METHODS AND COMPOSITIONS FOR THE TREATMENT OF AMYLOIDOSIS

Abstract

Methods and compositions for the treatment or prevention of amyloidosis are provided. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof. Such methods and compositions may be employed to reduce, prevent, degrade and/or eliminate amyloid formation in the lysosome and/or extracellularly.

Description

TECHNICAL FIELD

The present invention relates to compositions and methods suitable for the prevention or treatment of amyloidosis. For instance, catabolic enzymes are provided to reduce, prevent, or eliminate amyloid formation.

DESCRIPTION OF TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: ULPI_034_01US_SeqList_ST25.txt, date recorded: Oct. 21, 2016, file size: 146 kilobytes).

BACKGROUND

Amyloids are insoluble fibrous protein aggregates sharing specific structural traits, e.g., a beta-pleated sheet. They arise from at least 18 inappropriately folded versions of proteins and polypeptides present naturally in the body. These misfolded structures alter their proper configuration such that they erroneously interact with one another or other cell components forming insoluble amyloid fibrils. They have been associated with the pathology of more than 20 serious human diseases. Abnormal accumulation of these amyloid fibrils in organs may lead to amyloidosis, and may play a role in various neurodegenerative disorders, as well as other disorders.

The formation of these fibrils involves a passage through the lysosome where the acidic environment allows the formation of the protein aggregates. The amyloids are then released from the cell by exocytosis or by cell lysis.

Trying to eliminate specific fibrils has been the objective of significant research on amyloidosis but without success. Current treatment of amyloidosis involves chemotherapy agents or steroids, such as melphalan and dexamethasone. However, such treatment is not appropriate for all patients and is not effective in many cases due to its specificity. Therefore, there is a great need for alternatives that may safely and effectively prevent or treat diseases associated with amyloidosis.

The present invention solves the problem of how to prevent and stop the formation of excessive amyloids which have a very deleterious activity in the body. The present invention also solves the problem of specificity, and is applicable to different sources of amyloids and not restricted to a specific disease. The present invention also helps the degradation of already formed fibrils by keeping the lysosome more functional and ready to digest fibrils through endocytosis.

SUMMARY OF THE INVENTION

The present invention provides methods of treating or preventing amyloidosis in a subject. In some embodiments, the methods comprise administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof.

In some embodiments, the catabolic enzyme is selected from the group consisting of protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L. In some embodiments, the catabolic enzyme acts to prevent the formation of and/or degrade amyloid within the lysosome, i.e., intralysomally. In other embodiments, the catabolic enzyme acts to prevent the formation of and/or degrade amyloid outside the cell, i.e., extracellularly.

In some embodiments, the catabolic enzyme comprises a PPCA polypeptide, or a biologically active fragment thereof. In some embodiments, the PPCA polypeptide comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 2, 43, or 45, or a biologically active fragment thereof. In some embodiments, the PPCA polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 43, or 45, or a biologically active fragment thereof.

In some embodiments, the methods comprise administering a composition comprising a vector, wherein the vector comprises a nucleotide sequence encoding at least one catabolic enzyme of the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the catabolic enzyme is PPCA or a biologically active fragment thereof. In some embodiments, the administration of the PPCA catabolic enzyme comprises administration of a vector encoding a nucleotide sequence having at least 85% identity to SEQ ID NO: 1, 42, or 44. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 1, 42, or 44.

In some embodiments, the catabolic enzyme comprises a NEU1 polypeptide, or a biologically active fragment thereof. In some embodiments, the NEU1 polypeptide comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 4, or a biologically active fragment thereof. In some embodiments, the NEU1 polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or a biologically active fragment thereof.

In some embodiments, the administration of the NEU1 catabolic enzyme comprises administration of a vector encoding a nucleotide sequence having at least 85% identity to SEQ ID NO: 3. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 3.

In some embodiments, the catabolic enzyme comprises a TPP1 polypeptide, or a biologically active fragment thereof. In some embodiments, the TPP1 polypeptide comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 6, or a biologically active fragment thereof. In some embodiments, the TPP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or a biologically active fragment thereof.

In some embodiments, the administration of the TPP1 catabolic enzyme comprises administration of a vector encoding a nucleotide sequence having at least 85% identity to SEQ ID NO: 5. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 5.

In some embodiments, at least two catabolic enzymes are administered to the subject. In some embodiments, the at least two catabolic enzymes are selected from protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L.

In some embodiments, the at least two catabolic enzymes comprise PPCA and NEU1.

In some embodiments, the catabolic enzyme is targeted to the cell lysosome. In other embodiments, the catabolic enzyme is modified to remain outside the cell, i.e., the enzyme is modified to act extracellularly.

In some embodiments, the catabolic enzyme prevents the accumulation of and/or degrades amyloid in the cell lysosome. In other embodiments, the catabolic enzyme prevents the accumulation of and/or degrades amyloid outside the cell, i.e., extracellularly.

In some embodiments, the present invention provides a composition comprising at least two catabolic enzymes, wherein the composition comprises at least one catabolic enzyme that is targeted to the cell lysosome and at least one catabolic enzyme that remains outside the cell. In some embodiments, the catabolic enzymes are selected from protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L. In an exemplary embodiment, the present invention provides a composition comprising at least two catabolic enzymes, wherein the composition comprises a PPCA catabolic enzyme that is targeted to the cell lysosome and a PPCA catabolic enzyme that remains outside the cell.

In some embodiments, the methods further comprise the administration of one or more additional drugs for treating or preventing amyloidosis. In some embodiments, the one or more additional drugs is/are selected from melphalan, dexamethasone, prednisone, bortezomib, lenalidomide, vincristine, doxorubicin, and cyclophosphamide.

In some embodiments, the methods further comprise the administration of one or more drugs that acidifies the lysosome. In some embodiments, the drug that acidifies the lysosome is selected from an acidic nanoparticle, a catecholamine, a β-adrenergic receptor agonist, an adenosine receptor agonist, a dopamine receptor agonist, an activator of the cystic fibrosis transmembrane conductance regulator (CFTR), cyclic adenosine monophosphate (cAMP), a cAMP analog, and an inhibitor of glycogen synthase kinase-3 (GSK-3).

In some embodiments, the methods further comprise the administration of one or more drugs that modulates the lysosome. In an exemplary embodiment, the drug is Z-phenylalanyl-alanyl-diazomethylketone (PADK) or a PADK analog, or a pharmaceutically acceptable salt or ester thereof. In some embodiments, the PADK analog is selected from Z-L-phenylalanyl-D-alanyl-diazomethylketone (PdADK), Z-D-phenylalanyl-L-alanyl-diazomethylketone (dPADK), and Z-D-phenylalanyl-D-alanyl-diazomethylketone (dPdADK).

In some embodiments, the methods further comprise the administration of one or more drugs that promotes autophagy. In an exemplary embodiment, the drug is selected from an activator of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), an inhibitor of Lysine (K)-specific demethylase 1A (LSD1) , an agonist of Peroxisome proliferator-activated receptor (PPAR), an activator of Transcription factor EB (TFEB), an inhibitor of mechanistic target of rapamycin (mTOR), and an inhibitor of glycogen synthase kinase-3 (GSK3).

In some embodiments, the subject is further treated with stem cell transplantation.

In some embodiments, the administration is parenteral. In some embodiments, the administration is intramuscular, intraperitoneal, or intravenous.

In some embodiments, any one of the compositions and drugs provided herein comprise a pharmaceutically acceptable carrier.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In some embodiments, the amyloidosis is light-chain (AL) amyloidosis.

In some embodiments, the AL amyloidosis involves one or more organs selected from the heart, the kidneys, the nervous system, and the gastrointestinal tract.

In some embodiments, the amyloidosis is amyloid-beta (Aβ) amyloidosis.

In some embodiments, the Aβ amyloidosis involves one or more organs selected from the brain, the nervous system, and/or involves various muscles, e.g., muscles of the arms and legs. In some embodiments, the Aβ amyloidosis is associated with Alzheimer's disease. In some embodiments, the Aβ amyloidosis is associated with cerebral amyloid angiopathy. In some embodiments, the Aβ amyloidosis is associated with Lewy body dementia. In some embodiments, the Aβ amyloidosis is associated with inclusion body myositis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B shows the aggregation of synthetic Aβ42 peptide and Aβ15-36 peptide (negative control) monitored by Thioflavin-T (THT). FIG. 1A. Aggregation at physiological conditions. FIG. 1B. Aggregation at acidic pH.

FIG. 2A-B shows the aggregation of synthetic Aβ42 peptide in vitro over a 24 hour time period as detected by western blot. FIG. 2A. 12% Bis-Tris gel, reducing conditions, probed with 6E10, a commercially available purified anti-β-amyloid antibody that is reactive to amino acid residues 1-16 of beta amyloid. FIG. 2B. 18% Tris-Glycine gel, reducing conditions, probed with 6E10.

FIG. 3A-D show that cathepsin A (interchangeably referred to herein as Cath A or PPCA) prevents the aggregation of Aβ42 amyloid species. FIG. 3A. Activation of 90 ng cathepsin A by cathepsin L (full black circles). FIG. 3B. Activation of 450 ng cathepsin A by cathepsin L. FIG. 3C. Preventive effect of 90 ng PPCA on Aβ42 aggregation and the inhibition of PPCA by the serine protease inhibitor, PMSF (phenylmethylsulfonyl fluoride) FIG. 3D Preventive effect of 450 ng PPCA on Aβ42 aggregation. Aβ42 peptides were aggregated alone (open circles), with two concentrations of Cath A (open squares) and with combination of Cath A+inhibitor PMSF (open triangles). Cath A only (full squares) and inhibitor PMSF only (full triangles) were incubated with THT reagent and served as negative controls.

FIG. 4A-B shows that Cath A (i.e., PPCA) prevents the aggregation of Aβ42 amyloid species in a dose-dependent manner. FIG. 4A. Graph showing Aβ42 aggregation over 2 hours at pH 5, 37° C. with varying PPCA concentrations (7 ng to 900 ng) as measured by THT. Aβ42 aggregation was measured alone and with serial dilutions of PPCA. Lines are labeled for clarity. FIG. 4B. Bar graph showing end-point (2 hrs) Aβ42 aggregation.

FIG. 5 shows that Cath A (i.e., PPCA) prevents the aggregation of both high and lower molecular weight species of Aβ42 amyloid. Treatment of 0.9 μg Aβ42 monomer with 500 ng PPCA is shown over a time period of 2 hours on an 18% Tris-Glycine gel, under reducing conditions, probed with 6E10.

FIG. 6A-D show that cathepsin B (Cath B) prevents the aggregation of Aβ42 amyloid. FIG. 6A. Activation of 90 ng cathepsin B and its inhibition by the protease inhibitor E64. FIG. 6B. Activation of 450 ng cathepsin B and its inhibition by E64. FIG. 6C. Preventive effect of 90 ng cathepsin B on Aβ42 aggregation and the lack inhibition by E64. FIG. 6D. Preventive effect of 450 ng cathepsin B on Aβ42 aggregation and the lack inhibition by E64. Aβ42 peptides were aggregated alone (open circles), with two concentrations of Cath B (open squares) and with combination of Cath B+inhibitor E64 (open triangles). Cath B only (full squares) and inhibitor E64 only (full triangles) were incubated with THT reagent and served as negative controls.

FIG. 7A-B shows that cathepsin B moderately prevents the aggregation of Aβ42 amyloid species in a dose-dependent manner. FIG. 7A. Graph showing Aβ42 aggregation over 2 hours at pH 5, 37° C. with varying cathepsin B concentrations (7 ng to 900 ng) as measured by THT. Aβ42 aggregation was measured alone and with serial dilutions of cathepsin B. FIG. 7B. Bar graph showing end-point (2 hrs) Aβ42 aggregation.

FIG. 8 shows that cathepsin B prevents the aggregation of both low molecular weight species of Aβ42 amyloid and degrades Aβ42 in a time dependent manner. Treatment of 0.9 μg Aβ42 monomer with 200 ng cathepsin B is shown over a time period of 2 hours on an 18% Tris-Glycine gel, under reducing conditions, probed with 6E10

FIG. 9 shows that cathepsin D prevents the aggregation of Aβ42 amyloid as monitored by THT. Aβ42 peptides were aggregated alone (empty circles) and with cathepsin D (empty squares) over period of 2 hours. Cathepsin D alone (triangles) was incubated with THT reagent and served as a negative control.

FIG. 10 shows a western blot demonstrating that PPCA, cathepsin B, PPCA plus cathepsin B, and cathepsin D degrade high molecular weight oligomers/fibrils of Aβ42 amyloid. Cathepsin D degrades low molecular oligomers and completely eliminates Aβ42 monomers.

FIG. 11 shows a western blot demonstrating a comparison in the detection of Aβ42 oligomers and fibrils using an oligomer specific A11 antibody. Aβ42 peptides were subjected to 7 day aggregation protocols specific for oligomers and fibrils. Reduction of oligomer form in fibril formation (line 9) indicates transition of oligomers into fibril form, which is not detected by oligomer specific A11 antibody.

FIG. 12 shows a western blot demonstrating a comparison in the detection of Aβ42 oligomers and fibrils using an oligomer and fibril specific E610 antibody. Aβ42 peptides were subjected to 7 day aggregation protocols specific for oligomers and fibrils. Fibril formation was not detected in the oligomer specific protocol at day 7 (line 4). Reduction of oligomer form and appearance of fibril form (smear on line 9) was detected in the fibril formation protocol.

FIG. 13 shows a western blot illustrating the enzymatic degradation of Aβ42 oligomers as probed by the oligomer specific A11 antibody. Lines 1-6 contain day 9 oligomers aggregated at pH 7.0 at 25° C. and additionally treated overnight at 37° C. in enzyme specific pH. Lines 1-3 are not treated with enzymes. Lines 4-6 represent treatment with 90 ng of cathepsin A, B, and D, respectively. Line 8 contains day 9 oligomers aggregated at pH 7.0 at 25° C. Line 9 contains monomers at pH 7.0. Degradation of oligomers by 90 ng of cathepsin A is shown in line 4. 2 μg of material was loaded on each line.

FIG. 14 shows a western blot illustrating the enzymatic degradation of Aβ42 fibrils as probed by the oligomer and fibril specific antibody E610. Lines 1-6 contain day 9 fibrils aggregated at pH 7.0 at 25° C. and additionally treated overnight at 37° C. in enzyme specific pH. Lines 1-3 are not treated with enzymes. Lines 4-6 represent treatment with 90 ng of cathepsin A, B, and D, respectively. Line 8 contains day 9 fibers aggregated at pH 7.0 at 25° C. Line 9 contains monomers at pH 7.0. Degradation of fibers and oligomers by 90 ng of cathepsin A is shown in line 4. Degradation of fibers by 90 ng of cathepsin B is shown in line 5. 2 μg of material was loaded on each line.

FIG. 15 shows a human Aβ42 specific ELISA used to monitor the degradation of Aβ42 monomers with cathepsin A. Treatment of Aβ42 monomers with 90 ng of cathepsin A (striped bars) showed degradation from the C-terminus at various time points (0, 10, 30, 60, 120 min), which is reflected in loss of C-terminal capture by capturing antibody and in effect loss of fluorescent signal. In contrast, Aβ42 monomers not treated with cathepsin A showed lack of C-terminal degradation (solid bars), which is reflected in efficient antibody capture and strong fluorescent signal. An inhibitor of amyloid aggregation, phenol red was used in both cases to prevent peptide aggregation, which could affect capture by the C-terminal antibody in ELISA.

FIG. 16A-B show aggregation of Aβ40 and Aβ42 measured by THT assay. Aβ40, Aβ42, and Aβ16 were co-incubated with ThT for 2 h at 37° C. to measure the kinetics of aggregation. Aβ42 aggregates more efficiently and faster than Aβ40. FIG. 16A. Graphical representation aggregation of Aβ peptides on a single scale. FIG. 16B. Graphical representation of Aβ40 aggregation on a separate scale.

FIG. 17A-C show that simultaneous incubation of Aβ40, Cath A, and THT shows no change in Aβ40 aggregation. Increasing concentrations of Cath A were co-incubated with 15 μM Aβ40 and 2 mM ThT for 2 h at 37° C. to measure how Cath A affected the kinetics of Aβ40 aggregation. FIG. 17A. 900 ng Cath A was co-incubated with Aβ40 and THT. FIG. 17B. 1000 ng Cath A was co-incubated with Aβ40 and THT. FIG. 17C. 2250 ng Cath A was co-incubated with Aβ40 and THT.

FIG. 18A-C show that Aβ40 pre-incubated with Cath A leads to loss of its aggregation potential as revealed by lack of THT fluorescence. Aβ40 and 2500 ng Cath A were first incubated for 30′, 1 h, and 2 h at 37° C. (FIGS. 18A, 18B, and 18C, respectively). Reactions were then co-incubated with ThT for 2 h at 37° C. to measure how Cath A affected the kinetics of Aβ40 aggregation.

FIG. 19A-B show detection of cleavage of Aβ40 C-terminal end using a C-terminal capture antibody. Aβ40 peptide was incubated for 2 h at 37° C. at pH 5 with varying concentrations of Cath A. The reaction was transferred to an ELISA plate pre-coated with a C-terminal capture antibody and was co-incubated with N-terminal detection antibody overnight at 4° C. Error bars are referring to the standard deviation in the OD values. FIG. 19A. Recovery rate of undigested Aβ40 in samples treated with increased concentrations of Cath A. FIG. 19B. Mean absorbance at 450 nm of samples in ELISA wells treated with increased concentrations of Cath A.

FIG. 20A-C show aggregation and degradation of Aβ40 amyloid measured by Western Blot. FIG. 20A. Aggregation into amyloid species. Aβ40 was incubated in either Fibril Buffer or Oligomer buffer at RT for 0-9 days. 2 μg of Aβ40 were loaded per lane on an 18% Tris-Glycine gel and transferred to a PVDF membrane. The blot was probed with an Anti-Aβ40 C-terminal primary antibody (G2-10). Aβ40 incubated with Cath A during fibril formation prevents aggregation. Aβ40 was co-incubated with Cath A in fibril buffer at RT for 0-9 days. To observe high molecular weight bands the gel in FIG. 20B was run on a 7.5% Tris-glycine gel and to see the low molecular weight bands gel in FIG. 20C was run on an 18% Tris-glycine gel. 2 μg of Aβ40 were loaded into each lane. Each gel was transferred to a PVDF membrane and probed with an Anti-Aβ40 C-terminal primary antibody (G2-10).

DETAILED DESCRIPTION

As shown herein, the present inventors have discovered that various catabolic enzymes can be used to prevent the formation of and/or degrade various types of amyloid oligomers and fibrils. Because these oligomers and fibrils can contribute to the development of a variety of amyloid-associated diseases and disorders, the present invention is directed to methods and compositions for the treatment or prevention of amyloidosis in a subject.

Amyloids are insoluble fibrous protein aggregates sharing specific structural traits. The deposition of normally soluble proteins in this insoluble form can lead to cell death and tissue degeneration. To date, 18 different proteins and polypeptides have been identified in disease-associated amyloid deposits. See Westermark et al. (“Nomenclature of amyloid fibril proteins. Report from the meeting of the International Nomenclature Committee on Amyloidosis, Aug. 8-9, 1998. Part 1.” Amyloid. 1999 March; 6(1):63-6), which is incorporated by reference in its entirety. The amyloid fibrils are long, straight, unbranched filaments about 40-120 Å in diameter, which bind to physiological dyes such as Congo red and thioflavine T and are resistant to protease digestion.

