters discussed below; but, in any event, is that amount which establishes a level of a therapeutic or combination of therapeutics effective for treating a subject, such as a human subject, to prevent or reduce neurodegeneration. An effective amount means that amount alone or with multiple doses, necessary to delay the onset of, inhibit completely or lessen the progression of or halt altogether the onset or progression of the condition being treated. When administered to a subject, effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight; concurrent treatment; frequency of treatment; and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. The doses of the therapeutic molecules administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

In certain aspects of the invention, a tetanus toxin C fragment conjugated to biodegradable nanoparticles may be formulated to allow slow release such that a therapeutic molecule is released over a period of time, preferably a predetermined period of time. The slow release formulation may be formulated to release the therapeutic molecule over a predetermined period of time. Such treatment allows continuous delivery of the therapeutic molecule and reduces the number of doses required. Such slow release formulations are known to those of ordinary skill in the art. For example, the rate of release from the tetanus toxin C fragment conjugate of the invention can be controlled by selecting the release rate properties of the nanoparticles that are conjugated to tetanus toxin C fragment. According to aspects of the invention, methods for determining neurodegeneration in vivo are provided. A tetanus toxin C fragment conjugate is administered to a subject and retrograde axonal transport is measured. Axonal transport may be measured using methods such as magnetic resonance imaging (MRI). In some embodiments, axonal transport may be measured quantitatively. A "neurodegenerative disorder" is defined herein as a condition in which there is progressive loss of neurons in the nervous system. One of ordinary skill in the art would be familiar with the target area in the brain of a subject affected by a neurodegenerative disorder and therefore in need of treatment according to the instant invention. Examples of neurodegenerative disorders include a broad group of chronic neurodegenerative conditions such as familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively). familial and sporadic Parkinson's disease, Huntington's disease, familial and sporadic Alzheimer's disease, fronto-temporal dementia, multiple sclerosis, spinocerebellar atrophies such as olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse Lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, striatonigral degeneration, torsion dystonia, familial tremor, Down's Syndrome, Gilles de Ia Tourette syndrome, Hallervorden-Spatz disease, familial and sporadic peripheral neuropathies, dementia pugilistica, AIDS dementia, age-related dementia, age- associated memory impairment, and amyloidosis-related neurodegenerative diseases such as those caused by the prion protein (PrP) which is associated with transmissible spongiform encephalopathies (e.g., Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, scrapie, and kuru), and those caused by excess cystatin C accumulation (hereditary cystatin C angiopathy). The present invention also has application to other conditions in which there is acute or sub-acute deterioration of the brain including not only stroke and trauma, but also cerebral edema, surgery-related brain injury, metabolic brain diseases such as Wernicke-Korsakoff s dementia, and acute peripheral nerve injury. These examples are not meant to be comprehensive but serve merely to illustrate potential applications for this invention. Most of the chronic neurodegenerative diseases are typified by onset during the middle adult years and lead to rapid degeneration of specific subsets of neurons within the nervous system, ultimately resulting in premature death.

Alzheimer's disease is one of the most important of the neurodegenerative diseases due to the high frequency of occurrence within the population and the fatal course of the disease. It is characterized by loss of function and death of nerve cells in several areas of the brain leading to loss of cognitive function such as memory and language. There are both familial and sporadic forms of Alzheimer's disease, and there are both early onset (presenile) and later onset (senile) forms of the disease; in all of these forms, there is considerable overlap in the general pathological findings. The cause of nerve cell death is unknown but the affected neurons are recognized by the presence of unusual helical protein filaments within the cells (neurofibrillary tangles), by extracellular deposition of another abnormal protein, beta amyloid, and by neuronal degeneration. There is also gross atrophy in cortical regions of brain, especially frontal and temporal lobes. A clear genetic predisposition has been found for presenile dementia. Familial autosomal dominant cases have been reported and the majority of individuals with trisomy 21 (Down's syndrome) develop presenile dementia after the age of 40.