As used herein, amyloidosis refers to a disease that results from accumulation of amyloids. Such diseases to be treated or prevented by the present invention include, but are not limited to, systemic AL amyloidosis, Alzheimer's Disease, Diabetes mellitus type 2, Parkinson's disease, Transmissible spongiform encephalopathy e.g. Bovine spongiform encephalopathy, Fatal Familial Insomnia, Huntington's Disease, Medullary carcinoma of the thyroid, Cardiac arrhythmias, Atherosclerosis, Rheumatoid arthritis, Aortic medial amyloid, Prolactinomas, Familial amyloid polyneuropathy, Hereditary non-neuropathic systemic amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice corneal dystrophy, Cerebral amyloid angiopathy, Cerebral amyloid angiopathy (Icelandic type), Sporadic Inclusion Body Myositis, Amyotrophic lateral sclerosis (ALS), Prion-related or Spongiform encephalopathies, such as Creutzfeld-Jacob, Dementia with Lewy bodies, Frontotemporal dementia with Parkinsonism, Spinocerebellar ataxias, Spinocerebellar ataxia, Spinal and bulbar muscular atrophy, Hereditary dentatorubral-pallidoluysian atrophy, Familial British dementia, Familial Danish dementia, Non-neuropathic localized diseases, such as in Type II diabetes mellitus, Medullary carcinoma of the thyroid, Atrial amyloidosis, Hereditary cerebral haemorrhage with amyloidosis, Pituitary prolactinoma, Injection-localized amyloidosis, Aortic medial amyloidosis, Hereditary lattice corneal dystrophy, Corneal amyloidosis associated with trichiasis, Cataract, Calcifying epithelial odontogenic tumors, Pulmonary alveolar proteinosis, Inclusion-body myositis, Cutaneous lichen amyloidosis, and Non-neuropathic systemic amyloidosis, such as AL amyloidosis, AA amyloidosis, Familial Mediterranean fever, Senile systemic amyloidosis, Familial amyloidotic polyneuropathy, Hemodialysis-related amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Finnish hereditary amyloidosis, Lysozyme amyloidosis, Fibrinogen amyloidosis, Icelandic hereditary cerebral amyloid angiopathy, familial amyloidosis, and systemic amyloidosis which occurs in multiple tissues, such as light-chain amyloidosis, and other various neurodegenerative disorders. In exemplary embodiments, the amyloidosis is light-chain (AL) amyloidosis. In further exemplary embodiments, the AL amyloidosis involves one or more organs selected from the heart, the kidneys, the nervous system, and the gastrointestinal tract.

In some embodiments, the present invention provides methods and compositions for the treatment or prevention of a disease associated with amyloidosis in a subject, wherein the disease is associated with the formation of amyloid-beta (Aβ or Abeta) peptides. These peptides result from the amyloid precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield amyloid-beta. In some embodiments, the disease associated with the formation of amyloid-beta is selected from Alzheimer's Disease, cerebral amyloid angiopathy, Lewy body dementia, and inclusion body myositis.

In alternative embodiments, the present invention provides methods and compositions for the treatment or prevention of a disease associated with amyloidosis in a subject, wherein the disease is not associated with the formation of amyloid beta, i.e., wherein the disease is a disease other than one associated with the formation of amyloid beta, e.g., a disease other than Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia, and inclusion body myositis.

In one embodiment, the disease associated with amyloidosis is light-chain (AL) amyloidosis. In another embodiment, the disease associated with amyloidosis is selected from Parkinson's Disease, Huntington's Disease, Rheumatoid arthritis, and a prion-related disease.

In some embodiments, the amyloidosis is a systemic amyloidosis. Systemic amyloidosis encompasses a complex group of diseases caused by tissue deposition of misfolded proteins that result in progressive organ damage.

As noted above, in some embodiments, the amyloidosis is light-chain (AL) amyloidosis (also known as, i.e. a.k.a., primary systemic amyloidosis (PSA) or primary amyloidosis). AL amyloidosis refers to a condition caused when a subject's antibody-producing cells do not function properly and produce abnormal protein fibers made of components of antibodies called light chains. In some embodiments, such light chains form amyloid deposits in one or more different organs which may cause or already caused damage to these organs. In some embodiments, the abnormal light chains are in blood and/or urine. In some embodiments, the abnormal light chains are “Bence Jones proteins”. In some embodiments, the AL amyloidosis affects the heart, peripheral nervous system, gastrointestinal tract, blood, lungs and/or skin. Clinical features of AL amyloidosis also may include a constellation of symptoms and organ dysfunction that can include cardiac, renal, and hepatic dysfunction, gastrointestinal involvement, neuropathies and macroglossia.

In some embodiments, the amyloidosis is AA amyloidosis (a.k.a. secondary amyloidosis, AA), caused by deposited proteins called serum amyloid A protein (SAA). In some embodiments, the SAA protein is mainly deposited in the liver, spleen and/or kidney. In some embodiments, the AA amyloidosis leads to nephrotic syndrome. In some embodiments, the AA amyloidosis is caused by autoimmune diseases (e.g., Rheumatoid arthritis, Ankylosing spondylitis, or Crohn's disease and ulcerative colitis), Chronic infections (e.g., Tuberculosis, Bronchiectasis, or Chronic osteomyelitis), autoinflammatory diseases (e.g., Familial Mediterranean fever (FMF), Muckle-Wells syndrome (MWS), Cancer (e.g., Hodgkin's lymphoma, Renal cell carcinoma), and/or Chronic foreign body reaction (e.g., Silicone-induced granulomatous reaction).

In some embodiments, the amyloidosis is familial amyloidosis. In some embodiments, the familial amyloidosis is ATTR amyloidosis (a.k.a. or senile systemic amyloidosis) which is due one or more inherited amyloidosis, such as a mutation in the transthyretin (TTR) gene that produces abnormal transthyretin protein. In some embodiments, the familial amyloidosis is caused by one or more mutation in apolipoprotein A-I (AApoAI), apolipoprotein A-II (AApoAII), gelsolin (AGel), fibrinogen (AFib), lysozyme (ALys), and/or Lect2.

In some embodiments, the amyloidosis is Beta-2 Microglobulin Amyloidosis (Abeta2m). Beta-2 microglobulin amyloidosis is caused by chronic renal failure and often occurs in patients who are on dialysis for many years. Amyloid deposits are made of the beta-2 microglobulin protein that accumulated in tissues, particularly around joints, when it cannot be excreted by the kidney because of renal failure.

In some embodiments, the amyloidosis is Localized Amyloidosis (ALoc). In some embodiments, localized amyloid deposits in the airway (trachea or bronchus), eye, or urinary bladder. In some embodiments, the ALoc is caused by local production of immunoglobulin light chains not originating in the bone marrow. In some embodiments, the ALoc is associated with endocrine proteins, or proteins produced in the skin, heart, and other sites. These usually do not become systemic.

In some embodiments, the amyloidosis occurs in the kidney of the subject. In some embodiments, the amyloidosis in the kidney is AA amyloidosis. In some embodiments, the AA amyloidosis leads to nephrotic syndrome. In some embodiments, the amyloidosis in the kidney is AL amyloidosis. In some embodiments, symptoms of kidney disease and renal failure associated with AL amyloidosis include, but are not limited to, fluid retention, swelling, and shortness of breath.

In some embodiments, the amyloidosis occurs in the heart of the subject. In some embodiments, the amyloidosis in the heart is AL amyloidosis. In some embodiments, the amyloidosis in the heart leads to heart failure and/or irregular heart beat.

In some embodiments, the amyloidosis occurs in the gastrointestinal tract of the subject. In some embodiments, symptoms of GI amyloidosis include, but are not limited to, esophageal reflux, constipation, nausea, abdominal pain, diarrhea, weight loss, and early satiety. In some embodiments, the amyloidosis occurs in the duodenum, stomach, colo-rectum, and/or esophagus.

In some embodiments, the treatment methods provided herein alleviate, reduce the severity of, or reduce the occurrence of, one or more of the symptoms associated with amyloidosis. Such symptoms include those symptoms associated with light-chain (AL) amyloidosis (primary systemic amyloidosis) and/or AA amyloidosis (secondary amyloidosis). In some embodiments, the symptoms include, but are not limited to, fluid retention, swelling, shortness of breath, fatigue, irregular heartbeat, numbness of hands and feet, rash, shortness of breath, swallowing difficulties, swollen arms or legs, esophageal reflux, constipation, nausea, abdominal pain, diarrhea, early satiety, stroke, gastrointestinal disorders, enlarged liver, diminished spleen function, diminished function of the adrenal and other endocrine glands, skin color change or growths, lung problems, bleeding and bruising problems, fatigue and weight loss, decreased urine output, diarrhea, hoarseness or changing voice, joint pain, and weakness. In some embodiments, the symptoms are those associated with amyloid-beta (Aβ) amyloidosis. In some embodiments, the symptoms include, but are not limited to, common symptoms of Alzheimer's disease, including memory loss, confusion, trouble understanding visual images and spatial relationships, and problems speaking or writing.

According to the methods of the present invention, the term “subject,” includes any subject that has, is suspected of having, or is at risk for having a disease or condition. Suitable subjects (or patients) include mammals, such as laboratory animals (e.g., mouse, rat, rabbit, guinea pig), farm animals, and domestic animals or pets (e.g., cat, dog). Non-human primates and human patients are also included. A subject “at risk” may or may not have detectable disease, and may or may not have displayed detectable disease prior to the prevention or treatment methods described herein. “At risk” denotes that a subject has one or more so-called risk factors, which are measurable parameters that correlate with development of any one of the diseases, disorders, conditions, or symptoms described herein,. A subject having one or more of these risk factors has a higher probability of developing any one of the diseases, disorders, conditions, or symptoms described herein than a subject without these risk factor(s). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human diagnosed as having amyloidosis or disease/symptom caused by or associated with amyloidosis. In some embodiments, the subject is a human suspected to have amyloidosis. In some embodiments, the subject is a human having high risk of developing amyloidosis. In some embodiments, the subject is an amyloidosis patient with one or more diseases/conditions/symptoms as described herein.

The terms “treating” and “treatment” as used herein refer to an approach for obtaining beneficial or desired results including clinical results, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. A treatment is usually effective to reduce at least one symptom of a condition, disease, disorder, injury or damage. Exemplary markers of clinical improvement will be apparent to persons skilled in the art. Examples include, but are not limited to, one or more of the following: decreasing the severity and/or frequency one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life, etc.

“Prophylaxis,” “prophylactic treatment,” “prevention,” or “preventive treatment” refers to preventing or reducing the occurrence or severity of one or more symptoms and/or their underlying cause, for example, prevention of a disease or condition in a subject susceptible to developing a disease or condition (e.g., at a higher risk, as a result of genetic predisposition, environmental factors, predisposing diseases or disorders, or the like).

The present invention provides methods of treating or preventing amyloidosis in a subject. In some embodiments, the methods comprise administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof. In some embodiments, the methods comprise increasing the expression, activity, and/or concentration of at least one catabolic enzyme in the subject. Increasing the expression, activity, and/or concentration of a given catabolic enzyme may be accomplished at the genomic DNA level, transcriptional level, post-transcriptional level, translational level, and/or post-translational level, including but not limited to, increasing the gene copy number, mRNA transcription rate, mRNA abundance, mRNA stability, protein translation rate, protein stability, protein modification, protein activity, protein complex activity, etc. Increasing the concentration of a given catabolic enzyme may further be accomplished by administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof. As used herein, the term catabolic enzyme refers not only to the natural form the enzyme, but also any purified, isolated, synthetic, recombinant, and functional variants, fragments, chimeras, and mutants of the natural enzyme.

In some embodiments, the at least one catabolic enzyme is selected from the non-limiting group consisting of protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L.

In some embodiments, the at least one catabolic enzyme is PPCA (a.k.a. Protective Protein Cathepsin A, PPGB, Carboxypeptidase C, EC 3.4.16.5, GSL, GLB2, Carboxypeptidase Y-Like Kininase, NGBE, carboxypeptidase-L, Protective Protein For Beta-Galactosidase (Galactosialidosis), deamidase, Beta-Galactosidase, Lysosomal Carboxypeptidase A, Beta-Galactosidase Protective Protein, Lysosomal Protective Protein, Protective Protein For Beta-Galactosidase, Urinary Kininase, EC 3.4.168, or Carboxypeptidase L) is classified both as a cathepsin and a carboxypeptidase.

In some embodiments, the at least one catabolic enzyme is PPCA. PPCA is a glycoprotein that associates with the lysosomal enzymes beta-galactosidase and neuraminidase to form a complex of high-molecular-weight multimers. The formation of this complex provides a protective role for stability and activity. It is protective for β-galactosidase and neuraminidase. In some embodiments, the PPCA can be a natural, synthetic, or recombinant protein. In some embodiments, the PPCA polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2, 43, or 45. In some embodiments, the PPCA polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 43, or 45.

In some embodiments, the at least one catabolic enzyme is Neuraminidase 1 (NEU1, a.k.a. sialidase 1, lysosomal sialidase, EC 3.2.1.18, Acetylneuraminyl Hydrolase, SIAL1, Lysosomal Sialidase, exo-alpha-sialidase, NANH, sialidase-1, or G9 Sialidase) is a lysosomal neuraminidase enzyme. NEU1 is an enzyme that cleaves terminal sialic acid residues from substrates such as glycoproteins and glycolipids. In some embodiments, the NEU1 can be a natural, synthetic, or recombinant protein. In some embodiments, the NEU1 polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4. In some embodiments, the NEU1 polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the at least one catabolic enzyme is Tripeptidyl peptidase 1 (TPP1, Spinocerebellar Ataxia, Autosomal Recessive 7, CLN2, SCAR7, Growth-Inhibiting Protein 1, Cell Growth-Inhibiting Gene 1 Protein, Lysosomal Pepstatin Insensitive Protease, Tripeptidyl Aminopeptidase, Tripeptidyl-Peptidase 1, LPIC, Lysosomal Pepstatin-Insensitive Protease, or EC 3.4.14.9). TPP1 is an enzyme that cleaves N-terminal tripeptides from substrates and has weaker endopeptidase activity. In some embodiments, the TPP1 can be a natural, synthetic, or recombinant protein. In some embodiments, the TPP1 polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 6. In some embodiments, the TPP1 polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the at least one catabolic enzyme is Cathepsin B (a.k.a. EC 3.4.22.1, CPSB, Amyloid Precursor Protein Secretase, Cysteine Protease, APPS, APP secretase, or EC 3.4.22). Cathepsin B is a lysosomal cysteine protease composed of a dimer of disulfide-linked heavy and light chains, both produced from a single protein precursor. In some embodiments, the Cathepsin B can be a natural, synthetic, or recombinant protein. In some embodiments, the Cathepsin B polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8, 47, 49, 51, 53, 55, or 57. In some embodiments, the Cathepsin B polypeptide comprises the amino acid sequence of SEQ ID NO: 8, 47, 49, 51, 53, 55, or 57.

In some embodiments, the at least one catabolic enzyme is Cathepsin D (a.k.a. EC 3.4.23.5, CTSD). Cathepsin D refers is a lysosomal acid protease active in intracellular protein breakdown. In some embodiments, the Cathepsin D can be a natural, synthetic, or recombinant protein. In some embodiments, the Cathepsin D polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 68. In some embodiments, the Cathepsin D polypeptide comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the Cathepsin D polypeptide harbors one or more modifications relative to the amino acid sequence of SEQ ID NO: 68. In certain embodiments, the Cathepsin D polypeptide comprises the amino acid sequence of SEQ ID NO: 68, wherein the polypeptide harbors a modification at an amino acid position selected from position 58 (A to V), position 229 (F to I), position 282 (G to R), and position 383 (W to C).

In some embodiments, the at least one catabolic enzyme is Cathepsin E (a.k.a. EC 3.4.23.34, CTSE). Cathepsin E is a lysosomal aspartyl protease. In some embodiments, the Cathepsin E can be a natural, synthetic, or recombinant protein. In some embodiments, the Cathepsin E polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 69, 70, or 71. In some embodiments, the Cathepsin E polypeptide comprises the amino acid sequence of SEQ ID NO: 69, 70, or 71. In some embodiments, the Cathepsin E polypeptide harbors one or more modifications relative to the amino acid sequence of SEQ ID NO: 69, 70, or 71. In certain embodiments, the Cathepsin E polypeptide comprises the amino acid sequence of SEQ ID NO: 69, wherein the polypeptide harbors a modification at an amino acid position selected from position 82 (I to V) and position 329 (T to I).

In some embodiments, the at least one catabolic enzyme is Cathepsin K (a.k.a. EC 3.4.22.38, CTSO, Pycnodysostosis, PYCD, Cathepsis O, PKND, Cathepsin X). Cathepsin K is a lysosomal cysteine protease involved in bone remodeling and resorption, defined by its high specificity for kinins. In some embodiments, the Cathepsin K can be a natural, synthetic, or recombinant protein. In some embodiments, the Cathepsin K polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 10. In some embodiments, the Cathepsin K polypeptide comprises the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the at least one catabolic enzyme is Cathepsin L (a.k.a. MEP, CTSL, EC 3.4.22.15, CATL, Major Excreted Protein). Cathepsin L is a lysosomal endopeptidase enzyme which is involved in the initiation of protein degradation. Its substrates include collagen and elastin, as well as alpha-1 protease inhibitor, a major controlling element of neutrophil elastase activity. In some embodiments, the Cathepsin L can be a natural, synthetic, or recombinant protein. In some embodiments, the Cathepsin L polypeptide comprises an amino acid sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 12, 59, 61, 63, 65, or 67. In some embodiments, the Cathepsin L polypeptide comprises the amino acid sequence of SEQ ID NO: 12, 59, 61, 63, 65, or 67.

In some embodiments, the administration comprises the administration of a nucleotide sequence encoding at least one catabolic enzyme of the present invention.

As used herein, the terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, “nucleic acid fragment”, “nucleotide sequence,” and “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5′-monophosphate form) are referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

As used herein, the term “chimeric” or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid or a protein sequence that links at least two heterologous polynucleotides or two heterologous polypeptides into a single macromolecule, or that re-arranges one or more elements of at least one natural nucleic acid or protein sequence. For example, the term “recombinant” can refer to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence. It is recognized that a genetic regulatory element of the present invention comprises a synthetic nucleotide sequence. In some embodiments, the synthetic nucleotide sequence shares little or no extended homology to natural sequences. Extended homology in this context generally refers to 100% sequence identity extending beyond about 25 nucleotides of contiguous sequence. A synthetic genetic regulatory element of the present invention comprises a synthetic nucleotide sequence.

As used herein, an “isolated” or “purified” nucleic acid molecule or polynucleotide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or polynucleotide as found in its naturally occurring environment. Thus, an isolated or purified nucleic acid molecule or polynucleotide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

In some embodiments, the methods comprise administering to the subject a composition comprising an expression vector (interchangeably referred to herein as a vector), wherein the vector comprises a polynucleotide sequence encoding at least one catabolic enzyme. In some embodiments, the methods comprise administering to the subject a composition comprising at least one expression vector comprising an expression cassette of coding genes.

In some embodiments, the expression vector is a viral vector. Accordingly, in the some embodiments, the methods of the present invention comprise administering to the subject a composition comprising at least one viral vector comprising a polynucleotide sequence encoding at least one catabolic enzyme. In some embodiments, the expression cassette, the expression vector, or the viral vector further comprises one or more nucleotide sequences encoding a signal peptide. In some embodiments, the signal peptide is an intralysosomal localization peptide.

A nucleotide sequence encoding at least one catabolic enzyme can be delivered to a subject through any suitable delivery system, such as those described by Rolland (Pharmaceutical Gene Delivery Systems, ISBN: 978-0-8247-4235-5, 2003), which is incorporated by reference in its entirety. In some embodiments, the delivery system is a viral system, a physical system, and/or a chemical system.

In some embodiments, the delivery system to deliver a nucleotide sequence encoding at least one catabolic enzyme is a viral system. In some embodiments, an adenovirus vector is used (see, Thrasher et al., Gene therapy: X-SCID transgene leukaemologenicity. Nature. 2006; 443(7109): E5-E6; Zhang et al., Adenoviral and adeno-associated viral vectors-mediated neuronal gene transfer to cardiovascular control regions of the rat brain. Int J Med Sci. 2013; 10(5): 607-616). In some embodiments, an adeno-associated vector is used (see, Teramato et al., Crisis of adenoviruses in human gene therapy. Lancet. 2000; 355(9218): 1911-1912, Okada et al., Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors. Otol Neurotol. 2012; 33(4): 655-659). In some embodiments, a retroviral vector is used (see, Anson et al., The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genet Vaccines Ther. 2004; 2(1): 9; Frederic D. Retroviral integration and human gene therapy. J Clin Invest. 2007; 117(8): 2083-2086). In some embodiments, a lentivirus vector is used (see, Goss et al., Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion. Gene Ther. 2001; 8(7): 551-556; Real et al., Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression. Appl Microbiol Biotechnol. 2011; 91(6): 1581-91.). In some embodiments, a herpes simplex virus vector is used (see, Lachmann R H, Efstathiou S. The use of herpes simplex virus-based vectors for gene delivery to the nervous system. Mol Med Today. 1997; 3(9): 404-411; Liu S, Dai M, You L, Zhao Y. Advance in herpes simplex viruses for cancer therapy. Sci China Life Sci. 2013; 56(4): 298-305). In some embodiments, a poxvirus vector is used (see, Moss B. Reflections on the early development of poxvirus vectors. Vaccine. 2013; 31(39): 4220-4222). Each of the references is incorporated herein by reference in its entirety.

In some embodiments, the delivery system to deliver a nucleotide sequence encoding at least one catabolic enzyme of the invention is a physical system. In some embodiments, the physical systems include, but are not limited to jet injection, biolistics, electroporation, hydrodynamic injection, and ultrasound (see, Sirsi et al. Advances in ultrasound mediated gene therapy using microbubble contrast agents. Theranostics. 2012; 2(12): 1208-1222.; Naldini et al., In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259): 263-267; Panje et al., Ultrasound-mediated gene delivery with cationic versus neutral microbubbles: Effect of DNA and microbubble dose on in vivo transfection efficiency. Theranostics. 2012; 2(11): 1078-1091; Gao et al., Cationic liposome-mediated gene transfer. Gene Ther. 1995; 2(10): 710-722; Orio et al., Electric field orientation for gene delivery using high-voltage and low-voltage pulses. J Membr Biol. 2012; 245(10): 661-666.) Each of the references is incorporated herein by reference in its entirety.

In some embodiments, the delivery system to deliver a nucleotide sequence encoding at least one catabolic enzyme of the invention is a chemical system. The chemical systems include, but are not limited to calcium phosphate precipitation, liposomes and polymeric carriers. In some embodiments, the chemical system is based on calcium phosphate precipitation, such as calcium phosphate nano-composite particles encapsulating DNA (see, Nouri et al. Calcium phosphate-mediated gene delivery using simulated body fluid (SBF). Int J Pharm. 2012; 434(1-2): 199-208; Bhakta et al. Magnesium phosphate nanoparticles can be efficiently used in vitro and in vivo as non-viral vectors for targeted gene delivery. J Biomed Nanotechnol. 2009; 5(1): 106-114).

In some embodiments, the chemical system to deliver a nucleotide sequence encoding at least one catabolic enzyme of the invention is based on liposomes. In some embodiments, the liposomes are nano-sized. In some embodiments, liposomes conjugated with polyethylene glycol (PEG) and/or other molecules such as ligands and peptides can be used (see, Yang et al. Cationic nucleolipids as efficient siRNA carriers. Org Biomol Chem. 2011; 1(9): 291-296).

In some embodiments, the chemical system to deliver a nucleotide sequence encoding at least one catabolic enzyme of the invention is based on polymeric carriers. In some embodiments, the polymeric carriers are conjugated to the gene to be delivered. In some embodiments, the polymeric carriers include, but are not limited to chitosan, polyethylenimine (PEI), polylysine, polyarginine, polyamino ester, Polyamidoamine Dendrimers (PAMAM), Poly (lactide-co-glycolide), and PLL, such as those described in Choi et al., Enhanced transfection efficiency of PAMAM dendrimer by surface modification with 1-arginine. J Control Release. 2004; 3(99): 445-456; Pfeifer et al., Poly(ester-anhydride):poly(beta-amino ester) micro- and nanospheres: DNA encapsulation and cellular transfection. Int J Pharm. 2005; 304(1-2): 210-219; Anderson et al., Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters). Mol Ther. 2005; 3(11): 426-434; Hwang et al., Effects of structure of beta-cyclodextrin-containing polymers on gene delivery. Bioconjugate Chem. 2001; 2(12): 280-290; Kean et al., Trimethylated chitosans as non-viral gene delivery vectors: cytotoxicity and transfection efficiency. J Control Release. 2005; 3(103): 643-653.

In some embodiments, administration of a catabolic enzyme comprises the administration of at least one catabolic enzyme polypeptide or fragment thereof of the present invention. As used herein, the terms “polypeptide” and “protein” are used interchangeably herein.

The invention also envisions and encompasses the use of functional variants or fragments of the intralysosomal catabolic enzyme described herein. As used herein, the phrase “a biologically active variant” or “functional variant” with respect to a protein refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence, while still maintains substantial biological activity of the reference sequence. The variant can have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. The following table shows exemplary conservative amino acid substitutions.

	Very Highly -	Highly Conserved
Original	Conserved	Substitutions (from the	Conserved Substitutions
Residue	Substitutions	Blosum90 Matrix)	(from the Blosum65 Matrix)
Ala	Ser	Gly, Ser, Thr	Cys, Gly, Ser, Thr, Val
Arg	Lys	Gln, His, Lys	Asn, Gln, Glu, His, Lys
Asn	Gln; His	Asp, Gln, His, Lys, Ser, Thr	Arg, Asp, Gln, Glu, His, Lys, Ser, Thr
Asp	Glu	Asn, Glu	Asn, Gln, Glu, Ser
Cys	Ser	None	Ala
Gln	Asn	Arg, Asn, Glu, His, Lys, Met	Arg, Asn, Asp, Glu, His, Lys, Met, Ser
Glu	Asp	Asp, Gln, Lys	Arg, Asn, Asp, Gln, His, Lys, Ser
Gly	Pro	Ala	Ala, Ser
His	Asn; Gln	Arg, Asn, Gln, Tyr	Arg, Asn, Gln, Glu, Tyr
Ile	Leu; Val	Leu, Met, Val	Leu, Met, Phe, Val
Leu	Ile; Val	Ile, Met, Phe, Val	Ile, Met, Phe, Val
Lys	Arg; Gln; Glu	Arg, Asn, Gln, Glu	Arg, Asn, Gln, Glu, Ser,
Met	Leu; Ile	Gln, Ile, Leu, Val	Gln, Ile, Leu, Phe, Val
Phe	Met; Leu; Tyr	Leu, Trp, Tyr	Ile, Leu, Met, Trp, Tyr
Ser	Thr	Ala, Asn, Thr	Ala, Asn, Asp, Gln, Glu, Gly, Lys, Thr
Thr	Ser	Ala, Asn, Ser	Ala, Asn, Ser, Val
Trp	Tyr	Phe, Tyr	Phe, Tyr
Tyr	Trp; Phe	His, Phe, Trp	His, Phe, Trp
Val	Ile; Leu	Ile, Leu, Met	Ala, Ile, Leu, Met, Thr

Alternatively, a variant can have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the reference polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the reference polynucleotide. As used herein, a “reference” polynucleotide comprises a nucleotide sequence produced by the methods disclosed herein. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site directed mutagenesis but which still comprise genetic regulatory element activity. Generally, variants of a particular polynucleotide or nucleic acid molecule, or polypeptide of the invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more sequence identity to that particular polynucleotide/polypeptides as determined by sequence alignment programs and parameters as described elsewhere herein.

In some embodiments, a gene that can hybridize with the nucleic acid sequences encoding the catabolic enzymes of the present invention under stringent hybridization conditions can be used. The terms “stringency” or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na+ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or “conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures are well known in the art and are described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are hybridization in 0.25 M Na₂HPO₄ buffer (pH 7.2) containing 1 mM Na₂EDTA, 0.5-20% sodium dodecyl sulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by a wash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C. to 65° C.

The definition of each catabolic enzyme includes sequences having high similarity or identity to the nucleic acid sequences and/or polypeptide sequences of the specific catabolic enzymes mentioned herein. As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).

The invention also includes biologically active fragments of the catabolic enzymes described herein. These biologically active fragments may comprise at least 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or more amino acid residues and retain one or more activities associated with the catabolic enzymes described herein. Such fragments may be obtained by deletion mutation, by recombinant techniques that are routine and well-known in the art, or by enzymatic digestion of the catabolic enzyme(s) of interest using any of a number of well-known proteolytic enzymes. The invention further includes nucleic acid molecules which encode the above described variant enzymes and enzyme fragments.

In some embodiments, the methods comprise administering to the subject a composition comprising a therapeutically effective amount or prophylactically effective amount of at least one catabolic enzyme. The term “therapeutically effective amount” as used herein, refers to the level or amount of one or more catabolic enzymes needed to treat amyloidosis, or reduce or prevent injury or damage, optionally without causing significant negative or adverse side effects. A “prophylactically effective amount” refers to an amount of a catabolic enzyme sufficient to prevent or reduce severity of a future disease or condition associated with amyloidosis when administered to a subject who is susceptible and/or who may develop amyloidosis or a condition associated with amyloidosis.

In some embodiments, instead of or in addition to administering a polynucleotide sequence encoding a catabolic enzyme of the present invention, the methods comprise administering a composition comprising a polypeptide comprising a catabolic enzyme of the present invention or a biologically active fragment thereof directly to the subject in need.

In some embodiments, the catabolic enzyme is targeted to the intralysosomal space. In some embodiments, the catabolic enzyme to be administered comprises one or more signals which help with sorting the polypeptide to lysosome. In some embodiments, the signal can be a lysosomal localization signal polypeptide, a monosaccharide (including derivatives), a polysaccharide, or combinations thereof.

In some embodiments, the signal is mannose-6 phosphate. A catabolic enzyme comprising a mannose-6 phosphate can be targeted to lysosomes with the help of a mannose-6 phosphate receptor.

In some embodiments, the signal is not dependent on mannose-6 phosphate. In some embodiments, the signal is a signal peptide. In some embodiments, the signal peptide is located at the N-terminal, the C-terminal, or elsewhere in the intralysosomal catabolic enzyme to be administered. In some embodiments, the signal peptides include, but are not limited to the DXXLL type (SEQ ID NO: 13), [DE]XXXL[LI] type (SEQ ID NO: 14), and YXXO type (SEQ ID NO: 15). See Bonifacino et al., Signals for sorting of transmembrane proteins to endosomes and lysosomes, Annu. Rev. Biochem. 72 (2003) 395-447; and Brualke et al. (Sorting of lysosomal proteins, Biochimica et Biophysica Acta 1793 (2009) 605-614), each of which is incorporated by reference in its entirety.

In some embodiments, the signal peptides belong to the DXXLL type, such as those identified in MPR300/CI-MPR (, SEQ ID NO: 16), MPR46/CD-MPR (, SEQ ID NO: 17), Sortilin (, SEQ ID NO: 18), SorLA/SORL1 (, SEQ ID NO: 19), GGA1 (1) (, SEQ ID NO: 20), GGA1 (2) (, SEQ ID NO: 21), GGA2 (, SEQ ID NO: 22), and GGA3 (, SEQ ID NO: 23).

In some embodiments, the signal peptides belong to the [DE]XXXL[LI] type, such as those identified in LIMP-II (, SEQ ID NO: 24), NPC1 (, SEQ ID NO: 25), Mucolipin-1 (, SEQ ID NO: 26), Sialin (, SEQ ID NO: 27), GLUT8 (, SEQ ID NO: 28), Invariant chain (Ii) (1) (, SEQ ID NO: 29), and Invariant chain (Ii) (2) (, SEQ ID NO: 30).

In some embodiments, the signal peptides belong to the YXXO type, such as those identified in LAMP-1 (, SEQ ID NO: 31), LAMP-2A (, SEQ ID NO: 32), LAMP-2B (, SEQ ID NO: 33), LAMP-2C (, SEQ ID NO: 34), CD63 (, SEQ ID NO: 35), CD68 (, SEQ ID NO: 36), Endolyn (, SEQ ID NO: 37), DC-LAMP (, SEQ ID NO: 38), Cystinosin (, SEQ ID NO: 39), Sugar phosphate exchanger 2 (, SEQ ID NO: 40), and acid phosphatase (, SEQ ID NO: 41).

In some embodiments, the catabolic enzyme is targeted to remain outside the cell, i.e., the enzyme is modified to act extracellularly. In some embodiments, the catabolic enzyme to be administered lacks one or more signals that would otherwise target the polypeptide to the lysosome. In some embodiments, the catabolic enzyme lacks one or more mannose-6 phosphate (i.e., M6P) signals, thereby precluding entry of the catabolic enzyme into the cell. In some embodiments, the catabolic enzyme is recombinantly engineered to lack one or more mannose-6 phosphate signal. Not bound by any theory, it is generally understood in the art that reduced M6P content lowers the binding affinity of a recombinant enzyme for M6P receptors and decreases its cellular uptake and thereby allows the enzyme to remain outside the cell.

Methods for reducing the M6P content of a recombinant protein, e.g., a catabolic enzyme, are known in the art. See, e.g., U.S. Pat. No. 8,354,105, which is herein incorporated by reference in its entirety. In some embodiments, the mannose content of a recombinant catabolic enzyme may be reduced by manipulating the cell culture conditions such that the glycoprotein produced by the cell has low-mannose content. As used herein, the term “low-mannose content” refers to catabolic enzyme composition wherein less than about 20%, less than about 15%, less than about 10%, less than about 8%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or any values between any of these preceding ranges, or even at 0% of the enzymes in the composition have more than 4 mannose residues (i.e.. are species of M5 or greater).

In some embodiments, the present invention provides a composition comprising at least two catabolic enzymes, wherein the composition comprises at least one catabolic enzyme that is targeted to the cell lysosome and at least one catabolic enzyme that remains outside the cell. In some embodiments, the catabolic enzymes are selected from protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L. In an exemplary embodiment, the present invention provides a composition comprising at least two catabolic enzymes, wherein the composition comprises a PPCA catabolic enzyme that is targeted to the cell lysosome and a PPCA catabolic enzyme that remains outside the cell. In some embodiments, the ratio of the intralysosomal catabolic enzyme to the extracellular catabolic enzyme on a percentage basis within the composition is at least 5%:95%. In further embodiments, the ratio of the intralysosomal catabolic enzyme to the extracellular catabolic enzyme on a percentage basis within the composition is at least 10%:90%, at least 15%:85%, at least 20%:80%, at least 25%:75%, at least 30%:70%, at least 35%:65%, at least 40%:60%, at least 45%:55%, at least 50%:50%, at least 55%:45%, at least 60%:40%, at least 65%:35%, at least 70%:30%, at least 75%:25%, at least 80%:20%, at least 85%:15%, at least 90%:10%, or at least 95%:5%.

In some embodiments, the methods of the present invention comprise administering to the subject a composition comprising a therapeutically effective amount of at least two, three, or more catabolic enzymes. In some embodiments, the methods comprise increasing the expression, activity, and/or concentration of at least two, three, or more catabolic enzymes in the subject. In some embodiments, the methods comprise administering to the subject a composition comprising an expression cassette comprising one or more polynucleotide sequences encoding at least two, three, or more catabolic enzymes. In some embodiments, the methods comprise administering to the subject one or more expression cassettes comprising at least two, three or more polynucleotide sequences encoding at least two, three or more catabolic enzymes. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a first catabolic enzyme, and an expression cassette comprising a polynucleotide sequence encoding a second catabolic enzyme. In some embodiments, two or more catabolic enzymes are selected from the group consisting of protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L. In some embodiments, at least two catabolic enzymes are PPCA and NEU1.

In some embodiments, administration of the at least one catabolic enzyme is employed to prevent the formation of amyloid. In other embodiments, administration of the at least one catabolic enzyme is employed to degrade amyloid that has already formed. In some embodiments, administration of the at least one catabolic enzyme is employed to prevent the formation of one or more amyloid oligomers. In some embodiments, administration of the at least one catabolic enzyme is employed to prevent the formation of one or more amyloid fibrils. In some embodiments, administration of the at least one catabolic enzyme is employed to degrade one or more amyloid oligomers after it has already formed. In some embodiments, administration of the at least one catabolic enzyme is employed to degrade one or more amyloid fibrils after it has already formed.

In some embodiments, the methods of the present invention provided herein further comprise administering a composition (e.g. a pharmaceutical composition) comprising at least one catabolic enzyme or fragment thereof with at least one additional drug for treating or preventing amyloidosis.

In some embodiments, the at least one additional drug is a steroid. In some embodiments, the steroid is dexamethasone, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone or any combination thereof.

In some embodiments, the at least one additional drug is a non-steroid agent. In some embodiments, such non-steroid agent is diclofenac, flufenamic acid, flurbiprofen, diflunisal, detoprofen, diclofenac, etodolac, fenoprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium salicylate or any combination thereof.

In some embodiments, the at least one additional drug is a chemotherapy agent. In some embodiments, the chemotherapy agent is selected from the group consisting of cyclophosphamide (e.g., Cytoxan, Neosar) and melphalan (e.g., Alkeran).

In some embodiments, at least one additional drug is an anti-inflammatory medication, when the subject has inflammatory symptoms.

In some embodiments, the at least one additional drug is an antibiotic, when the subject has infection symptoms. In some embodiments, the infection is a chromic infection. In some embodiments, the infection is a microbial infection.

In some embodiments, the at least one additional drug is a Carbonic Anhydrase (CA) enzyme (e.g., CA-I, CA-II, CA-III, CA-IV, CA-V, CA-VI, and CA-VII) and/or agents that can increase the activity of a Carbonic Anhydrase enzyme in the subject.

In some embodiments, at least one additional drug is a disease modifying antirheumatic drug (DMARD). In some embodiments, the DMARD is cyclosporine, azathioprine, methotrexate, leflunomide, cyclophosphamide, hydroxychloroquine, sulfasalazine, D-penicillamine, minocycline, gold, or any combination thereof.

In some embodiments, the at least one additional drug is a recombinant protein. In some embodiments, the recombinant protein is ENBREL® (etanercept, a soluble TNF receptor) or REMICADE® (infliximab, a chimeric monoclonal anti-TNF antibody).

In some embodiments, the one or more additional drugs is/are selected from melphalan, dexamethasone, bortezomib, lenalidomide, vincristine, doxorubicin, cyclophosphamide and pomalidomide.

In some embodiments, the methods of the present invention further comprise the administration of one or more drugs that acidifies the lysosome. As used herein, drugs that acidify the lysosome are drugs capable of lowering the lysosomal pH of a target cell. Accordingly, in some embodiments, the present invention provides a method of treating or preventing amyloidosis in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof, wherein the subject is also administered one or more drugs that acidifies the lysosome. As described herein, when performing a combination therapy, the two or more drugs (e.g., a catabolic enzyme or a biologically active fragment thereof and a drug that acidifies the lysosome) can be administered simultaneously or sequentially in any order.

In some embodiments, the drug that acidifies the lysosome is selected from an acidic nanoparticle, a catecholamine, a β-adrenergic receptor agonist, an adenosine receptor agonist, a dopamine receptor agonist, an activator of the cystic fibrosis transmembrane conductance regulator (CFTR), cyclic adenosine monophosphate (cAMP), a cAMP analog, and an inhibitor of glycogen synthase kinase-3 (GSK-3).

In some embodiments, the drug that acidifies the lysosome is an acidic nanoparticle. Acidic nanoparticles have been shown to localize to lysosomes and reduce lysosomal pH. See Baltazar et al., 2012, PloS ONE 7(12): e49635 and Lee et al., 2015, Cell Rep. 12(9): 1430-44, both of which are herein incorporated by reference in their entireties. In some embodiments, the acidic nanoparticle is a polymeric acidic nanoparticle. In some embodiments, the polymeric acidic nanoparticle is a poly (DL-lactide-co-glycolide) (PLGA) acidic nanoparticle. In a specific embodiment, the PLGA acidic nanoparticle comprises PLGA Resomer RG 503 H. In some embodiments, the PLGA acidic nanoparticle comprises PLGA Resomer RG 502 H. In other embodiments, the polymeric acidic nanoparticle is a poly (DL-lactide) (PLA) acidic nanoparticle. In a specific embodiment, the PLA acidic nanoparticle comprises PLA Resomer R 203 S. In some embodiments, the acid number of the acidic nanoparticle is between about 0.5 mg KOH/g to about 8 mg KOH/g. In some embodiments, the acid number of the acidic nanoparticle is between about 1 mg KOH/g to about 6 mg KOH/g. In some embodiments, the acid number of the acidic nanoparticle is selected from about 1 mg KOH/g, about 2 mg KOH/g, about 3 mg KOH/g, about 4 mg KOH/g, about 5 mg KOH/g, or about 6 mg KOH/g. In a specific embodiment, the acid number of the acidic nanoparticle is about 3 mg KOH/g. In some embodiments, the nanoparticle size is about 50 nm to about 800 nm. In some embodiments, the nanoparticle size is about 100 nm to about 600 nm. In a specific embodiment, the nanoparticle size is about 350 nm to about 550 nm. In a further specific embodiment, the nanoparticle size is about 375 nm to about 400 nm. In an exemplary embodiment, the acidic nanoparticle is spherical. In some embodiments, the nanoparticles are targeting a specific transport process in the brain, which enhance drug transport through the blood-brain barrier (BBB). In some embodiments, such transport processes include, but are not limited to: (1) nanoparticles open TJs between endothelial cells or induce local toxic effect which leads to a localized permeabilization of the BBB allowing the penetration of the drug in a free form or conjugated with the nanoparticles; (2) nanoparticles pass through endothelial cell by transcytosis; (3) nanoparticles are transported through endothelial cells by endocytosis, where the content is released into the cell cytoplasm and then exocytosed in the endothelium abluminal side; and (4) a combination of several of the mechanisms. In some embodiments, the receptors targeted by nanoparticles are transferrin and low-density lipo-protein receptors. In some embodiments, the targeting can be achieved by peptides, proteins, or antibodies, which can be physically and/or chemically immobilized on the nanoparticles. In some embodiments, the nanoparticles are coated with one or more apolipoproteins, such as apolipoprotein AII, B, CII, E, and/or J (see, Kreuter et al., (2002, DOI: 10.1080/10611860290031877). For more nanoparticle-mediated brain drug delivery compositions and methods, see Saraiva et al. (Journal of Controlled Release, 2016, 235:34-37). Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the drug that acidifies the lysosome is a catecholamine. Catecholamines have been shown to reduce lysosomal pH. See Liu et al., 2008, Invest Ophthalmol Vis Sci. 49(2): 772-780, which is herein incorporated by reference in its entirety. In some embodiments, the catecholamine is selected from epinephrine, metanephrine, synephrine, norepinephrine, normetanephrine, octopamine or norphenephrine, dopamine, and dopa. In exemplary embodiment, the catecholamine is selected from epinephrine, norepinephrine, and dopamine.