Amyotrophic lateral sclerosis (ALS) is the most commonly diagnosed progressive motor neuron disease. The disease is characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord (Harrison's Principles of Internal Medicine, 1991 McGraw-Hill, Inc., New York; Tandan et al. Ann. Neurol, 18:271-280, 419-431 , 1985). Generally, the onset is between the third and sixth decade, typically in the sixth decade; ALS is uniformly fatal. Although some genetic bases have been demonstrated (e.g., mutations in superoxide dismutase gene on chromosome 21 ; see Rosen et al., Nature 362:59-62, 1993), these genetic abnormalities do not uniformly exist in ALS patients, and thus the full spectrum of causes of the disease is yet unknown.

In ALS motor neurons of the cerebral cortex, brainstem and anterior horns of the spinal cord are affected. The class of neurons affected is highly specific: motor neurons for ocular motility and sphincteric motor neurons of the spinal cord remain unaffected until very late in the disease. Death in ALS is generally due to respiratory failure secondary to profound generalized and diaphragmatic weakness. About 10% of ALS cases are inherited as an autosomal dominant trait with high penetrance after the sixth decade (Mulder et al. Neurology, 36:51 1-517, 1986; Horton et al. Neurology, 26:460-464, 1976). In almost all instances, sporadic and autosomal dominant familial ALS (FALS) are clinically similar (Mulder et al. Neurology, 36:51 1-517, 1986; Swerts et al., Genet. Hum, 24:247-255, 1976; Huisquinet et al., Genet. 18: 109-1 15, 1980). As noted, in some but not all FALS pedigrees the disease is caused by defects in the gene on chromosome 21q that encodes the cytosolic protein, Cu/Zn superoxide dismutase (Rosen et al., Nature 362:59-62, 1993). While a single drug has been approved by the F. D. A. for treatment of ALS, its effect is minimal at best; there is no primary therapy for ALS.

Parkinson's disease (paralysis agitans) is a common neurodegenerative disorder that appears in mid to late life. Familial and sporadic cases occur, although familial cases account for only 1 -2 percent of the observed cases. Patients frequently have nerve cell loss with reactive gliosis and formation of Lewy bodies in the substantia nigra and locus coeruleus of the brainstem. Similar changes are observed in the nucleus basalis of Meynert and, in the long term, the nerve cell loss may be quite widespread. As a class, the nigrostriatal dopaminergic neurons seem to be most affected. The disorder generally develops asymmetrically with tremors in one hand or leg and progresses into symmetrical loss of voluntary movement. Eventually, the patient becomes incapacitated by rigidity and tremors. In the advanced stages the disease is frequently accompanied by dementia. Diagnosis of both familial and sporadic cases of Parkinson's disease can only be made after the onset of the disease. While there are symptomatic therapies for Parkinson's disease, there is no primary treatment that slows the underlying neurodegeneration in this disease.

Huntington's disease is a progressive disease characterized by a movement disorder and dementia; it is always transmitted as an autosomal dominant trait. Individuals are asymptomatic until the middle adult years, although some patients show symptoms as early as age 15. Once symptoms appear, the disease is characterized by choreoathetotic movements and progressive dementia until death occurs 15-20 years after the onset of symptoms. Patients with Huntington's disease have progressive atrophy of the caudate nucleus and the structures of the basal ganglia. Atrophy of the caudate nucleus and the putamen is seen microscopically where there is an excessive loss of neural tissue. However, there are no morphologically distinctive cytopathological alterations.