In some embodiments, the drug that acidifies the lysosome is a β-adrenergic receptor agonist. β-adrenergic receptor agonists have been shown to reduce lysosomal pH. See Liu et al., 2008, Invest Ophthalmol Vis Sci. 49(2): 772-780. Examples of β-adrenergic receptor agonists may be found in US Patent Publication No. 2012/0329879, which is herein incorporated by reference in its entirety. In some embodiments, the β-adrenergic receptor agonist is selected from isoproterenol, metaproterenol, formoterol, salmeterol, salbutamol, albuterol, terbutaline, fenoterol, and vilanterol. In an exemplary embodiment, the β-adrenergic receptor agonist is isoproterenol.

In some embodiments, the drug that acidifies the lysosome is an adenosine receptor agonist. Adenosine receptor agonists have been shown to reduce lysosomal pH. See Liu et al., 2008, Invest Ophthalmol Vis Sci. 49(2): 772-780. In an exemplary embodiment, the adenosine receptor agonist is a non-specific adenosine receptor agonist or an A_2A adenosine receptor agonist. Examples of A_2A adenosine receptor agonists may be found in US Patent Publication No. 2012/0130481, which is herein incorporated by reference in its entirety. In some embodiments, the adenosine receptor agonist is selected from 5′-N-ethylcarboxamidoadenosine (NECA), CGS21680, 2-phenylaminoadenosine, 2-[para-(2carboxyethyl)phenyl]amino-5′N-ethylcarboxamidoadenosine, SRA-082, 5′-N-cyclopropylcarboxamidoadenosine, 5′N-methylcarboxamidoadenosine and PD-125944.

In some embodiments, the drug that acidifies the lysosome is a dopamine receptor agonist. Dopamine receptor agonists have been shown to reduce lysosomal pH. See Guha et al., 2014, Adv Exp Med Biol. 801: 105-111, which is herein incorporated by reference in its entirety. In some embodiments, the dopamine receptor agonist is selected from A68930, A77636, A86929, SKF81297, SKF82958, SKF38393, SKF89145, SKF89626, dihydrexidine, dinapsoline, dinoxyline, doxanthrine, fenoldopam, 6-Br-APB, stepholidine, CY-208243, 7,8-Dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline, cabergoline, and pergolide. In an exemplary embodiment, the dopamine receptor agonist is selected from A68930, A77636, and SKF81297. In a further exemplary embodiment, the dopamine receptor agonist is SKF81297, also known as 6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol.

In some embodiments, the drug that acidifies the lysosome is an activator of the cystic fibrosis transmembrane conductance regulator (CFTR). Activators of CFTR have been shown to reduce lysosomal pH. See Liu et al., 2012, Am J Physiol Cell Physiol 303: C160-9, which is herein incorporated by reference in its entirety. In some embodiments, the CFTR activator is selected from CFTR_Act01 to CFTR_Act17. See Ma et al., J Biol Chem 277: 37235-37241. In an exemplary embodiment, the CFTR activator is selected from CFTR_Act11 and CFTR_Act16, having the following structures:

In some embodiments, the CFTR activator is co-administered with forskolin.

In some embodiments, the drug that acidifies the lysosome is cAMP or a cAMP analog. cAMP and/or cAMP analogs have been shown to reduce lysosomal pH. See Liu et al., 2008, Invest Ophthalmol Vis Sci. 49(2): 772-780. For instance, the cell-permeable analogs chlorophenylthio-cAMP (cpt-cAMP) and 8-bromo-cAMP have the ability to lower lysosomal pH in cells. In some embodiments, cAMP and/or a cAMP analog may be administered in a cocktail comprising 3-isobutyl-1-methylxanthine (IBMX) and forskolin. For example, in one embodiment, a cocktail comprising IBMX, forskolin, and cpt-cAMP may be administered to acidify the lysosome. In some embodiments, the cAMP analog is selected from 9-pCPT-2-O-Me-cAMP, Rp-cAMPS, 8-Cl-cAMP, Dibutyryl cAMP, pCPT-cAMP, N6-monobutyryladenosine 3′,5′-cyclic monophosphate, and PDE inhibitors.

In some embodiments, the drug that acidifies the lysosome is an inhibitor of glycogen synthase kinase-3 (GSK-3). GSK-3 inhibitors have been shown to be effective in reducing the lysosomal pH. See Avrahami et al., 2013, Commun Integr Biol 6(5): e25179, which is herein incorporated by reference in its entirety. For instance, the competitive GSK-3 inhibitor, L803-mts, has been shown to facilitate acidification of the lysosome by inhibiting GSK-3 activity, which acts to impair lysosomal acidification. Accordingly, in one embodiment, the inhibitor of GSK-3 is the cell permeable peptide, L803-mts (SEQ ID NO: 72). Suitable GSK-3 inhibitors may be found in US Patent Publication Nos. 2013/0303441 and 2015/0004255, which are herein incorporated by reference in their entireties. In some embodiments, the GSK-3 inhibitor is selected from 2′Z,3′E)-6-bromoindirubin-3′-acetoxime, TDZD-8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl), NP-103, 2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, L803, L803-mts, and GF-109203X (2-[1-(3-Dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)malemide and pharmaceutically acceptable salts and mixtures thereof.

In some embodiments, the methods of the present invention further comprise the administration of one or more drugs that promotes autophagy. As used herein, drugs that promote autophagy can promote the intracellular degradation system that delivers cytoplasmic constituents to the lysosome. Accordingly, in some embodiments, the present invention provides a method of treating or preventing amyloidosis in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof, and one or more drugs that promotes autophagy. In some embodiments, the present invention provides a method of treating or preventing amyloidosis in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof, wherein the subject is also administered one or more drugs that acidifies the lysosome and/or endosome, and one or more drugs that promotes autophagy. In some embodiments, the drug that acidifies the lysosome and/or endosome, and the drug that promotes autophagy can be the same drug, or different drugs. As described herein, when performing a combination therapy, the drugs (e.g., a catabolic enzyme or a biologically active fragment thereof, a drug that acidifies the lysosome and/or endosome, and/or a drug that promotes autophagy) can be administered simultaneously or sequentially in any order. Without wishing to be bound by any particular theory, a treatment of therapeutic catabolic enzyme or a biologically active fragment thereof with an agent that can cause lysosome and/or endosome acidification and/or an agent that can promote autophagy is capable of lowering pH to optimal conditions for enzymatic proteolysis, and improving lysosomal proteolysis power.

In some embodiments, autophagy promoting reagents include, but are not limited to reagents that directly or indirectly promote autophagy such as TFEB activators, PPAR agonists, PGC-1α activators, LSD1 inhibitors, mTOR inhibitors, GSK3 inhibitors, etc.

In some embodiments, the drug promotes autophagy via activation of Transcription factor EB (TFEB) pathway. TFEB is a master gene for lysosomal biogenesis. It encodes a transcription factor that coordinates expression of lysosomal hydrolases, membrane proteins and genes involved in autophagy. TFEB overexpression in cultured cells induced lysosomal biogenesis and increased the degradation of complex molecules. TFEB is activated by PGC-1α and promotes reduction of htt aggregation and neurotoxicity.

In some embodiments, the drug that promotes autophagy via activation of TFEB pathway is an activator of TFEB. In some embodiments, such TFEB activator include, but are not limited to C1 (Song et al, 2016, Autophagy, 12(8):1372-1389), and 2-hydroxypropyl-β-cyclodextrin (Kilpatrick et al., 2015, PLOS ONE DOI:10.1371/journal.pone.0120819). Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the drug that promotes autophagy via activation of TFEB pathway is an agent that can activate peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α). In some embodiments, such activators of PGC-1α include, but are not limited to, pyrroloquinoline quinone, resveratrol, R-α-lipoic acid (ALA), ALA /acetyl-L-carnitine (ALC), flavonoids, isoflavones and derivatives (e.g., quercetin, daidzein, genistein, biochanin A, and formononetin). See, Das and Sharma 2015 (CNS & Neurological Disorders—Drug Targets, 2015, 14, 1024-1030.) Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the drug promotes autophagy via activation of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and/or Forehead box O3 (FOXO3). PGC-1α is a master regulator of mitochondrial biogenesis. PGC-1α interacts with the nuclear receptor PPAR-γ, which permits the interaction of this protein with multiple transcription factors. This protein can interact with, and regulate the activities of, cAMP response element-binding protein (CREB) and nuclear respiratory factors (NRFs). It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis, and is a major factor that regulates muscle fiber type determination. FOXO3 is a transcription factor that can be inhibited and translocated out of the nucleus on phosphorylation by protein such as Akt/PKB in the PI3K signaling pathway.

In some embodiments, a drug that promotes autophagy via PGC-1α and/or FOXO3 activation is an inhibitor of Lysine (K)-specific demethylase 1A (LSD1). LSD1 is a flavin-dependent monoamine oxidase, which can demethylate mono- and bi-methylated lysines. LSD1 has roles critical in embryogenesis and tissue-specific differentiation. In some embodiments, such LSD1 inhibitors include, but are not limited to, 1-(4-methyl-1-piperazinyl)-2-[[(1R*,2S*)-2-[4-phenylmethoxy)phenyl]cyclopropyl]amino]ethanone dihydrochloride (RN-1; Cui et al., 2015, Blood 2015 126:386-396), CBB1001-1009 (Wang et al., 2011, Cancer Res. 2011 Dec. 1; 71(23): 7238-7249.), TCP, Pargyline, CGC-11047, and Namolone (Pieroni et al., 2015, European Journal of Medicinal Chemistry 92 (2015) 377e386), phenelzine analogues (Prusevich et al., ACS Chem. Biol. 2014, 9, 1284-1293), and those described in WO2015156417, which is herein incorporated by reference in its entirety. In some embodiments, one or more LSD1 inhibitors are used. In some embodiments, both RN-1 and a LSD1 inhibitor described in WO2015156417 are used. WO2015156417 describes inhibitors of LSD1 represented by formula I:

wherein, A is an optionally substituted heterocyclic group, or an optionally substituted hydrocarbon group; B is a ring selected from

• (1) a 5- or 6-membered aromatic heterocycle optionally fused with an optionally substituted 5- or 6-membered ring, and
• (2) a benzene ring fused with an optionally substituted 5- or 6-membered ring, wherein the ring represented by B is optionally substituted, and binds, via two adjacent carbon atoms with one atom in between, to a group represented by the formula

and a group represented by the formula

• R¹, R², R³ and R⁴ are each independently a hydrogen atom, an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group;
• A and R¹ are optionally bonded with each other to form, together with the adjacent nitrogen atom, an optionally substituted cyclic group; and
• R² and R³ are optionally bonded with each other to form, together with the adjacent nitrogen atom, an optionally substituted cyclic group, or a salt thereof. Such LSD1 inhibitors are more specific with less side effect and good blood-brain barrier penetration.

In some embodiments, the LSD1 inhibitors are selected from the group consisting of the following compounds (compounds 1-30), and salts, stereoisomers, geometric isomers, tautomers, oxynitrides, enantiomers, diastereoisomers, racemates, prodrugs, solvates, metabolites, esters, and mixtures thereof:

In one embodiment, the LSD1 inhibitor to be co-administered with a catabolic enzyme of the present invention or a biologically active fragment thereof is compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any mixtures thereof.

In some embodiments, the drug is capable of modify the activity of a regulator or a co-activator of PGC-1α. Such regulators or co-activators of PGC-1α include, but are not limited to, Parkin Interacting Substrate (PARIS), Sirtuin 1 (SIRT1), 5′ AMP-activated protein kinase(AMPK), General control of amino acid synthesis protein 5 (GCN5), Nuclear respiratory factor 1, 2(NRF-1,2), Glycogen synthase kinase 3β (GSK3β), Peroxisome proliferator-activated receptor-α,β/δ,γ (PPAR-α,β/δ,γ), p38 mitogen-activated protein kinase (p38MAPK), Estrogen-related receptors (ERRs), myocyte enhancer factor-2 (MEF2), and Thyroid hormone receptor (TR), see Das and Sharma (CNS & Neurological Disorders—Drug Targets, 2015, 14, 1024-1030). Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the drug that promotes autophagy is a Peroxisome proliferator-activated receptor (PPAR) agonist. PPARs are nuclear receptor proteins that function as transcription factors regulating the expression of genes. They are critical in the regulation of cellular differentiation, development, and metabolism and tumorigenesis.

In some embodiments, the PPAR is selected from PPARα, PPARβ/δ, and PPARγ. In some embodiments, the PPAR agonist is a PPARα agonist, including but not limited to amphipathic carboxylic acids (e.g., clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate), fibrate, ureidofibrate, oxybenzylglycine, triazolone, agonists containing a 2,4-dihydo-3H-1,2,4 triazole-3-one (triazolone) core (e.g., LY518674), BMS-687453, Wy-14643, GW2331, GW 95798, LY518674, and GW590735.

In some embodiments, the PPAR agonist is a PPARβ/δ agonist, including but not limited to GW501516 (Brunmair; et al. Diabetologia. 49 (11): 2713-22), L-165041, compound 7 (Burdick et al., Cell Signal 2006, 18 (1), 9-20), thiazole, bisaryl substituted thiazoles, non-TZD compounds (e.g., L-165041), L-165041, compound 7 (Burdick et al., Cell Signal 2006, 18 (1), 9-20), 38c (Johnson et al., J Steroid Biochem Mol Biol 1997, 63 (1-3), 1-8), and oxazoles. Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the PPAR agonist is a PPARγ agonist, including but not limited to thiazolidinediones (TZDs or glitazones), glitazar, indenone, NSAIDs, dihydrocinnamate, β-carboxyethyl rhodamine, and those described in Corona and Duchen, 2016 (Free Radical Biology and Medicine, published online Jun. 23, 2016). In some embodiments, the PPARγ agonist is an endogenous or natural agonist. In some embodiments, the PPARγ agonist is a synthetic agonist. In some embodiments, the PPARγ agonist is selected from the group consisting of eicosanoids prostaglandin-A1, cyclopentenone prostaglandin 15-deoxy-Δ^12,14-Prostaglandin J2 (15D-PGJ2), unsaturated fatty acids such as linoleic acid and socosahexaenoic acid, nitroalkenes such as nitrated oleic acid and linoleic acid, oxidized phospholipids such as hexadecyl azelaoyl phosphatidylcholine and lysophosphatidic acid, non-steroidal anti-inflammatory drugs, such as flufenamic acid, ibuprofen, fenoprofen, and indomethacin, pioglitazone, GW0072, ciglitazone, troglitazone, rosiglitazone, isoglitazone, NC-2100 (Loiodice et al., Curr. Top. Med. Chem. 2011, 11(7):819-39), SB-236636, tesaglitazar, farglitazar, GW1929, compound 14c (Haigh et al., Bioorg Med Chem 1999, 7(5):821-30), SP1818, ragaglitazar, metaglidasen, balaglitazone, and INT131. Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the PPAR agonist binds to PPARα, PPARβ/δ, and PPARγ, such as bezafibrate, LY465608, indeglitazar, TIPP-204, GW693085, TIPP-401, and TIPP-703. In some embodiments, the PPAR agonist binds to PPARα and PPARγ, such as farglitazar, muraglitazar, tesaglitazar, GW409544, aleglitazar, MK-767, TAK-559, compound 18 (Kojo et al., J. Pharmacol Sci 2003, 93 (3), 347-55), compounds 68, 70, 72, 76 (Felts et al., J Med Chem 2008, 51 (16), 4911-9), metaglidasen, and S-2/S-4 (Suh et al., J Med Chem 2008, 51 (20), 6318-33). In some embodiments, the PPAR agonist binds to PPARβ and PPARγ, such as compound 23 (Martin et al., J Med Chem 2009, 52(21), 6835-50). More PPARs agonists are described in Nevin et al., 2011 (Current Medicinal Chemistry, 2011, 18, 5598-5623). Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the drug that promotes autophagy is an inhibitor of mechanistic target of rapamycin (mTOR). mTOR is a serine/threonine-specific protein kinase that belongs to the family of phosphatidylinositol-3 kinase (PI3K) related kinases (PIKKs), see Maiese et al. (Br J Clin Pharmacol, 82(5):1245-1266), which is herein incorporated by reference in its entirety. mTOR integrates the input from upstream pathways, including insulin, growth factors (such as IGF-1 and IGF-2), and amino acids, and also senses cellular nutrient, oxygen, and energy levels. In some embodiments, mTOR inhibitors include, but are not limited to, an antibody of mTOR, rapamycin and its analogs (e.g., temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), sirolimus, deforolimus), curcumin (Zhang et al., 2016, Oncotarget), curcumin analogs (Song et al. 2016, Autophagy, 12(8):1372-1389), ATP-competitive mTOR kinase inhibitors, mTOR/PI3K dual inhibitors (dactolisib, BGT226, SF1126, PKI-587 etc.), deptor (Maiese, Neural Regeneration Research. 2016; 11(3):372-385), and mTORC1/mTORC2 dual inhibitors (TORCdIs, such as sapanisertib (a.k.a. INK128), AZD8055, and AZD2014). Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the drug that promotes autophagy is an inhibitor of Glycogen synthase kinase 3 (GSK3). GSK3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. In some embodiments, the GSK3 inhibitor is ATP-competitive. In some embodiments, the GSK3 inhibitor is non-ATP competitive. In some embodiments, GSK3 inhibitors include, but are not limited to, an antibody of GSK3, metal cations (e.g., beryllium, copper, lithium, mercury, and tungsten), marine organism-derived drugs (e.g., 6-BIO, dibromocantharelline, hymenialdesine, indirubins, meridianins, manzamine A, palinurine, tricantine), aminopyrimidines (e.g., CT98014, CT98023, CT99021, and TWS119), ketamine, arylindolemaleimide (e.g., SB-216763 and SB-41528), thiazoles (e.g., AR-A014418 and AZD-1080), paullones (e.g., Alsterpaullone, Cazpaullone, Kenpaullone), thiadiazolidindiones (e.g., TDZD-8, NP00111, NP031115, and tideglusib), halomethylketones (e.g., HMK-32), certain peptides (L803-mts), SB415286, SB216763, and CT99021 (Stretton et al., 2015, Biochem. J. (2015) 470, 207-221; Marchand et al., 2015, The Journal of Biological Chemistry, 290(9):5592-5605). Each of the references mentioned herein is incorporated by reference in its entirety.

In some embodiments, the methods of the present invention further comprise the administration of one or more drugs that modulates the lysosome. In some embodiments, drugs that modulate the lysosome may be capable of decreasing the level of Rab5a, a marker of early endosomes. Accordingly, in some embodiments, the present invention provides a method of treating or preventing amyloidosis in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof, wherein the subject is also administered one or more drugs that modulates the lysosome. As described herein, when performing a combination therapy, the two or more drugs (e.g., a catabolic enzyme or a biologically active fragment thereof and a drug that modulates the lysosome) can be administered simultaneously or sequentially in any order

In some embodiments, the drug that modulates the lysosome is Z-phenylalanyl-alanyl-diazomethylketone (PADK) or a PADK analog, or a pharmaceutically acceptable salt or ester thereof. In some embodiments, the PADK analog is selected from Z-L-phenylalanyl-D-alanyl-diazomethylketone (PdADK), Z-D-phenylalanyl-L-alanyl-diazomethylketone (dPADK), and Z-D-phenylalanyl-D-alanyl-diazomethylketone (dPdADK). In some embodiments, the drug that modulates the lysosome is Z-phenylalanyl-phenylalanyl-diazomethylketone (PPDK) or a PPDK analog, or a pharmaceutically acceptable salt or ester thereof. An exemplary listing of suitable lysosome modulators may be found in US Patent Publication No. 2016/0136229, which is herein incorporated by reference in its entirety.