Although some of the characteristic mental depression and motor symptoms associated with Huntington's disease may be suppressed using tricyclic antidepressants and dopamine receptor antagonists, respectively, no therapy exists for slowing or preventing of the underlying disease process. Huntington's disease appears to map to a single gene on chromosome 4 that encodes a protein known as "huntingtin". The huntingtin gene in its mutant form contains pathological expansions of CAG repeats (see US Patent 5,686,288). A genetic test currently exists for the clinical assessment of disease risk in presymptomatic individuals with afflicted relatives but there is no primary therapy for Huntington's disease. Hallervorden-Spatz disease is a neurodegenerative disease that affects neurons in the region of the basal ganglia. Symptoms generally first appear during childhood or adolescence and the disease seems to be inherited in an autosomal recessive fashion. Patients show abnormalities in muscle tone and movement such a choreoathetosis and dystonia similar to that seen in parkinsonism. As the disease progresses there is increasing dementia. Death generally occurs approximately ten years after onset. There is no known presymptomatic diagnosis, cure or treatment for Hallervorden-Spatz disease. However, iron toxicity has recently been implicated in the progression of this disease (Greenfield, Neuropathology, W. Blackwood & J. A. N. Corsellis, Eds. (Edinborgh; T. and A. Constable, Ltd., 1976) pages 178-180). As a result of this implication, the chelating agent deferoxamine mesylate has been administered to patients. However, this therapeutic approach has shown no definite benefit (Harrison's Principles of Internal Medicine, Wilson et al. Eds., McGraw-Hill, Inc., New York, 1991). There is no primary treatment for this highly debilitating disease. A large group of neurodegenerative diseases are described as the "spinocerebellar atrophies" (SCA). These all entail progressive degeneration of various subsets of neurons in the cerebellum, brainstem and other regions of the neuraxis; most are inherited as dominant traits. Among these are the olivopontocerebellar atrophies (OPCA), which include a number of disorders characterized by a combination of cerebellar cortical degeneration, atrophy of the inferior olivary nuclei, and degeneration of the pontine nuclei in the basis pontis and middle cerebellar peduncles. Many of the spinocerebellar atrophies arise from expansions of CAG repeat domains in different types of neurons (for example, Orr et al., Nature Genetics 4:221-226, 1993). According to aspects of the invention, a tetanus toxin C fragment conjugate may be administered via any suitable route. In some embodiments, a tetanus toxin C fragment may be administered intramuscularly, intraperitoneally, systemically, intravascularly, or via infusion into the cerebrospinal fluid.

According to aspects of the invention, methods for treating neurodegeneration in a subject are provided. A tetanus toxin C fragment conjugate of the invention may be administered to a subject in an amount effective to treat neurodegeneration.

According to aspects of the invention, the terms "treating" and "treatment" include prophylaxis and therapy. When provided prophylactically, a treatment may be administered to a subject in advance of neurodegeneration (e.g., to a patient at risk of a neurodegenerative disorder), or upon the development of early signs of neurodegeneration in a patient. A prophylactic treatment serves to prevent, delay, or reduce the rate of onset of neurodegeneration or the appearance of symptoms associated with neurodegeneration. A prophylactic treatment may reduce the incidence and accelerate the recovery of neural and muscle function. When provided therapeutically, a treatment may be administered at (or shortly after) the onset of the appearance of symptoms of neurodegeneration. Therapy may include preventing, slowing, or stopping neurodegeneration, or certain symptoms associated with neurodegeneration. In some embodiments, a treatment may serve to reduce the severity and duration of neurodegeneration, or symptoms thereof. In some embodiments, treating a subject may involve halting or slowing the progression of neurodegeneration, or of one or more symptoms associated with neurodegeneration. In some embodiments, treating a subject may involve preventing, delaying, or slowing the onset or progression of long-term symptoms associated with neurodegeneration. Treatment, in another aspect of the invention, can include administering therapeutic molecules that produce RNA interference to reduce expression level and/or function level of polypeptides in the subject. The use of RNA interference or "RNAi" involves the use of double-stranded RNA (dsRNA) to block gene expression, (see, e.g., Sui, G, et al., Proc Natl. Acad. Sci U.S.A. 99:5515-5520,2002). Methods of applying RNAi strategies in embodiments of the invention are understood by one of ordinary skill in the art.

Methods in which small interfering RNA (siRNA) molecules are used to reduce the expression of polypeptides may be used. In one aspect, a cell is contacted with a siRNA molecule to produce RNA interference (RNAi) that reduces expression of one or more polypeptides. The siRNA molecule is directed against nucleic acids coding for the polypeptide (e.g., RNA transcripts including untranslated and translated regions) as is well known in the art. In a preferred aspect of the invention the polypeptide is mutant SODl . The expression level of the targeted polypeptide(s) can be determined using well known methods such as Western blotting for determining the level of protein expression and Northern blotting or RT-PCR for determining the level of mRNA transcript of the target gene.