In some embodiments, when performing a combination therapy, the two or more drugs can be administered simultaneously or sequentially in any order. In some embodiments, when at least two drugs are administered sequentially, the duration between the two administrations can be about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 2 days, three days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or more.

In some embodiments, the methods of the present invention further comprise a surgery to be performed on the subject. In some embodiments, the surgery is stem cell transplantation and/or organ transplantation. In some embodiments, the stem cell transplantation is autologous (e.g., stem cells derived from the subject).

In some embodiments, the methods further comprise providing a supportive treatment to the subject. In some embodiments, when the heart or kidneys of the subject are affected, the methods comprise taking a diuretic (water excretion pill), restricting the amount of salt in diet, and/or wearing elastic stockings and elevating their legs to help lessen the amount of swelling. In some embodiments, when the gastrointestinal tract is involved, dietary changes and certain medications can be tried to help symptoms of diarrhea and stomach fullness.

A pharmaceutical composition of the present invention can be administered to a patient by any suitable methods known in the art. In some embodiments, administration of a composition of the present invention may be carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, aerosolly (e.g., inhalation) or by application to mucous membranes.

In some embodiments, a pharmaceutical composition of the present invention further comprises a pharmaceutically-acceptable carrier. When the term “pharmaceutically acceptable” is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

Compositions intended for oral use may be prepared in either solid or fluid unit dosage forms. Fluid unit dosage form can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. An elixir is prepared by using a hydroalcoholic (e.g., ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.

Solid formulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.

Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl-p-hydroxy benzoate, one or more colouring agents, one or more flavoring agents or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and colouring agents, may also be present.

Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.

In some embodiments, the delivery systems suitable include time-release, delayed release, sustained release, or controlled release delivery systems. In some embodiments, a composition of the present invention can be delivered in a controlled release system, such as sustained-release matrices. Non-limiting examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 and Langer, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)], polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers, 22:547-556), non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP 133,988). In some embodiments, the composition may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, for example liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990). In some embodiments, the composition may be administered through subcutaneous injection.

In some embodiments, the release of the composition occurs in bursts. Examples of systems in which release occurs in bursts includes, e.g., systems in which the composition is entrapped in liposomes which are encapsulated in a polymer matrix, the liposomes being sensitive to specific stimuli, e.g., temperature, pH, light or a degrading enzyme and systems in which the composition is encapsulated by an ionically-coated microcapsule with a microcapsule core degrading enzyme.

In some embodiments, the release of the composition is gradual/continuous. Examples of systems in which release of the inhibitor is gradual and continuous include, e.g., erosional systems in which the composition is contained in a form within a matrix and effusional systems in which the composition is released at a controlled rate, e.g., through a polymer. Such sustained release systems can be e.g., in the form of pellets, or capsules.

Other embodiments of the compositions administered according to the invention incorporate particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, such as parenteral, pulmonary, nasal and oral. Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000). In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable diluent, excipient, carrier, or adjuvant.

In some embodiments, the dosage to be administered is not subject to defined limits, but it will usually be an effective amount, or a therapeutically/pharmaceutically effective amount. The term “effective amount” refers to the amount of one or more compounds that renders a desired treatment outcome. An effective amount may be comprised within one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. The term “therapeutically/pharmaceutically effective amount” as used herein, refers to the level or amount of one or more agents needed to treat a condition, or reduce or prevent injury or damage, optionally without causing significant negative or adverse side effects. It will usually be the equivalent, on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects. In some embodiments, the compositions may be formulated in a unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

In some embodiments, dosing regimen of a pharmaceutical composition of the present invention includes, without any limitation, the amount per dose, frequency of dosing, e.g., per day, week, or month, total amount per dosing cycle, dosing interval, dosing variation, pattern or modification per dosing cycle, maximum accumulated dosing, or warm up dosing, or any combination thereof.

In some embodiments, dosing regimen includes a pre-determined or fixed amount per dose in combination with a frequency of such dose. For example, dosing regimen includes a fixed amount per dose in combination with the frequency of such dose being administered to a subject.

In some embodiments, the at least one catabolic enzyme (e.g., PPCA, NEU1, TPP1, cathepsin B, cathepsin D, cathepsin E, cathepsin K, and/or cathepsin L) is administered at about 0.1 to 20 mg/kg daily, weekly, biweekly, monthly, or bi-monthly. In some embodiments, the at least one intralysosomal catabolic enzyme is administered at about 0.2 to 15 mg/kg, about 0.5 to 12 mg/kg, about 1 to 10 mg/kg, about 2 to 8 mg/kg, or about 4 to 6 mg/kg daily, weekly, biweekly, monthly, or bi-monthly.

Based on the suitable dosage, the at least one catabolic enzyme can be provided in various suitable unit dosages. For example, a catabolic enzyme can comprise a unit dosage for administration of one or multiple times per day, for 1-7 days per week, or for 1-31 times per month. Such unit dosages can be provided as a set for daily, weekly and/or monthly administration.

As will be appreciated by those skilled in the art, the duration of the treatment methods depends on the type of amyloidosis being treated, any underlying diseases associated with amyloidosis, the age and conditions of the subject, how the subject responds to the treatment, etc.

In some embodiments, a person having risk of developing amyloidosis (e.g., a person who is genetically predisposed or previously had amyloidosis or associated diseases) can also receive prophylactic treatment of the present invention to inhibit or delay the development of amyloidosis and/or associated diseases.

The pharmaceutical composition of the present invention may also alleviate, reduce the severity of, or reduce the occurrence of, one or more of the symptoms associated with amyloidosis. In some embodiments, the symptoms are those associated with light-chain (AL) amyloidosis (primary systemic amyloidosis) and/or AA amyloidosis (secondary amyloidosis). In some embodiments, the symptoms include, but are not limited to, fluid retention, swelling, shortness of breath, fatigue, irregular heartbeat, numbness of hands and feet, rash, shortness of breath, swallowing difficulties, swollen arms or legs, esophageal reflux, constipation, nausea, abdominal pain, diarrhea, early satiety, stroke, gastrointestinal disorders, enlarged liver, diminished spleen function, diminished function of the adrenal and other endocrine glands, skin color change or growths, lung problems, bleeding and bruising problems, decreased urine output, diarrhea, hoarseness or changing voice, joint pain, and weakness. In some embodiments, the symptoms are those associated with amyloid-beta (Aβ) amyloidosis. In some embodiments, the symptoms include, but are not limited to, common symptoms of Alzheimer's disease, including memory loss, confusion, trouble understanding visual images and spatial relationships, and problems speaking or writing.

In some embodiments, the methods further comprise monitoring the response of the subject after administration to avoid severe and/or fatal immune-mediated adverse reactions due to over-dosage. In some embodiments, the administration of a pharmaceutical composition of the present invention is modified, such as reduced, paused or terminated if the patient shows persistent adverse reactions. In some embodiments, the dosage is modified if the patient fails to respond within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks or more from administration of first dose.

In some embodiments, a pharmaceutical composition of the present invention can ameliorate, treat, and/or prevent one or more conditions or associated symptoms described herein in a clinically relevant, statistically significant and/or persistent fashion. In some embodiments, administration of a pharmaceutical composition of the present invention provides statistically significant therapeutic effect for ameliorating, treating, and/or preventing one or more symptoms of amyloidosis. In one embodiment, the statistically significant therapeutic effect is determined based on one or more standards or criteria provided by one or more regulatory agencies in the United States, e.g., FDA or other countries. In some embodiments, the statistically significant therapeutic effect is determined based on results obtained from regulatory agency approved clinical trial set up and/or procedure.

In some embodiments, the statistically significant therapeutic effect is determined based on a patient population of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some embodiments, the statistically significant therapeutic effect is determined based on data obtained from randomized and double blinded clinical trial set up. In some embodiments, the statistically significant therapeutic effect is determined based on data with a p value of less than or equal to about 0.05, 0.04, 0.03, 0.02 or 0.01. In some embodiments, the statistically significant therapeutic effect is determined based on data with a confidence interval greater than or equal to 95%, 96%, 97%, 98% or 99%. In some embodiments, the statistically significant therapeutic effect is determined on approval of Phase III clinical trial of the methods provided by the present invention, e.g., by FDA in the US.

In some embodiment, the statistically significant therapeutic effect is determined by a randomized double blind clinical trial of a patient population of at least 50, 100, 200, 300 or 350; treated with a pharmaceutical composition of the present invention, but not in combination with any other agent. In some embodiment, the statistically significant therapeutic effect is determined by a randomized clinical trial of a patient population of at least 50, 100, 200, 300 or 350 and using any commonly accepted criteria for amyloidosis symptoms assessment.

In general, statistical analysis can include any suitable method permitted by a regulatory agency, e.g., FDA in the US or China or any other country. In some embodiments, statistical analysis includes non-stratified analysis, log-rank analysis, e.g., from Kaplan-Meier, Jacobson-Truax, Gulliken-Lord-Novick, Edwards-Nunnally, Hageman-Arrindel and Hierarchical Linear Modeling (HLM) and Cox regression analysis.

The invention also provides packaged pharmaceutical compositions or kits. In some embodiments, the packaged pharmaceutical compositions or kits include a therapeutically effective amount of an intralysosomal catabolic enzyme or a formulation comprising an intralysosomal catabolic enzyme of the present invention described herein. In some embodiments, the compound or formulation can increase the expression, activity, and/or concentration of at least one intralysosomal catabolic enzyme in a subject when the composition is administered to the subject. In some embodiments, the packaged pharmaceutical compositions or kits further comprise in combination with a label or insert advising that the pharmaceutical compound or formulation be administered in combination with a second agent for treating or preventing amyloidosis described herein.

In some embodiments, the packaged pharmaceutical compositions or kits further comprise a therapeutically effective amount of a second agent described herein. In some embodiments, the packaged pharmaceutical compositions or kits is packaged in combination with a label or insert advising that the second agent be administered in combination with the intralysosomal catabolic enzyme or the formulation comprising an intralysosomal catabolic enzyme, or the compound or formulation that can increase the expression, activity, and/or concentration of at least one intralysosomal catabolic enzyme in a subject.

As used herein, the term “label or insert” includes, but is not limited to all written, electronic, or spoken communication with the subject, or with any person substantially responsible for the care of the subject, regarding the administration of the compositions of the present invention. An insert may further include information regarding co-administration of the compositions of the present invention with other compounds or compositions. Additionally, an insert may include instructions regarding administration of the compositions of the present invention before, during, or after a meal, or with/without food.

The following examples illustrate various aspects of the invention. The examples should, of course, be understood to be merely illustrative of only certain embodiments of the invention and not to constitute limitations upon the scope of the invention.

EXAMPLES

Example 1

Degradative Effects of Intralysosomal Catabolic Enzymes on Synthetic Amyloid Species

In this example, an in vitro study is performed to illustrate that intralysosomal enzymes such as PPCA (i.e., cathepsin A), cathepsin B, cathepsin D, and/or cocktail mixtures of two or more intralysosomal enzymes can be used for the treatment of amyloidosis. Without being bound by theory, it is hypothesized that delivery of PPCA, cathepsin B, cathepsin D, and other intralysosomal enzymes to lysosomes can assist in the degradation of abnormally accumulated amyloid species, e.g., Aβ-amyloid species before they can be transported into the extracellular space by exocytosis and be deposited as amyloid plaques.

This in vitro study shows the degradative effects of PPCA, cathepsin B, and cathepsin D on synthetic Aβ-amyloid species in a test tube.

First, in vitro aggregation assays of Aβ-amyloid species using synthetic Aβ-peptides is performed via a Thioflavin-T (THT) assay and western blot. FIG. 1 shows the aggregation of synthetic Aβ42 peptide and Aβ15-36 peptide (negative control) monitored by Thioflavin-T (THT) at physiological conditions (FIG. 1A) or an acidic pH (FIG. 1B). FIG. 2 shows the aggregation of Aβ42 amyloid species over time 24 hours as detected by western blot.

Second, prevention of the aggregation of synthetic Aβ-amyloid species by proteolytic degradation using PPCA, cathepsin B, and cathepsin D is tested via a Thioflavin-T (THT) assay and western blot. FIG. 3 shows that cathepsin A (i.e., PPCA) prevents the aggregation of Aβ42 amyloid. FIG. 4 shows that PPCA prevents the aggregation of Aβ42 amyloid in a dose dependent manner. FIG. 5 shows that PPCA prevents the aggregation of both high and low molecular weight species of Aβ42 amyloid. FIG. 6 shows that cathepsin B prevents the aggregation of Aβ42 amyloid. FIG. 7 shows that cathepsin B moderately prevents the aggregation of Aβ42 amyloid in a dose dependent manner. FIG. 8 shows that cathepsin B prevents the aggregation of low molecular weight species of Aβ42 amyloid and degrades Aβ42 monomers in a time-dependent manner. FIG. 9 shows that cathepsin B prevents the aggregation of Aβ42 amyloid.

Lastly, the ability of PPCA, cathepsin B, and cathepsin D to degrade pre-formed synthetic Aβ-amyloid species was tested. FIG. 10 shows that PPCA, cathepsin B, PPCA plus cathepsin B, and cathepsin D degrade high molecular weight oligomers/fibrils of Aβ42 amyloid. Cathepsin D degrades low molecular oligomers and completely eliminates Aβ42 monomers.

Example 1 Summary:

Experiments in Example 1 were designed to determine (1) whether the selected intralysosomal catabolic enzymes can prevent aggregation/formation of Aβ amyloid species (called prevention) and (2) whether the selected intralysosomal catabolic enzymes can degrade already pre-formed Aβ amyloid species (called degradation). Example 1 experiments have shown that Aβ42 amyloid species can be aggregated in vitro using synthetic Aβ42 peptides, and that this process can be monitored by THT assay (FIG. 1) and/or western blot analysis (FIG. 2). The THT assay allows for the monitoring of dynamic changes in Aβ42 aggregation upon treatment with degradative enzymes.

Data obtained from the experiments of Example 1 reveal that PPCA can efficiently prevent formation of Aβ42 amyloid species as shown by THT assay (FIG. 3, FIG. 4) and western blot (FIG. 5), as well as degrade already pre-formed amyloid species (FIG. 10). Prevention of amyloid formation and degradation by PPCA was efficient, reproducible and showed concentration dependent dynamics (FIG. 4). Data obtained from experiments with cathepsin B showed moderate reduction in amyloid species formation as measured by THT (FIG. 6). Western blot analysis revealed that cathepsin B prevents aggregation of low molecular weight Aβ42 species and degrades Aβ42 monomers in a time dependent manner (FIG. 8). Experiments with the use of cathepsin D revealed strong prevention of aggregation of Aβ42 species, measured by THT (FIG. 9). Cathepsin D also showed degradation of low molecular oligomers in pre-aggregated amyloid species and complete elimination Aβ42 monomers (FIG. 10).

Example 2

Degradation of Aβ42 Oligomers and Fibrils by Cathepsin A, B, and D

In this example, two protocols specific for oligomer and fibril formation were applied to aggregate amyloid material to investigate which forms of Aβ42 species can be degraded by cathepsin A (PPCA), cathepsin B and cathepsin D. Aggregated oligomers and fibrils were then subjected to an enzymatic treatment followed by western blot analysis.

Initially, oligomers and fibrils were aggregated for a period of 7 days and material collected at different time points (days: 0, 1, 3 and 7) was subjected to SDS-PAGE electrophoresis followed by western blot analysis. In FIG. 11, Aβ42 oligomers and Aβ42 fibrils were probed with oligomer specific antibody (A11), which does not recognize monomeric and fibril Aβ42 species. Various forms of oligomers were positively detected on western blot carrying material aggregated using both, oligomer formation and fibril formation protocols. A significant reduction in oligomer forms was observed at day 7 of fibril formation procedure (FIG. 11, line 9), indicating a time dependent transition from oligomers to fibrils, undetectable by A11 antibody. In FIG. 12, the same material as shown in FIG. 11 was probed with E610 antibody, which is specific for both oligomers and fibrils of Aβ42. A lack of fibrils at day 7 was observed when oligomer formation protocol was applied (FIG. 12, line 4) and a strong appearance of fibrils at day 7 when fibril formation protocol was applied.

To study enzymatic degradation of oligomer species, Aβ42 oligomers were first aggregated for 9 days at pH 7.0 at 25° C. and then additionally incubated overnight at 37° C. in various pH, optimal for each of enzymes used in the study (pH 5.0 Cathepsin A, B and pH 3.5 Cathepsin D), with and without addition of enzymes. Western blot was probed with oligomer specific A11 antibody (FIG. 13). Additional overnight aggregation of oligomers was observed at pH 5.0 as indicated by presence of higher molecular weight oligomers (lines 1, 2, 4, and 5) when compared to control line 9 (incubation for 9 days at 25° C.). In contrast, this aggregation was not observed for oligomers incubated overnight at pH 3.5. Overnight treatment of oligomers with 90 ng of cathepsin A at pH 5.0 and 37° C. resulted in degradation of the lowest oligomer band (line 4). Treatment of oligomers with 90 ng of cathepsin B and D did not reveal changes in intensity or size of oligomer band (lines 5, 6).

To study enzymatic degradation of fibril species, Aβ42 fibrils were first aggregated for 9 days at pH 7.0 at 25° C. and then additionally incubated overnight at 37 C in various pH, optimal for each of enzymes used in the study (pH 5.0 cathepsin A, B and pH 3.5 cathepsin D), with and without addition of enzymes. Western blot was probed with oligomer specific E610 antibody (FIG. 14). Additional overnight aggregation of fibrils was observed in all pHs applied, as indicated by the presence of stronger/darker smear (lines 1, 2, 3) when compared to control line 9 (incubation for 9 days at 25° C.). Overnight treatment of fibrils with 90 ng of cathepsin A at pH 5.0 and 37° C. resulted in reduction/degradation of the fibril smear as well as degradation of oligomer species (line 4 compared to line 1). Overnight treatment of fibrils with 90 ng of cathepsin B at pH 5.0 and 37° C. resulted in weak reduction/degradation of the fibril smear (line 5 compared to line 2). Overnight treatment of fibrils with 90 ng of cathepsin D at pH 3.5 and 37° C. did not result in visible reduction/degradation of fibril smear or oligomer bands.

Example 3

Degradation of Aβ42 Monomers by Cathepsin A Monitored by ELISA

The purpose of this example is to assess whether cathepsin A can degrade Aβ42 peptides (monomers).

In this example, an enzymatic treatment of peptides with 90 ng of cathepsin A was carried out for 0-2 hr at 37° C. and pH 5.0. An identical experiment without the addition of cathepsin A was performed in parallel. In both cases, phenol red, an inhibitor of Aβ aggregation was used to prevent peptide aggregation into higher molecular weight species of amyloid. The effects of supplementation or lack of cathepsin A on Aβ42 monomers were measured using commercially available ELISA (SensoLyte® Anti-Human β-Amyloid (1-42) Quantitative ELISA, Colorimetric) at various time points (0, 10, 30, 60, 120 min). Sensolite ELISA consists of two antibodies: C-terminal capture antibody, which recognizes specifically human Aβ42 peptide but not Aβ40 or Aβ41 and N-terminal detection antibody. Because Cathepsin A is a carboxyl peptidase, Aβ42 monomers, if degraded, will be degraded from their C-terminus. This degradation would result in a lack of C-terminal amino acid 42 and in consequence lack of capture by C-terminus specific antibody, which should be visualized as a loos of fluorescent signal in ELISA. The ELISA read out for samples treated with cathepsin A revealed a loss of fluorescent signal already within first 10 min of treatment indicating degradation of Aβ42 monomers from the C-terminus by cathepsin A (FIG. 15). Samples without supplementation of cathepsin A showed a strong fluorescent signal in ELISA indicating lack of C-terminal degradation in the absence of enzyme and thus efficient capture of Aβ42 monomers by C-terminus antibody.

Example 4

Degradation of Aβ40 Amyloid Species by Cath A

Aggregation experiments showed that Aβ40 amyloid species can be aggregated in vitro using synthetic Aβ40 peptides, and that this process can be monitored by THT assay (FIG. 16). When compared with aggregation of Aβ42 peptides, Aβ40 showed much slower and less efficient rate of aggregation (FIG. 16A).

Additional experiments were performed where THT assay was used to monitor dynamic changes in Aβ42 & Aβ40 aggregation upon treatment with degradative enzyme Cath A (FIG. 17). Initial experiment aimed to measure the effect of Cath A treatment on aggregation of both Aβ42 & Aβ40 peptides in real time. To achieve this, Cath A was simultaneously incubated with corresponding peptides and THT reagent in separate reactions at conditions optimal for Cath A proteolysis. The above experiment revealed that in contrast to Aβ42 (FIG. 17A), aggregation of Aβ40 amyloid is not affected by Cath A, in applied experimental settings, even when high concentration of enzyme is used (FIG. 17B, C). Second experiment was carried out to investigate whether the result of the initial experiment is due to lack of proteolysis of Aβ40 by Cath A or whether the speed of such proteolysis is slower than the speed of Aβ40 aggregation and therefore no changes in THT fluorescence could be observed. In this experiment Aβ40 peptide was first incubated with Cath A for up to two hours in conditions optimal for Cath A proteolysis and followed by incubation with THT to measure aggregation. Obtained data revealed that Aβ40 peptide did not aggregate after pre-incubation with Cath A, proving its proteolysis (FIG. 18).

To prove that observed loss of aggregation by Aβ40 peptide is caused by carboxypeptidase activity of Cath A, Aβ40 peptide was incubated for two hours at 37° C. at pH 5 with varying concentrations of Cath A. Subsequently, the reaction was transferred to an ELISA plate pre-coated with a C-terminal capture antibody, specifically for Aβ40 peptide only and was co-incubated with N-terminal detection antibody overnight at 4°. The results have shown progressively reduced binding of Aβ40 peptide to C-terminal capture antibody with increasing concentration of Cath A (FIG. 19). This proves that C-terminus of Aβ40 peptide was removed by caboxyterminal activity of Cath A.

Aggregation of Aβ40 peptide into amyloid species was also monitored using Western Blot technique (FIG. 20A). We were able to aggregate Aβ40 into high molecular weight fibrils but not oligomeric forms using aggregation process taking up to 9 days. An experiment was carried out in which Aβ40 was simultaneously incubated Cath A for up to 9 days during the process of fibril formation. Obtained results revealed that Cath A significantly prevents formation of high molecular weight fibrils due to its proteolytic action on Aβ40 amyloid (FIG. 20B). Reduction of levels of monomeric Aβ40 form was also observed in this experiment (FIG. 20C).

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and the application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features set forth and as follows in the scope of the appended claims.