As used herein, a "siRNA molecule" is a double stranded RNA molecule (dsRNA) consisting of a sense and an antisense strand or a single stranded molecule that has a dsRNA component, for example a section of the molecule that hybridizes to itself (e.g., a "hairpin" structure). The antisense strand of the siRNA molecule is a complement of the sense strand (Tuschl, T. et al., 1999, Genes & Dev., 13:3191-3197; Elbashir, S.M. et al., 2001 , EMBO J., 20:6877-6888; incorporated herein by reference). In one embodiment the last nucleotide at the 3' end of the antisense strand may be any nucleotide and is not required to be complementary to the region of the target gene. The siRNA molecule may be 19-23 nucleotides in length and form a hairpin structure. In one preferred embodiment the siRNA molecule includes a two nucleotide 3' overhang on the sense strand. In a second preferred embodiment the two nucleotide overhang is thymidine-thymidine (TT). The siRNA molecule corresponds to at least a portion of a target gene transcript. In one embodiment the siRNA molecule corresponds to a region selected from a cDNA target gene beginning between 50 to 100 nucleotides downstream of the start codon. In a preferred embodiment the first nucleotide of the siRNA molecule is a purine.

The siRNA molecules can be plasmid-based. In a preferred method, a nucleic acid sequence that encodes a polypeptide is amplified using the well known technique of polymerase chain reaction (PCR). The use of the entire polypeptide encoding sequence is not necessary; as is well known in the art, a portion of the polypeptide encoding sequence is sufficient for RNA interference. The PCR fragment is inserted into a vector using routine techniques well known to those of skill in the art. In one aspect the nucleotide encoding sequence is the coding sequence of mutant SODl . Combinations of the foregoing can be expressed from a single vector or from multiple vectors introduced into cells, including by administration to a subject.

In one aspect of the invention a mammalian vector comprising any suitable nucleotide sequences is provided. The mammalian vectors include but are not limited to the pSUPER RNAi vectors (Brummelkamp, T.R. et al., 2002, Science, 296:550-553, incorporated herein by reference). In one embodiment a nucleotide coding sequence can be inserted into the mammalian vector using restriction sites, creating a stem-loop structure. In a second embodiment, the mammalian vector may comprise the polymerase-III Hl -RNA gene promoter. The polymerase-III Hl-RNA promoter produces a RNA transcript lacking a polyadenosine tail and has a well-defined start of transcription and a termination signal consisting of five thymidines (T5) in a row. The cleavage of the transcript at the termination site occurs after the second uridine and yields a transcript resembling the ends of synthetic siRNAs containing two 3' overhanging T or U nucleotides. The antisense strand of the siRNA molecule hybridizes to the corresponding region of the mRNA of the target gene. Preferred systems for mRNA expression in mammalian cells are those such as pSUPER RNAi system as described in Brummelkamp et al. (2002). Other examples include but are not limited to pSUPER.neo, pSUPER.neo+gfp, pSUPER.puro, BLOCK-iT T7-TOPO linker, pcDNA1.2/V5-GW/lacZ, pENTR/U6, pLenti6-GW/U6-laminshrna, and pLenti6/BLOCK-iT-DEST. These vectors are available from suppliers such as Invitrogen®, and one of skill in the art is able to obtain and use them routinely.

Examples

Example 1

Tetanus Toxin C Fragment and Delivery to Motor Neurons:

Tetanus toxin C fragment binds to motor neurons. MRI contrast agents such as gadolinium, iron core nanoparticles, and biodegradable nanoparticles are directly conjugated to tetanus toxin C fragment (Figure 2). The conjugated compositions are delivered to motor neurons.

Synthesis of Gd-TTC: Amine-reactive chemistry was used to conjugate gadolinium (Gd) to the tetanus toxin

C fragment (Figure 3). A gadolinium protein labeling kit was used for preparation of Gd- TTC according to manufacturer's directions. Briefly, 4 mg TTC protein was dissolved in 1.4mL 0.2M carbonate buffer (pH 8.9, final protein concentration 2.86mg/mL). 393uL of IM sodium acetate containing IM sodium hydroxide and 6.66uL IM GdC13 were added to 7.3μmol of an amine-reactive gadolinium chelate in sequence. The chelate was vortexed for approximately 2 minutes and allowed to incubate for 5 minutes at RT. lOOuL of the subsequent chelate was incubated with 4mg of the TTC protein under gentle stirring for 2 hours. Subsequent product was dialyzed with PBS for 2 hours (RT), 2 hours (RT), and overnight (4°C) against a 10 kDa dialysis cassette (Pierce Biotechnology). Dialysis medium was exchanged between each dialysis period. Protein was lyophilized and stored at -2O⁰C until use.