SEQUENCE LISTING
SEQ ID NO: 1 Human PPCA mRNA, variant 1 mRNA
1    agagtgcacc cgaatccacg ggctcggagg cagcagccat ctctcggcca tagggcaggc
61   cagctggcgc cgggggctat tttgggcggc gggcaatgat ggtgaccgca aggcgacctt
121  gtaaggcatt tcccccctga ctcccttccc cgagcctctg cccgggggtc ctagcgccgc
181  tttctcagcc atcccgccta caacttagcc gtccacaaca ggatcatctg atcgcgtgcg
241  cccgggctac gatctgcgag gcccgcggac cttgacccgg cattgaccgc caccgccccc
301  caggtccgta gggaccaaag aaggggcggg aggaagactg tcacgtggcg ccggagttca
361  cgtgactcgt acacatgact tccagtcccc gggcgcctcc tggagagcaa ggacgcgggg
421  gagcagagat gatccgagcc gcgccgccgc cgctgttcct gctgctgctg ctgctgctgc
481  tgctagtgtc ctgggcgtcc cgaggcgagg cagcccccga ccaggacgag atccagcgcc
541  tccccgggct ggccaagcag ccgtctttcc gccagtactc cggctacctc aaaggctccg
601  gctccaagca cctccactac tggtttgtgg agtcccagaa ggatcccgag aacagccctg
661  tggtgctttg gctcaatggg ggtcccggct gcagctcact agatgggctc ctcacagagc
721  atggcccctt cctggtccag ccagatggtg tcaccctgga gtacaacccc tattcttgga
781  atctgattgc caatgtgtta tacctggagt ccccagctgg ggtgggcttc tcctactccg
841  atgacaagtt ttatgcaact aatgacactg aggtcgccca gagcaatttt gaggcccttc
901  aagatttctt ccgcctcttt ccggagtaca agaacaacaa acttttcctg accggggaga
961  gctatgctgg catctacatc cccaccctgg ccgtgctggt catgcaggat cccagcatga
1021 accttcaggg gctggctgtg ggcaatggac tctcctccta tgagcagaat gacaactccc
1081 tggtctactt tgcctactac catggccttc tggggaacag gctttggtct tctctccaga
1141 cccactgctg ctctcaaaac aagtgtaact tctatgacaa caaagacctg gaatgcgtga
1201 ccaatcttca ggaagtggcc cgcatcgtgg gcaactctgg cctcaacatc tacaatctct
1261 atgccccgtg tgctggaggg gtgcccagcc attttaggta tgagaaggac actgttgtgg
1321 tccaggattt gggcaacatc ttcactcgcc tgccactcaa gcggatgtgg catcaggcac
1381 tgctgcgctc aggggataaa gtgcgcatgg accccccctg caccaacaca acagctgctt
1441 ccacctacct caacaacccg tacgtgcgga aggccctcaa catcccggag cagctgccac
1501 aatgggacat gtgcaacttt ctggtaaact tacagtaccg ccgtctctac cgaagcatga
1561 actcccagta tctgaagctg cttagctcac agaaatacca gatcctatta tataatggag
1621 atgtagacat ggcctgcaat ttcatggggg atgagtggtt tgtggattcc ctcaaccaga
1681 agatggaggt gcagcgccgg ccctggttag tgaagtacgg ggacagcggg gagcagattg
1741 ccggcttcgt gaaggagttc tcccacatcg cctttctcac gatcaagggc gccggccaca
1801 tggttcccac cgacaagccc ctcgctgcct tcaccatgtt ctcccgcttc ctgaacaagc
1861 agccatactg atgaccacag caaccagctc cacggcctga tgcagcccct cccagcctct
1921 cccgctagga gagtcctctt ctaagcaaag tgcccctgca ggccgggttc tgccgccagg
1981 actgccccct tcccagagcc ctgtacatcc cagactgggc ccagggtctc ccatagacag
2041 cctgggggca agttagcact ttattcccgc agcagttcct gaatggggtg gcctggcccc
2101 ttctctgctt aaagaatgcc ctttatgatg cactgattcc atcccaggaa cccaacagag
2161 ctcaggacag cccacaggga ggtggtggac ggactgtaat tgatagattg attatggaat
2221 taaattgggt acagcttcaa aaaaaaaaaa aaaa
SEQ ID NO: 2 Human PPCA Polypeptide, variant 1 protein
MTSSPRAPPGEQGRGGAEMIRAAPPPLFLLLLLLLLLVSWASRG
EAAPDQDEIQRLPGLAKQPSFRQYSGYLKGSGSKHLHYWFVESQKDPENSPVVLWLNG
GPGCSSLDGLLTEHGPFLVQPDGVTLEYNPYSWNLIANVLYLESPAGVGFSYSDDKFY
AINDTEVAQSNFEALQDFFRLFPEYKNNKLFLIGESYAGIYIPTLAVLVMQDPSMNLQ
GLAVGNGLSSYEQNDNSLVYFAYYHGLLGNRLWSSLQTHCCSQNKCNFYDNKDLECVT
NLQEVARIVGNSGLNIYNLYAPCAGGVPSHFRYEKDIVVVQDLGNIFIRLPLKRMWHQ
ALLRSGDKVRMDPPCINTTAASTYLNNPYVRKALNIPEQLPQWDMCNFLVNLQYRRLY
RSMNSQYLKLLSSQKYQILLYNGDVDMACNFMGDEWFVDSLNQKMEVQRRPWLVKYGD
SGEQIAGFVKEFSHIAFLTIKGAGHMVPTDKPLAAFTMFSRFLNKQPY
SEQ ID NO: 3 Human NEU1 mRNA
1    gagctacttg aagaccaatt agagtccggg aagcgcggcg gggcctccag accggggcgg
61   gcttaagggt gacatctgcg ctttaaaggg tccgggtcag ctgactcccg actctgtgga
121  gtctagctgc cagggtcgcg gcagctgcgg ggagagatga ctggggagcg acccagcacg
181  gcgctcccgg acagacgctg ggggccgcgg attctgggct tctggggagg ctgtagggtt
241  tgggtgtttg ccgcgatctt cctgctgctg tctctggcag cctcctggtc caaggctgag
301  aacgacttcg gtctggtgca gccgctggtg accatggagc aactgctgtg ggtgagcggg
361  agacagatcg gctcagtgga caccttccgc atcccgctca tcacagccac tccgcggggc
421  actcttctcg cctttgctga ggcgaggaaa atgtcctcat ccgatgaggg ggccaagttc
481  atcgccctgc ggaggtccat ggaccagggc agcacatggt ctcctacagc gttcattgtc
541  aatgatgggg atgtccccga tgggctgaac cttggggcag tagtgagcga tgttgagaca
601  ggagtagtat ttcttttcta ctccctttgt gctcacaagg ccggctgcca ggtggcctct
661  accatgttgg tatggagcaa ggatgatggt gtttcctgga gcacaccccg gaatctctcc
721  ctggatattg gcactgaagt gtttgcccct ggaccgggct ctggtattca gaaacagcgg
781  gagccacgga agggccgcct catcgtgtgt ggccatggga cgctggagcg ggacggagtc
841  ttctgtctcc tcagcgatga tcatggtgcc tcctggcgct acggaagtgg ggtcagcggc
901  atcccctacg gtcagcccaa gcaggaaaat gatttcaatc ctgatgaatg ccagccctat
961  gagctcccag atggctcagt cgtcatcaat gcccgaaacc agaacaacta ccactgccac
1021 tgccgaattg tcctccgcag ctatgatgcc tgtgatacac taaggccccg tgatgtgacc
1081 ttcgaccctg agctcgtgga ccctgtggta gctgcaggag ctgtagtcac cagctccggc
1141 attgtcttct tctccaaccc agcacatcca gagttccgag tgaacctgac cctgcgatgg
1201 agcttcagca atggtacctc atggcggaaa gagacagtcc agctatggcc aggccccagt
1261 ggctattcat ccctggcaac cctggagggc agcatggatg gagaggagca ggccccccag
1321 ctctacgtcc tgtatgagaa aggccggaac cactacacag agagcatctc cgtggccaaa
1381 atcagtgtct atgggacact ctgagctgtg ccactgccac aggggtattc tgccttcagg
1441 actctgcctt caggaacacg ggtctgtaga gggtctgctg gagacgcctg aaagacagtt
1501 ccatcttcct ttagactcca gccttggcaa aatcaccttc cctttaccag ggaaatcact
1561 tcctttagga ctgaaagcta ggcgtcctct cccacaaaaa agtcctgccc tcatctgaga
1621 atactgtctt tccatatggc taagtgtggc cccaccaccc tctctgccct cccgggacat
1681 tgattggtcc tgtcttgggc aggtctagtg agctgtagaa ttgaatcaat gtgaactcag
1741 ggaactgggg aaggctgagc ctcctctttg gtgttgcggt aagataaccg acagggctgg
1801 tgaaagtccc cagatggcag gatatttggt ttcagagtaa ggactaggtg caccaccatg
1861 actgactatc aatcaaaatg tttgtaactt aaaattttta atgaaggata atgaatattt
1921 gtagagtctc tatggttctg tcaatgcaca tcttcgtgtc tgttttcctc atgtatcctt
1981 gtgagcctgg gtgagttctg gggagagacc tgatgtgcgt actgcctgtg aaaatctgac
2041 tttggcaaat caaatcctct tttccttttg aaaaaaaaaa aaaaaaaa
SEQ ID NO: 4 Human NEU1 Polypeptide
10         20         30         40         50
MTGERPSTAL PDRRWGPRIL GFWGGCRVWV FAAIFLLLSL AASWSKAEND
60         70         80         90        100
FGLVQPLVTM EQLLWVSGRQ IGSVDTFRIP LITATPRGTL LAFAEARKMS
110        120        130        140        150
SSDEGAKFIA LRRSMDQGST WSPTAFIVND GDVPDGLNLG AVVSDVETGV
160        170        180        190        200
VFLFYSLCAH KAGCQVASTM LVWSKDDGVS WSTPRNLSLD IGTEVFAPGP
210        220        230        240        250
GSGIQKQREP RKGRLIVCGH GTLERDGVFC LLSDDHGASW RYGSGVSGIP
260        270        280        290        300
YGQPKQENDF NPDECQPYEL PDGSVVINAR NQNNYHCHCR IVLRSYDACD
310        320        330        340        350
TLRPRDVTFD PELVDPVVAA GAVVTSSGIV FFSNPAHPEF RVNLTLRWSF
360        370        380        390        400
SNGTSWRKET VQLWPGPSGY SSLATLEGSM DGEEQAPQLY VLYEKGRNHY
410
TESISVAKIS VYGTL
SEQ ID NO: 5 Human TPP1 mRNA
1    ggtggtggaa tatagagctc atgtgatccg tcacatgaca gcagatccgc ggaagggcag
61   aatgggactc caagcctgcc tcctagggct ctttgccctc atcctctctg gcaaatgcag
121  ttacagcccg gagcccgacc agcggaggac gctgccccca ggctgggtgt ccctgggccg
181  tgcggaccct gaggaagagc tgagtctcac ctttgccctg agacagcaga atgtggaaag
241  actctcggag ctggtgcagg ctgtgtcgga tcccagctct cctcaatacg gaaaatacct
301  gaccctagag aatgtggctg atctggtgag gccatcccca ctgaccctcc acacggtgca
361  aaaatggctc ttggcagccg gagcccagaa gtgccattct gtgatcacac aggactttct
421  gacttgctgg ctgagcatcc gacaagcaga gctgctgctc cctggggctg agtttcatca
481  ctatgtggga ggacctacgg aaacccatgt tgtaaggtcc ccacatccct accagcttcc
541  acaggccttg gccccccatg tggactttgt ggggggactg caccgttttc ccccaacatc
601  atccctgagg caacgtcctg agccgcaggt gacagggact gtaggcctgc atctgggggt
661  aaccccctct gtgatccgta agcgatacaa cttgacctca caagacgtgg gctctggcac
721  cagcaataac agccaagcct gtgcccagtt cctggagcag tatttccatg actcagacct
781  ggctcagttc atgcgcctct tcggtggcaa ctttgcacat caggcatcag tagcccgtgt
841  ggttggacaa cagggccggg gccgggccgg gattgaggcc agtctagatg tgcagtacct
901  gatgagtgct ggtgccaaca tctccacctg ggtctacagt agccctggcc ggcatgaggg
961  acaggagccc ttcctgcagt ggctcatgct gctcagtaat gagtcagccc tgccacatgt
1021 gcatactgtg agctatggag atgatgagga ctccctcagc agcgcctaca tccagcgggt
1081 caacactgag ctcatgaagg ctgccgctcg gggtctcacc ctgctcttcg cctcaggtga
1141 cagtggggcc gggtgttggt ctgtctctgg aagacaccag ttccgcccta ccttccctgc
1201 ctccagcccc tatgtcacca cagtgggagg cacatccttc caggaacctt tcctcatcac
1261 aaatgaaatt gttgactata tcagtggtgg tggcttcagc aatgtgttcc cacggccttc
1321 ataccaggag gaagctgtaa cgaagttcct gagctctagc ccccacctgc caccatccag
1381 ttacttcaat gccagtggcc gtgcctaccc agatgtggct gcactttctg atggctactg
1441 ggtggtcagc aacagagtgc ccattccatg ggtgtccgga acctcggcct ctactccagt
1501 gtttgggggg atcctatcct tgatcaatga gcacaggatc cttagtggcc gcccccctct
1561 tggctttctc aacccaaggc tctaccagca gcatggggca ggactctttg atgtaacccg
1621 tggctgccat gagtcctgtc tggatgaaga ggtagagggc cagggtttct gctctggtcc
1681 tggctgggat cctgtaacag gctggggaac acccaacttc ccagctttgc tgaagactct
1741 actcaacccc tgaccctttc ctatcaggag agatggcttg tcccctgccc tgaagctggc
1801 agttcagtcc cttattctgc cctgttggaa gccctgctga accctcaact attgactgct
1861 gcagacagct tatctcccta accctgaaat gctgtgagct tgacttgact cccaacccta
1921 ccatgctcca tcatactcag gtctccctac tcctgcctta gattcctcaa taagatgctg
1981 taactagcat tttttgaatg cctctccctc cgcatctcat ctttctcttt tcaatcaggc
2041 ttttccaaag ggttgtatac agactctgtg cactatttca cttgatattc attccccaat
2101 tcactgcaag gagacctcta ctgtcaccgt ttactctttc ctaccctgac atccagaaac
2161 aatggcctcc agtgcatact tctcaatctt tgctttatgg cctttccatc atagttgccc
2221 actccctctc cttacttagc ttccaggtct taacttctct gactactctt gtcttcctct
2281 ctcatcaatt tctgcttctt catggaatgc tgaccttcat tgctccattt gtagattttt
2341 gctcttctca gtttactcat tgtcccctgg aacaaatcac tgacatctac aaccattacc
2401 atctcactaa ataagacttt ctatccaata atgattgata cctcaaatgt aagatgcgtg
2461 atactcaaca tttcatcgtc caccttccca accccaaaca attccatctc gtttcttctt
2521 ggtaaatgat gctatgcttt ttccaaccaa gccagaaacc tgtgtcatct tttcacccca
2581 ccttcaatca acaagtcctc aatcaacaag tcctactgac tgcacatctt aaatatatct
2641 ttatcagtcc acaagtcctt ccaattatat ttcccaagta tatctagaac ttatccactt
2701 atatccccac tgctactacc ttagtttagg gctatattct cttgaaaaaa agtgtcctta
2761 cttcctgcca atccccaagt catcttccag agtaaaatgc aaatcccatc aggccacttg
2821 gatgaaaacc cttcaaggat tactggatag aattcaggct ttcccctcca gcccccaatc
2881 atagctcaca aaccttcctt gctatttgtt cttaagtaaa aaatcatttt tcctcctccc
2941 tccccaaacc ccaaggaact ctcactcttg ctcaagctgt tccgtcccct taccacccct
3001 gatacaactg ccaggttaat ttccagaatt cttgcaagac tcagttcaga agtcaccttc
3061 tttcgtgaat gttttgattc cctgaggcta ctttattttg gtatggctga aaaatcctag
3121 attttctaaa caaaacctgt ttgaatcttg gttctgatat ggactaggag agagactggg
3181 tcaagtaagc ttatctccct gaggctgttt cctcgtctgt taagtgtgaa tatcaatacc
3241 tgcctttcat aatcaccagg gaataaagtg gaataatgtt gataacagtg cttggcacct
3301 ggaagtaggt ggcagatgtt aacgcccttc ctcccttgca ctgcgccccc tgtgcctacc
3361 tctagcattg taacgaccac gtagtattga aatggccagt ttacttgtct gccttccttt
3421 ccaagaccgt tggtgcctag aggactagaa tcgtgtccta tttaactttg tgttcccagg
3481 tcctagctca ggagttggca aataagaatt aaatgtctgc tacaccgaaa accaaaaaaa
SEQ ID NO: 6 Human TPP1 Polypeptide
10         20         30         40         50
MGLQACLLGL FALILSGKCS YSPEPDQRRT LPPGWVSLGR ADPEEELSLT
60         70         80         90        100
FALRQQNVER LSELVQAVSD PSSPQYGKYL TLENVADLVR PSPLTLHTVQ
110        120        130        140        150
KWLLAAGAQK CHSVITQDFL TCWLSIRQAE LLLPGAEFHH YVGGPTETHV
160        170        180        190        200
VRSPHPYQLP QALAPHVDFV GGLHRFPPTS SLRQRPEPQV TGTVGLHLGV
210        220        230        240        250
TPSVIRKRYN LTSQDVGSGT SNNSQACAQF LEQYFHDSDL AQFMRLFGGN
260        270        280        290        300
FAHQASVARV VGQQGRGRAG IEASLDVQYL MSAGANISTW VYSSPGRHEG
310        320        330        340        350
QEPFLQWLML LSNESALPHV HTVSYGDDED SLSSAYIQRV NTELMKAAAR
360        370        380        390        400
GLTLLFASGD SGAGCWSVSG RHQFRPTFPA SSPYVTTVGG TSFQEPFLIT
410        420        430        440        450
NEIVDYISGG GFSNVFPRPS YQEEAVTKFL SSSPHLPPSS YFNASGRAYP
460        470        480        490        500
DVAALSDGYW VVSNRVPIPW VSGTSASTPV FGGILSLINE HRILSGRPPL
510        520        530        540        550
GFLNPRLYQQ HGAGLFDVTR GCHESCLDEE VEGQGFCSGP GWDPVTGWGT
560
PNFPALLKTL LNP
SEQ ID NO: 7 Human Cathepsin B mRNA, variant 1
1    ggggcggggc cgggagggta cttagggccg gggctggccc aggctacggc ggctgcaggg
61   ctccggcaac cgctccggca acgccaaccg ctccgctgcg cgcaggctgg gctgcaggct
121  ctcggctgca gcgctgggtg gatctaggat ccggcttcca acatgtggca gctctgggcc
181  tccctctgct gcctgctggt gttggccaat gcccggagca ggccctcttt ccatcccctg
241  tcggatgagc tggtcaacta tgtcaacaaa cggaatacca cgtggcaggc cgggcacaac
301  ttctacaacg tggacatgag ctacttgaag aggctatgtg gtaccttcct gggtgggccc
361  aagccacccc agagagttat gtttaccgag gacctgaagc tgcctgcaag cttcgatgca
421  cgggaacaat ggccacagtg tcccaccatc aaagagatca gagaccaggg ctcctgtggc
481  tcctgctggg ccttcggggc tgtggaagcc atctctgacc ggatctgcat ccacaccaat
541  gcgcacgtca gcgtggaggt gtcggcggag gacctgctca catgctgtgg cagcatgtgt
601  ggggacggct gtaatggtgg ctatcctgct gaagcttgga acttctggac aagaaaaggc
661  ctggtttctg gtggcctcta tgaatcccat gtagggtgca gaccgtactc catccctccc
721  tgtgagcacc acgtcaacgg ctcccggccc ccatgcacgg gggagggaga tacccccaag
781  tgtagcaaga tctgtgagcc tggctacagc ccgacctaca aacaggacaa gcactacgga
841  tacaattcct acagcgtctc caatagcgag aaggacatca tggccgagat ctacaaaaac
901  ggccccgtgg agggagcttt ctctgtgtat tcggacttcc tgctctacaa gtcaggagtg
961  taccaacacg tcaccggaga gatgatgggt ggccatgcca tccgcatcct gggctgggga
1021 gtggagaatg gcacacccta ctggctggtt gccaactcct ggaacactga ctggggtgac
1081 aatggcttct ttaaaatact cagaggacag gatcactgtg gaatcgaatc agaagtggtg
1141 gctggaattc cacgcaccga tcagtactgg gaaaagatct aatctgccgt gggcctgtcg
1201 tgccagtcct gggggcgaga tcggggtaga aatgcatttt attctttaag ttcacgtaag
1261 atacaagttt cagacagggt ctgaaggact ggattggcca aacatcagac ctgtcttcca
1321 aggagaccaa gtcctggcta catcccagcc tgtggttaca gtgcagacag gccatgtgag
1381 ccaccgctgc cagcacagag cgtccttccc cctgtagact agtgccgtag ggagtacctg
1441 ctgccccagc tgactgtggc cccctccgtg atccatccat ctccagggag caagacagag
1501 acgcaggaat ggaaagcgga gttcctaaca ggatgaaagt tcccccatca gttcccccag
1561 tacctccaag caagtagctt tccacatttg tcacagaaat cagaggagag acggtgttgg
1621 gagccctttg gagaacgcca gtctcccagg ccccctgcat ctatcgagtt tgcaatgtca
1681 caacctctct gatcttgtgc tcagcatgat tctttaatag aagttttatt ttttcgtgca
1741 ctctgctaat catgtgggtg agccagtgga acagcgggag acctgtgcta gttttacaga
1801 ttgcctcctt atgacgcggc tcaaaaggaa accaagtggt caggagttgt ttctgaccca
1861 ctgatctcta ctaccacaag gaaaatagtt taggagaaac cagcttttac tgtttttgaa
1921 aaattacagc ttcaccctgt caagttaaca aggaatgcct gtgccaataa aagttttctc
1981 caacttgaag tctactctga tgggatctca gatcctttgt cactgcctat agacttgtag
2041 ctgctgtctc tctttgtccc tgcagagaat cacgtcctgg aactgcatgt tcttgcgact
2101 cttgggactt catcttaact tctcgctgcc ccagccatgt tttcaaccat ggcatccctc
2161 ccccaattag ttccctgtca tcctcgtcaa ccttctctgt aagtgcctgg taagcttgcc
2221 cttgcttaag aactcaaaac atagctgtgc tctatttttt tgttgttgtt gtgactgaca
2281 gagtgagatt ccgtctccca ggctggagtg cagtggcgcc ttctcagctc actgcaacct
2341 gcagcctcct agattcaagc gattctcctg cttcagcctt ccgagtagct gggatgacag
2401 gcactcacca atatgcctgg gtaatttttg tatttttaag tacatacagg atttcaccat
2461 gttggccagg ctagtttcaa actcccggcc tcaggtggtc tgcctgcctc agcctcccaa
2521 agtgttggga ttacaggcgt gagccactgg gccctgcctg tattttttat cagccacaaa
2581 tccagcaaca agctgaggat tcagctcata aaacaggctt ggtgtcttgg tgatctcaca
2641 taaccaagat gctaccccgt ggggaaccac atccccctgg atgccctcca gccttggttt
2701 gggctggagt cagggcctgt atacagtatt ttgaatttgt atgccactgg tttgcattgc
2761 tggtcaggaa ctctagtgct ttgcatagcc ctggtttaga aacatgttat agcagttctt
2821 ggtatagagc aaactagaag aaccagcaat cattccactg tcctgccaag gtacacctca
2881 gtactcccct tcccaactga agtggtatga ggctagctct ttccaaaagc attcaagttt
2941 ggcttctgat gtgactcaga atttaggaac cagatgctag atcaaataag ctctgaaaat
3001 ctgaggaaca ttgtaggaaa ggtttgttaa gcatctctta agtgccatga tgagcataac
3061 agccggccgt cgtggctcac gcctgtaatc ccagcacttt gggaggccaa ggtgggagga
3121 tgacaaggtc aggagttcaa gaccagcctg gccaacatgc tgaaacctca cctctactaa
3181 aaatacaaaa attagctggg catggtggca catgcctgta atcccagcta cttgggaggc
3241 tgaggcagga gaatcgcttg aacccgggag gcggaggttg cagtgagcca agacagtgcc
3301 agtgcactcc agcctcggtg acagcgcaag gctccgtctc aataattaaa aaaaaaaaaa
3361 aaaaaaaaaa ggccgggcgc agtggctcaa gcctgtaatc ccagcacttt gggaggctga
3421 ggcgggcaga tcacctgagg tcaggagttt tgagatcagc cttggcaaca cggtgaaacc
3481 ccatctctac taaaaataca aaattagcca agcatgctgg cacatgcctg taatcccagc
3541 tactcgggag gctgaggtac gagaatcgct tgaacctggg aggcagagga tgcagtgagc
3601 cgagatcacg ccattgcact ccagcctggg ggacaagagt gaatctgtgt ctcaccaaaa
3661 aaaaaaagaa aaagaaagat gcttaacaaa ggttaccata agccacaaat tcataaccac
3721 ttatccttcc agtttcaagt agaatatatt cataacctca ataaagttct ccctgctccc
3781 aaa
SEQ ID NO: 8 Human Cathepsin B Polypeptide, variant 1
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 9 Human Cathepsin K mRNA
1    acacatgctg catacacaca gaaacactgc aaatccactg cctccttccc tcctccctac
61   ccttccttct ctcagcattt ctatccccgc ctcctcctct tacccaaatt ttccagccga
121  tcactggagc tgacttccgc aatcccgatg gaataaatct agcacccctg atggtgtgcc
181  cacactttgc tgccgaaacg aagccagaca acagatttcc atcagcagga tgtgggggct
241  caaggttctg ctgctacctg tggtgagctt tgctctgtac cctgaggaga tactggacac
301  ccactgggag ctatggaaga agacccacag gaagcaatat aacaacaagg tggatgaaat
361  ctctcggcgt ttaatttggg aaaaaaacct gaagtatatt tccatccata accttgaggc
421  ttctcttggt gtccatacat atgaactggc tatgaaccac ctgggggaca tgaccagtga
481  agaggtggtt cagaagatga ctggactcaa agtacccctg tctcattccc gcagtaatga
541  caccctttat atcccagaat gggaaggtag agccccagac tctgtcgact atcgaaagaa
601  aggatatgtt actcctgtca aaaatcaggg tcagtgtggt tcctgttggg cttttagctc
661  tgtgggtgcc ctggagggcc aactcaagaa gaaaactggc aaactcttaa atctgagtcc
721  ccagaaccta gtggattgtg tgtctgagaa tgatggctgt ggagggggct acatgaccaa
781  tgccttccaa tatgtgcaga agaaccgggg tattgactct gaagatgcct acccatatgt
841  gggacaggaa gagagttgta tgtacaaccc aacaggcaag gcagctaaat gcagagggta
901  cagagagatc cccgagggga atgagaaagc cctgaagagg gcagtggccc gagtgggacc
961  tgtctctgtg gccattgatg caagcctgac ctccttccag ttttacagca aaggtgtgta
1021 ttatgatgaa agctgcaata gcgataatct gaaccatgcg gttttggcag tgggatatgg
1081 aatccagaag ggaaacaagc actggataat taaaaacagc tggggagaaa actggggaaa
1141 caaaggatat atcctcatgg ctcgaaataa gaacaacgcc tgtggcattg ccaacctggc
1201 cagcttcccc aagatgtgac tccagccagc caaatccatc ctgctcttcc atttcttcca
1261 cgatggtgca gtgtaacgat gcactttgga agggagttgg tgtgctattt ttgaagcaga
1321 tgtggtgata ctgagattgt ctgttcagtt tccccatttg tttgtgcttc aaatgatcct
1381 tcctactttg cttctctcca cccatgacct ttttcactgt ggccatcagg actttccctg
1441 acagctgtgt actcttaggc taagagatgt gactacagcc tgcccctgac tgtgttgtcc
1501 cagggctgat gctgtacagg tacaggctgg agattttcac ataggttaga ttctcattca
1561 cgggactagt tagctttaag caccctagag gactagggta atctgacttc tcacttccta
1621 agttcccttc tatatcctca aggtagaaat gtctatgttt tctactccaa ttcataaatc
1681 tattcataag tctttggtac aagtttacat gataaaaaga aatgtgattt gtcttccctt
1741 ctttgcactt ttgaaataaa gtatttatct cctgtctaca gtttaataaa tagcatctag
1801 tacacattca aaaaaaaaaa aaaaa
SEQ ID NO: 10 Human Cathepsin K Polypeptide
10         20         30         40         50
MWGLKVLLLP VVSFALYPEE ILDTHWELWK KTHRKQYNNK VDEISRRLIW
60         70         80         90        100
EKNLKYISIH NLEASLGVHT YELAMNHLGD MTSEEVVQKM TGLKVPLSHS
110        120        130        140        150
RSNDTLYIPE WEGRAPDSVD YRKKGYVTPV KNQGQCGSCW AFSSVGALEG
160        170        180        190        200
QLKKKTGKLL NLSPQNLVDC VSENDGCGGG YMTNAFQYVQ KNRGIDSEDA
210        220        230        240        250
YPYVGQEESC MYNPTGKAAK CRGYREIPEG NEKALKRAVA RVGPVSVAID
260        270        280        290        300
ASLTSFQFYS KGVYYDESCN SDNLNHAVLA VGYGIQKGNK HWIIKNSWGE