Protein Gel Electrophoresis:

The Gd-TTC conjugate was analyzed using protein gel electrophoresis (Figure 4). ~20ug/lane of each sample was loaded onto a 15% SDS gel. The gel was stained with Coomassie blue stain overnight. The gel was destained until bands were visible, and photographed. The gel shows that the conjugation was successful, there is not a large amount of unlabeled protein, and that there is a distribution of the number of Gd molecules on the TTC.

Flow Cytometry Analysis:

Flow cytometry analysis was used to measure the cellular uptake of fluorescein conjugated to tetanus toxin C fragment or bovine serum albumin (BSA) (Figure 5). NHS- FITC was incubated with TTC or BSA in different molar ratios (FITC:TTC, or FITC:biotin) for 2 hrs under gentle shaking. The molar ratios were 1 : 1 , 8: 1 , and 24: 1 FITCTTC, or

FITC:biotin. A similar amine-reactive chemistry was used to prepare the conjugates as that used to conjugate Gd to the TTC as described above. Protein was separated from unbound FITC by a gravity size separation column. Protein was incubated on cells in suspension for 30 minutes, washed by centrifugation 3x with PBS, and analyzed on flow cytometry. The upper 5% of unlabeled cells were gated. Values were plotted on a graph as the value of cells in each sample in the 5% of control gate.

The flow cytometry shows that TTC still maintains specificity for neurons. The observed dual peaks may be a result of heterogenous cell population in the cells (larger and smaller cells).

The larger BSA shift on the 24: 1 ratio may be due to unconjugated FITC removal that was not 100% successful (i.e. not all washed out).

MRI relaxivity measurements:

Gd-TTC and Gd-BSA were synthesized and diluted in 1 :2 linear dilutions in a 96-well plate (20OuL per well). Space between wells and surrounding wells were filled with PBS to preserve a uniform magnetic field. The plate was imaged at 1.5T using inversion recovery sequences for measurement of Tl with inversion times varied between 20-4000ms. Average signal intensity was quantified using ImageJ by identifying a region of interest (ROI) in each well at each inversion time. Intensity data was fit to the following equation using Matlab. Relaxivity values were determined by fitting to the above relationship (Figure 6).

In vivo Studies: Animals were injected on one side with TTC, or Gd-TTC (5OuL injection, 20mg/mL, lmg total injection). 48 hours post-injection, the animals were sacrificed. Transcardial perfusion with PBS was performed, then repeated with 4% paraformaldehyde. The samples were fixed overnight in 4% paraformaldehyde at 4°C. Lumbar sections were sectioned with a cryostat. Samples were incubated in blocking solution for 1 h. Primary antibody was added and incubated overnight at 4°C (rabbit polyclonal or mouse monoclonal for TTC, NeuN was mouse conjugated to FITC). Samples were washed 3x in PBS and a secondary antibody added and incubated for 2h at RT. Samples were washed 3x in PBS. NeuN was incubated with rabbit polyclonal as a secondary antibody. Samples were incubated in secondary antibody for 2h at RT. DAPI mounting medium was added to mount the samples. The results are shown in Figures 7A-D.

Three 25uL injections containing Gd-TTC (20mg/mL) were administered into the quadriceps of mice (right side only). MRI sequence was performed 24-30 hrs post injection. The animals were anesthetized using isoflurane, and surface MRI coil 9.4T MRI Tl -weighted sequences obtained. The results are shown in Figures 8A-8E.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All references disclosed herein are incorporated by reference in their entirety.

We claim:

Claims

Claims

1. A conjugate comprising tetanus toxin C fragment (TTC) directly conjugated to a magnetic resonance imaging (MRI) contrast agent, iron core nanoparticle, or biodegradable nanoparticle.

2. The conjugate of claim 1 , wherein the MRI contrast agent-TTC conjugate, iron core nanoparticle-TTC conjugate or biodegradable nanoparticle-TTC conjugate binds to neuron cells.

3. The conjugate of claim 2, wherein the MRI contrast agent-TTC conjugate, iron core nanoparticle-TTC conjugate or biodegradable nanoparticle-TTC conjugate binds to neuron cells with high affinity.

4. The conjugate of claim 1 , wherein the MRI contrast agent-TTC conjugate, iron core nanoparticle-TTC conjugate or biodegradable nanoparticle-TTC conjugate is retrogradely transported from the neuronal periphery to the central nervous system.

5. The conjugate of claim 4, wherein the conjugate retains similar retrograde transport properties as the tetanus toxin C fragment alone.

6. The conjugate of any of claims 1 -5, wherein the MRI contrast agent, iron core nanoparticle or biodegradable nanoparticle is directly conjugated to the tetanus toxin C fragment by amine reactive chemistry.

7. The conjugate of any of claims 1-6, wherein the MRI contrast agent is gadolinium.

8. The conjugate of any of claims 1-7, wherein a therapeutic molecule is attached to or incorporated into the MRI contrast agent-TTC conjugate, the iron core nanoparticle-TTC conjugate, or the biodegradable nanoparticle-TTC conjugate.

9. The conjugate of claim 8, wherein the biodegradable nanoparticle encapsulates the therapeutic molecule.

10. The conjugate of claim 8, wherein the biodegradable nanoparticle is formulated to release the therapeutic molecule over a predetermined time.

1 1. A composition comprising the conjugate of any of claims 1 -7.

12. The composition of claim 1 1, further comprising a therapeutic molecule.

13. The composition of claim 1 1 or claim 12, further comprising a pharmaceutically acceptable carrier.

14. A composition comprising the conjugate of any of claims 8- 10.

15. The composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. The conjugate of any of claims 8-10 or the composition of any of claims 12-15, wherein the therapeutic molecule is a RNAi molecule.

17. The conjugate or composition of claim 16, wherein the RNAi molecule is a siRNA molecule or a vector that expresses a RNAi molecule.

18. The conjugate or composition of claim 16 or claim 17, wherein the RNAi molecule is targeted to mutant SODl .

19. A method of determining neurodegeneration of a subject comprising administering to a subject a conjugate comprising tetanus toxin C fragment (TTC) directly conjugated to a MRl contrast agent, iron core nanoparticle, or biodegradable nanoparticle, wherein said conjugate is retrogradely transported to the central nervous system, and measuring retrograde axonal transport of the conjugate in the subject to determine neurodegeneration.

20. The method of claim 19, wherein retrograde axonal transport is measured quantitatively.

21. The method of claim 19, wherein the conjugate is administered intramuscularly, intraperitoneal^, systemically, intravascularly, or via infusion into the cerebrospinal fluid.

22. The method of claim 19, wherein the conjugate binds neuron cells.

23. The method of claim 22, wherein the conjugate binds neuron cells with high affinity.

24. The method of any of claims 19-23, wherein the conjugate is retrogradely transported from the neuronal periphery to the central nervous system.

25. The method of any of claims 19-24, wherein the MRI contrast agent is gadolinium.

26. The method of any of claims 19-25, wherein the neurodegeneration is a result of amyotrophic lateral sclerosis.

27. The method of any of claims 19-26, wherein the conjugate further comprises a therapeutic molecule.

28. The method of claim 27, wherein the conjugate delivers the therapeutic molecule to motor neurons.

29. The method of claim 27, wherein the therapeutic molecule is attached to or incorporated into the MRI contrast agent-TTC conjugate, the iron core nanoparticle-TTC conjugate, or the biodegradable nanoparticle-TTC conjugate.

30. The method of claim 27, wherein the biodegradable nanoparticle encapsulates the therapeutic molecule.

31. The method of claim 27, wherein the biodegradable nanoparticle is formulated to release the therapeutic molecule over a predetermined time.

32. The method of any of claims 27-31 , wherein the therapeutic molecule is a RNAi molecule.

33. The method of claim 32, wherein the RNAi molecule is a siRNA molecule or a vector that expresses a RNAi molecule.

34. The method of claim 32 or claim 33, wherein the RNAi molecule is targeted to mutant SODl .

35. A method for treating neurodegeneration in a subject comprising administering to a subject the conjugate or the composition of any of claims 8-10 or

14-18, wherein the conjugate or composition is administered in an amount effective to treat neurodegeneration, and wherein said therapeutic molecule is retrogradely transported to the central nervous system.

36. The method of claim 35, wherein the neurodegeneration is a result of amyotrophic lateral sclerosis.

37. A conjugate of any of claims 1-10 or 16-18 prepared by the process of amine reactive chemistry.

38. A composition comprising the conjugate of claim 37.