310        320
NWGNKGYILM ARNKNNACGI ANLASFPKM
SEQ ID NO: 11 Human Cathepsin L mRNA, variant 1
1    ggcggtgccg gccgaaccca gacccgaggt tttagaagca gagtcaggcg aagctgggcc
61   agaaccgcga cctccgcaac cttgagcggc atccgtggag tgcgcctgcg cagctacgac
121  cgcagcagga aagcgccgcc ggccaggccc agctgtggcc ggacagggac tggaagagag
181  gacgcggtcg agtaggtgtg caccagccct ggcaacgaga gcgtctaccc cgaactctgc
241  tggccttgag gtggggaagc cggggagggc agttgaggac cccgcggagg cgcgtgactg
301  gttgagcggg caggccagcc tccgagccgg gtggacacag gttttaaaac atgaatccta
361  cactcatcct tgctgccttt tgcctgggaa ttgcctcagc tactctaaca tttgatcaca
421  gtttagaggc acagtggacc aagtggaagg cgatgcacaa cagattatac ggcatgaatg
481  aagaaggatg gaggagagca gtgtgggaga agaacatgaa gatgattgaa ctgcacaatc
541  aggaatacag ggaagggaaa cacagcttca caatggccat gaacgccttt ggagacatga
601  ccagtgaaga attcaggcag gtgatgaatg gctttcaaaa ccgtaagccc aggaagggga
661  aagtgttcca ggaacctctg ttttatgagg cccccagatc tgtggattgg agagagaaag
721  gctacgtgac tcctgtgaag aatcagggtc agtgtggttc ttgttgggct tttagtgcta
781  ctggtgctct tgaaggacag atgttccgga aaactgggag gcttatctca ctgagtgagc
841  agaatctggt agactgctct gggcctcaag gcaatgaagg ctgcaatggt ggcctaatgg
901  attatgcttt ccagtatgtt caggataatg gaggcctgga ctctgaggaa tcctatccat
961  atgaggcaac agaagaatcc tgtaagtaca atcccaagta ttctgttgct aatgacaccg
1021 gctttgtgga catccctaag caggagaagg ccctgatgaa ggcagttgca actgtggggc
1081 ccatttctgt tgctattgat gcaggtcatg agtccttcct gttctataaa gaaggcattt
1141 attttgagcc agactgtagc agtgaagaca tggatcatgg tgtgctggtg gttggctacg
1201 gatttgaaag cacagaatca gataacaata aatattggct ggtgaagaac agctggggtg
1261 aagaatgggg catgggtggc tacgtaaaga tggccaaaga ccggagaaac cattgtggaa
1321 ttgcctcagc agccagctac cccactgtgt gagctggtgg acggtgatga ggaaggactt
1381 gactggggat ggcgcatgca tgggaggaat tcatcttcag tctaccagcc cccgctgtgt
1441 cggatacaca ctcgaatcat tgaagatccg agtgtgattt gaattctgtg atattttcac
1501 actggtaaat gttacctcta ttttaattac tgctataaat aggtttatat tattgattca
1561 cttactgact ttgcattttc gtttttaaaa ggatgtataa atttttacct gtttaaataa
1621 aatttaattt caaatgtagt ggtggggctt ctttctattt ttgatgcact gaatttttgt
1681 gtaataaaga acataattgg gctctaagcc ataaaaaaaa aaaaaaaaaa
SEQ ID NO: 12 Human Cathepsin L Polypeptide, variant 1
MNPTLILAAFCLGIASATLIFDHSLEAQWTKWKAMHNRLYGMNE
EGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMISEEFRQVMNGFQNRKPRK
GKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLIS
LSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYS
VANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDH
GVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
SEQ ID NO: 13
DXXLL
SEQ ID NO: 14
[DE]XXXL[LI]
SEQ ID NO: 15
YXX
SEQ ID NO: 16, MPR300/CI-MPR
SFHDDSDEDLL
SEQ ID NO: 17, MPR46/CD-MPR
EESEERDDHLL
SEQ ID NO: 18 Sortilin
GYHDDSDEDLL
SEQ ID NO: 19 SorLA/SORL1
ITGFSDDVPMV
SEQ ID NO: 20 GGA1 (1)
ASVSLLDDELM
SEQ ID NO: 21 GGA1 (2)
ASSGLDDLDLL
SEQ ID NO: 22, GGA2
VQNPSADRNLL
SEQ ID NO: 23, GGA3
NALSWLDEELL
SEQ ID NO: 24, LIMP-II
DERAPLI
SEQ ID NO: 25, NPC1
TERERLL
SEQ ID NO: 26, Mucolipin-1
SETERLL
SEQ ID NO: 27, Sialin
TDRTPLL
SEQ ID NO: 28, GLUT8
EETQPLL
SEQ ID NO: 29, Invariant chain (Ii) (1)
DDQRDLI
SEQ ID NO: 30, Invariant chain (Ii) (2)
NEQLPML
SEQ ID NO: 31, LAMP-1
GYQTI
SEQ ID NO: 32, LAMP-2A
GYEQF
SEQ ID NO: 33, LAMP-2B
GYQTL
SEQ ID NO: 34, LAMP-2C
GYQSV
SEQ ID NO: 35, CD63
GYEVM
SEQ ID NO: 36, CD68
AYQAL
SEQ ID NO: 37, Endolyn
NYHTL
SEQ ID NO: 38, DC-LAMP
GYQRI
SEQ ID NO: 39, Cystinosin
GYDQL
SEQ ID NO: 40, Sugar phosphate exchanger 2
GYKEI
SEQ ID NO: 41, acid phosphatase
GYRHV
SEQ ID NO: 42, Human PPCA, variant 2 mRNA
1    agagtgcacc cgaatccacg ggctcggagg cagcagccat ctctcggcca tagggcaggc
61   cagctggcgc cgggggctat tttgggcggc gggcaatgat ggtgaccgca aggcgacctt
121  gtaaggcatt tcccccctga ctcccttccc cgagcctctg cccgggggtc ctagcgccgc
181  tttctcagcc atcccgccta caacttagcc gtccacaaca ggatcatctg atcgcgtgcg
241  cccgggctac gatctgcgag gcccgcggac cttgacccgg cattgaccgc caccgccccc
301  caggtccgta gggaccaaag aaggggcggg aggaagactg tcacgtggcg ccggagttca
361  cgtgactcgt acacatgact tccagtcccc gggcgcctcc tggagagcaa ggacgcgggg
421  gagcagaggt gagctggcac cggaggctgg aggggatccc cgagcccggg atcgatgatc
481  cgagccgcgc cgccgccgct gttcctgctg ctgctgctgc tgctgctgct agtgtcctgg
541  gcgtcccgag gcgaggcagc ccccgaccag gacgagatcc agcgcctccc cgggctggcc
601  aagcagccgt ctttccgcca gtactccggc tacctcaaag gctccggctc caagcacctc
661  cactactggt ttgtggagtc ccagaaggat cccgagaaca gccctgtggt gctttggctc
721  aatgggggtc ccggctgcag ctcactagat gggctcctca cagagcatgg ccccttcctg
781  gtccagccag atggtgtcac cctggagtac aacccctatt cttggaatct gattgccaat
841  gtgttatacc tggagtcccc agctggggtg ggcttctcct actccgatga caagttttat
901  gcaactaatg acactgaggt cgcccagagc aattttgagg cccttcaaga tttcttccgc
961  ctctttccgg agtacaagaa caacaaactt ttcctgaccg gggagagcta tgctggcatc
1021 tacatcccca ccctggccgt gctggtcatg caggatccca gcatgaacct tcaggggctg
1081 gctgtgggca atggactctc ctcctatgag cagaatgaca actccctggt ctactttgcc
1141 tactaccatg gccttctggg gaacaggctt tggtcttctc tccagaccca ctgctgctct
1201 caaaacaagt gtaacttcta tgacaacaaa gacctggaat gcgtgaccaa tcttcaggaa
1261 gtggcccgca tcgtgggcaa ctctggcctc aacatctaca atctctatgc cccgtgtgct
1321 ggaggggtgc ccagccattt taggtatgag aaggacactg ttgtggtcca ggatttgggc
1381 aacatcttca ctcgcctgcc actcaagcgg atgtggcatc aggcactgct gcgctcaggg
1441 gataaagtgc gcatggaccc cccctgcacc aacacaacag ctgcttccac ctacctcaac
1501 aacccgtacg tgcggaaggc cctcaacatc ccggagcagc tgccacaatg ggacatgtgc
1561 aactttctgg taaacttaca gtaccgccgt ctctaccgaa gcatgaactc ccagtatctg
1621 aagctgctta gctcacagaa ataccagatc ctattatata atggagatgt agacatggcc
1681 tgcaatttca tgggggatga gtggtttgtg gattccctca accagaagat ggaggtgcag
1741 cgccggccct ggttagtgaa gtacggggac agcggggagc agattgccgg cttcgtgaag
1801 gagttctccc acatcgcctt tctcacgatc aagggcgccg gccacatggt tcccaccgac
1861 aagcccctcg ctgccttcac catgttctcc cgcttcctga acaagcagcc atactgatga
1921 ccacagcaac cagctccacg gcctgatgca gcccctccca gcctctcccg ctaggagagt
1981 cctcttctaa gcaaagtgcc cctgcaggcc gggttctgcc gccaggactg cccccttccc
2041 agagccctgt acatcccaga ctgggcccag ggtctcccat agacagcctg ggggcaagtt
2101 agcactttat tcccgcagca gttcctgaat ggggtggcct ggccccttct ctgcttaaag
2161 aatgcccttt atgatgcact gattccatcc caggaaccca acagagctca ggacagccca
2221 cagggaggtg gtggacggac tgtaattgat agattgatta tggaattaaa ttgggtacag
2281 cttcaaaaaa aaaaaaaaaa
SEQ ID NO: 43, Human PPCA, variant 2 protein
10         20         30         40         50
MIRAAPPPLF LLLLLLLLLV SWASRGEAAP DQDEIQRLPG LAKQPSFRQY
60         70         80         90        100
SGYLKGSGSK HLHYWFVESQ KDPENSPVVL WLNGGPGCSS LDGLLTEHGP
110        120        130        140        150
FLVQPDGVTL EYNPYSWNLI ANVLYLESPA GVGFSYSDDK FYATNDTEVA
160        170        180        190        200
QSNFEALQDF FRLFPEYKNN KLFLTGESYA GIYIPTLAVL VMQDPSMNLQ
210        220        230        240        250
GLAVGNGLSS YEQNDNSLVY FAYYHGLLGN RLWSSLQTHC CSQNKCNFYD
260        270        280        290        300
NKDLECVTNL QEVARIVGNS GLNIYNLYAP CAGGVPSHFR YEKDTVVVQD
310        320        330        340        350
LGNIFTRLPL KRMWHQALLR SGDKVRMDPP CTNTTAASTY LNNPYVRKAL
360        370        380        390        400
NIPEQLPQWD MCNFLVNLQY RRLYRSMNSQ YLKLLSSQKY QILLYNGDVD
410        420     430 440 450
MACNFMGDEW FVDSLNQKME VQRRPWLVKY GDSGEQIAGF VKEFSHIAFL
460        470 480
TIKGAGHMVP TDKPLAAFTM FSRFLNKQPY
SEQ ID NO: 44, Human PPCA, variant 3 mRNA
1    agagtgcacc cgaatccacg ggctcggagg cagcagccat ctctcggcca tagggcaggc
61   cagctggcgc cgggggctat tttgggcggc gggcaatgat ggtgaccgca aggcgacctt
121  gtaaggcatt tcccccctga ctcccttccc cgagcctctg cccgggggtc ctagcgccgc
181  tttctcagcc atcccgccta caacttagcc gtccacaaca ggatcatctg atcgcgtgcg
241  cccgggctac gatctgcgag gcccgcggac cttgacccgg cattgaccgc caccgccccc
301  caggtccgta gggaccaaag aaggggcggg aggaagactg tcacgtggcg ccggagttca
361  cgtgactcgt acacatgact tccagtcccc gggcgcctcc tggagagcaa ggacgcgggg
421  gagcagagat gatccgagcc gcgccgccgc cgctgttcct gctgctgctg ctgctgctgc
481  tgctagtgtc ctgggcgtcc cgaggcgagg cagcccccga ccaggacgag atccagcgcc
541  tccccgggct ggccaagcag ccgtctttcc gccagtactc cggctacctc aaaggctccg
601  gctccaagca cctccactac tggtttgtgg agtcccagaa ggatcccgag aacagccctg
661  tggtgctttg gctcaatggg ggtcccggct gcagctcact agatgggctc ctcacagagc
721  atggcccctt cctgattgcc aatgtgttat acctggagtc cccagctggg gtgggcttct
781  cctactccga tgacaagttt tatgcaacta atgacactga ggtcgcccag agcaattttg
841  aggcccttca agatttcttc cgcctctttc cggagtacaa gaacaacaaa cttttcctga
901  ccggggagag ctatgctggc atctacatcc ccaccctggc cgtgctggtc atgcaggatc
961  ccagcatgaa ccttcagggg ctggctgtgg gcaatggact ctcctcctat gagcagaatg
1021 acaactccct ggtctacttt gcctactacc atggccttct ggggaacagg ctttggtctt
1081 ctctccagac ccactgctgc tctcaaaaca agtgtaactt ctatgacaac aaagacctgg
1141 aatgcgtgac caatcttcag gaagtggccc gcatcgtggg caactctggc ctcaacatct
1201 acaatctcta tgccccgtgt gctggagggg tgcccagcca ttttaggtat gagaaggaca
1261 ctgttgtggt ccaggatttg ggcaacatct tcactcgcct gccactcaag cggatgtggc
1321 atcaggcact gctgcgctca ggggataaag tgcgcatgga ccccccctgc accaacacaa
1381 cagctgcttc cacctacctc aacaacccgt acgtgcggaa ggccctcaac atcccggagc
1441 agctgccaca atgggacatg tgcaactttc tggtaaactt acagtaccgc cgtctctacc
1501 gaagcatgaa ctcccagtat ctgaagctgc ttagctcaca gaaataccag atcctattat
1561 ataatggaga tgtagacatg gcctgcaatt tcatggggga tgagtggttt gtggattccc
1621 tcaaccagaa gatggaggtg cagcgccggc cctggttagt gaagtacggg gacagcgggg
1681 agcagattgc cggcttcgtg aaggagttct cccacatcgc ctttctcacg atcaagggcg
1741 ccggccacat ggttcccacc gacaagcccc tcgctgcctt caccatgttc tcccgcttcc
1801 tgaacaagca gccatactga tgaccacagc aaccagctcc acggcctgat gcagcccctc
1861 ccagcctctc ccgctaggag agtcctcttc taagcaaagt gcccctgcag gccgggttct
1921 gccgccagga ctgccccctt cccagagccc tgtacatccc agactgggcc cagggtctcc
1981 catagacagc ctgggggcaa gttagcactt tattcccgca gcagttcctg aatggggtgg
2041 cctggcccct tctctgctta aagaatgccc tttatgatgc actgattcca tcccaggaac
2101 ccaacagagc tcaggacagc ccacagggag gtggtggacg gactgtaatt gatagattga
2161 ttatggaatt aaattgggta cagcttcaaa aaaaaaaaaa aaaaaaaa
SEQ ID NO: 45, Human PPCA, variant 3 protein
MTSSPRAPPGEQGRGGAEMIRAAPPPLFLLLLLLLLLVSWASRG
EAAPDQDEIQRLPGLAKQPSFRQYSGYLKGSGSKHLHYWFVESQKDPENSPVVLWLNG
GPGCSSLDGLLTEHGPFLIANVLYLESPAGVGFSYSDDKFYATNDTEVAQSNFEALQD
FFRLFPEYKNNKLFLTGESYAGIYIPTLAVLVMQDPSMNLQGLAVGNGLSSYEQNDNS
LVYFAYYHGLLGNRLWSSLQTHCCSQNKCNFYDNKDLECVTNLQEVARIVGNSGLNIY
NLYAPCAGGVPSHFRYEKDTVVVQDLGNIFTRLPLKRMWHQALLRSGDKVRMDPPCTN
TTAASTYLNNPYVRKALNIPEQLPQWDMCNFLVNLQYRRLYRSMNSQYLKLLSSQKYQ
ILLYNGDVDMACNFMGDEWFVDSLNQKMEVQRRPWLVKYGDSGEQIAGFVKEFSHIAF
LTIKGAGHMVPTDKPLAAFTMFSRFLNKQPY
SEQ ID NO: 46 Human Cathepsin B mRNA, variant 2
1    ggggcggggc cgggagggta cttagggccg gggctggccc aggctacggc ggctgcaggg
61   ctccggcaac cgctccggca acgccaaccg ctccgctgcg cgcaggctgg gctgcaggct
121  ctcggctgca gcgctgggct ggtgtgcagt ggtgcgacca cggctcacgg cagcctcagc
181  cacccagatg taagcgatct ggttcccacc tcagcctccc gagtagtgtc ttcaggccta
241  tggagagcag cttgcgtggg ctgggcctgc agtacctggt ttgcatagat gattggcagg
301  tggatctagg atccggcttc caacatgtgg cagctctggg cctccctctg ctgcctgctg
361  gtgttggcca atgcccggag caggccctct ttccatcccc tgtcggatga gctggtcaac
421  tatgtcaaca aacggaatac cacgtggcag gccgggcaca acttctacaa cgtggacatg
481  agctacttga agaggctatg tggtaccttc ctgggtgggc ccaagccacc ccagagagtt
541  atgtttaccg aggacctgaa gctgcctgca agcttcgatg cacgggaaca atggccacag
601  tgtcccacca tcaaagagat cagagaccag ggctcctgtg gctcctgctg ggccttcggg
661  gctgtggaag ccatctctga ccggatctgc atccacacca atgcgcacgt cagcgtggag
721  gtgtcggcgg aggacctgct cacatgctgt ggcagcatgt gtggggacgg ctgtaatggt
781  ggctatcctg ctgaagcttg gaacttctgg acaagaaaag gcctggtttc tggtggcctc
841  tatgaatccc atgtagggtg cagaccgtac tccatccctc cctgtgagca ccacgtcaac
901  ggctcccggc ccccatgcac gggggaggga gataccccca agtgtagcaa gatctgtgag
961  cctggctaca gcccgaccta caaacaggac aagcactacg gatacaattc ctacagcgtc
1021 tccaatagcg agaaggacat catggccgag atctacaaaa acggccccgt ggagggagct
1081 ttctctgtgt attcggactt cctgctctac aagtcaggag tgtaccaaca cgtcaccgga
1141 gagatgatgg gtggccatgc catccgcatc ctgggctggg gagtggagaa tggcacaccc
1201 tactggctgg ttgccaactc ctggaacact gactggggtg acaatggctt ctttaaaata
1261 ctcagaggac aggatcactg tggaatcgaa tcagaagtgg tggctggaat tccacgcacc
1321 gatcagtact gggaaaagat ctaatctgcc gtgggcctgt cgtgccagtc ctgggggcga
1381 gatcggggta gaaatgcatt ttattcttta agttcacgta agatacaagt ttcagacagg
1441 gtctgaagga ctggattggc caaacatcag acctgtcttc caaggagacc aagtcctggc
1501 tacatcccag cctgtggtta cagtgcagac aggccatgtg agccaccgct gccagcacag
1561 agcgtccttc cccctgtaga ctagtgccgt agggagtacc tgctgcccca gctgactgtg
1621 gccccctccg tgatccatcc atctccaggg agcaagacag agacgcagga atggaaagcg
1681 gagttcctaa caggatgaaa gttcccccat cagttccccc agtacctcca agcaagtagc
1741 tttccacatt tgtcacagaa atcagaggag agacggtgtt gggagccctt tggagaacgc
1801 cagtctccca ggccccctgc atctatcgag tttgcaatgt cacaacctct ctgatcttgt
1861 gctcagcatg attctttaat agaagtttta ttttttcgtg cactctgcta atcatgtggg
1921 tgagccagtg gaacagcggg agacctgtgc tagttttaca gattgcctcc ttatgacgcg
1981 gctcaaaagg aaaccaagtg gtcaggagtt gtttctgacc cactgatctc tactaccaca
2041 aggaaaatag tttaggagaa accagctttt actgtttttg aaaaattaca gcttcaccct
2101 gtcaagttaa caaggaatgc ctgtgccaat aaaagttttc tccaacttga agtctactct
2161 gatgggatct cagatccttt gtcactgcct atagacttgt agctgctgtc tctctttgtc
2221 cctgcagaga atcacgtcct ggaactgcat gttcttgcga ctcttgggac ttcatcttaa
2281 cttctcgctg ccccagccat gttttcaacc atggcatccc tcccccaatt agttccctgt
2341 catcctcgtc aaccttctct gtaagtgcct ggtaagcttg cccttgctta agaactcaaa
2401 acatagctgt gctctatttt tttgttgttg ttgtgactga cagagtgaga ttccgtctcc
2461 caggctggag tgcagtggcg ccttctcagc tcactgcaac ctgcagcctc ctagattcaa
2521 gcgattctcc tgcttcagcc ttccgagtag ctgggatgac aggcactcac caatatgcct
2581 gggtaatttt tgtattttta agtacataca ggatttcacc atgttggcca ggctagtttc
2641 aaactcccgg cctcaggtgg tctgcctgcc tcagcctccc aaagtgttgg gattacaggc
2701 gtgagccact gggccctgcc tgtatttttt atcagccaca aatccagcaa caagctgagg
2761 attcagctca taaaacaggc ttggtgtctt ggtgatctca cataaccaag atgctacccc
2821 gtggggaacc acatccccct ggatgccctc cagccttggt ttgggctgga gtcagggcct
2881 gtatacagta ttttgaattt gtatgccact ggtttgcatt gctggtcagg aactctagtg
2941 ctttgcatag ccctggttta gaaacatgtt atagcagttc ttggtataga gcaaactaga
3001 agaaccagca atcattccac tgtcctgcca aggtacacct cagtactccc cttcccaact
3061 gaagtggtat gaggctagct ctttccaaaa gcattcaagt ttggcttctg atgtgactca
3121 gaatttagga accagatgct agatcaaata agctctgaaa atctgaggaa cattgtagga
3181 aaggtttgtt aagcatctct taagtgccat gatgagcata acagccggcc gtcgtggctc
3241 acgcctgtaa tcccagcact ttgggaggcc aaggtgggag gatgacaagg tcaggagttc
3301 aagaccagcc tggccaacat gctgaaacct cacctctact aaaaatacaa aaattagctg
3361 ggcatggtgg cacatgcctg taatcccagc tacttgggag gctgaggcag gagaatcgct
3421 tgaacccggg aggcggaggt tgcagtgagc caagacagtg ccagtgcact ccagcctcgg
3481 tgacagcgca aggctccgtc tcaataatta aaaaaaaaaa aaaaaaaaaa aaggccgggc
3541 gcagtggctc aagcctgtaa tcccagcact ttgggaggct gaggcgggca gatcacctga
3601 ggtcaggagt tttgagatca gccttggcaa cacggtgaaa ccccatctct actaaaaata
3661 caaaattagc caagcatgct ggcacatgcc tgtaatccca gctactcggg aggctgaggt
3721 acgagaatcg cttgaacctg ggaggcagag gatgcagtga gccgagatca cgccattgca
3781 ctccagcctg ggggacaaga gtgaatctgt gtctcaccaa aaaaaaaaag aaaaagaaag
3841 atgcttaaca aaggttacca taagccacaa attcataacc acttatcctt ccagtttcaa
3901 gtagaatata ttcataacct caataaagtt ctccctgctc ccaaa
SEQ ID NO: 47 Human Cathepsin B Polypeptide, variant 2
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 48 Human Cathepsin B mRNA, variant 3
1    ggggcggggc cgggagggta cttagggccg gggctggccc aggctacggc ggctgcaggg
61   ctccggcaac cgctccggca acgccaaccg ctccgctgcg cgcaggctgg gctgcaggct
121  ctcggctgca gcgctgggtg tcttcaggcc tatggagagc agcttgcgtg ggctgggcct
181  gcagtacctg gtttgcatag atgattggca ggtgggcagc acggggaagg acctgtgagt
241  ggccaacctg gttcaggtgg atctaggatc cggcttccaa catgtggcag ctctgggcct
301  ccctctgctg cctgctggtg ttggccaatg cccggagcag gccctctttc catcccctgt
361  cggatgagct ggtcaactat gtcaacaaac ggaataccac gtggcaggcc gggcacaact
421  tctacaacgt ggacatgagc tacttgaaga ggctatgtgg taccttcctg ggtgggccca
481  agccacccca gagagttatg tttaccgagg acctgaagct gcctgcaagc ttcgatgcac
541  gggaacaatg gccacagtgt cccaccatca aagagatcag agaccagggc tcctgtggct
601  cctgctgggc cttcggggct gtggaagcca tctctgaccg gatctgcatc cacaccaatg
661  cgcacgtcag cgtggaggtg tcggcggagg acctgctcac atgctgtggc agcatgtgtg
721  gggacggctg taatggtggc tatcctgctg aagcttggaa cttctggaca agaaaaggcc
781  tggtttctgg tggcctctat gaatcccatg tagggtgcag accgtactcc atccctccct
841  gtgagcacca cgtcaacggc tcccggcccc catgcacggg ggagggagat acccccaagt
901  gtagcaagat ctgtgagcct ggctacagcc cgacctacaa acaggacaag cactacggat
961  acaattccta cagcgtctcc aatagcgaga aggacatcat ggccgagatc tacaaaaacg
1021 gccccgtgga gggagctttc tctgtgtatt cggacttcct gctctacaag tcaggagtgt
1081 accaacacgt caccggagag atgatgggtg gccatgccat ccgcatcctg ggctggggag
1141 tggagaatgg cacaccctac tggctggttg ccaactcctg gaacactgac tggggtgaca
1201 atggcttctt taaaatactc agaggacagg atcactgtgg aatcgaatca gaagtggtgg
1261 ctggaattcc acgcaccgat cagtactggg aaaagatcta atctgccgtg ggcctgtcgt
1321 gccagtcctg ggggcgagat cggggtagaa atgcatttta ttctttaagt tcacgtaaga
1381 tacaagtttc agacagggtc tgaaggactg gattggccaa acatcagacc tgtcttccaa
1441 ggagaccaag tcctggctac atcccagcct gtggttacag tgcagacagg ccatgtgagc
1501 caccgctgcc agcacagagc gtccttcccc ctgtagacta gtgccgtagg gagtacctgc
1561 tgccccagct gactgtggcc ccctccgtga tccatccatc tccagggagc aagacagaga
1621 cgcaggaatg gaaagcggag ttcctaacag gatgaaagtt cccccatcag ttcccccagt
1681 acctccaagc aagtagcttt ccacatttgt cacagaaatc agaggagaga cggtgttggg
1741 agccctttgg agaacgccag tctcccaggc cccctgcatc tatcgagttt gcaatgtcac
1801 aacctctctg atcttgtgct cagcatgatt ctttaataga agttttattt tttcgtgcac
1861 tctgctaatc atgtgggtga gccagtggaa cagcgggaga cctgtgctag ttttacagat
1921 tgcctcctta tgacgcggct caaaaggaaa ccaagtggtc aggagttgtt tctgacccac
1981 tgatctctac taccacaagg aaaatagttt aggagaaacc agcttttact gtttttgaaa
2041 aattacagct tcaccctgtc aagttaacaa ggaatgcctg tgccaataaa agttttctcc
2101 aacttgaagt ctactctgat gggatctcag atcctttgtc actgcctata gacttgtagc
2161 tgctgtctct ctttgtccct gcagagaatc acgtcctgga actgcatgtt cttgcgactc
2221 ttgggacttc atcttaactt ctcgctgccc cagccatgtt ttcaaccatg gcatccctcc
2281 cccaattagt tccctgtcat cctcgtcaac cttctctgta agtgcctggt aagcttgccc
2341 ttgcttaaga actcaaaaca tagctgtgct ctattttttt gttgttgttg tgactgacag
2401 agtgagattc cgtctcccag gctggagtgc agtggcgcct tctcagctca ctgcaacctg
2461 cagcctccta gattcaagcg attctcctgc ttcagccttc cgagtagctg ggatgacagg
2521 cactcaccaa tatgcctggg taatttttgt atttttaagt acatacagga tttcaccatg
2581 ttggccaggc tagtttcaaa ctcccggcct caggtggtct gcctgcctca gcctcccaaa
2641 gtgttgggat tacaggcgtg agccactggg ccctgcctgt attttttatc agccacaaat
2701 ccagcaacaa gctgaggatt cagctcataa aacaggcttg gtgtcttggt gatctcacat
2761 aaccaagatg ctaccccgtg gggaaccaca tccccctgga tgccctccag ccttggtttg
2821 ggctggagtc agggcctgta tacagtattt tgaatttgta tgccactggt ttgcattgct
2881 ggtcaggaac tctagtgctt tgcatagccc tggtttagaa acatgttata gcagttcttg
2941 gtatagagca aactagaaga accagcaatc attccactgt cctgccaagg tacacctcag
3001 tactcccctt cccaactgaa gtggtatgag gctagctctt tccaaaagca ttcaagtttg
3061 gcttctgatg tgactcagaa tttaggaacc agatgctaga tcaaataagc tctgaaaatc
3121 tgaggaacat tgtaggaaag gtttgttaag catctcttaa gtgccatgat gagcataaca
3181 gccggccgtc gtggctcacg cctgtaatcc cagcactttg ggaggccaag gtgggaggat
3241 gacaaggtca ggagttcaag accagcctgg ccaacatgct gaaacctcac ctctactaaa
3301 aatacaaaaa ttagctgggc atggtggcac atgcctgtaa tcccagctac ttgggaggct
3361 gaggcaggag aatcgcttga acccgggagg cggaggttgc agtgagccaa gacagtgcca
3421 gtgcactcca gcctcggtga cagcgcaagg ctccgtctca ataattaaaa aaaaaaaaaa
3481 aaaaaaaaag gccgggcgca gtggctcaag cctgtaatcc cagcactttg ggaggctgag
3541 gcgggcagat cacctgaggt caggagtttt gagatcagcc ttggcaacac ggtgaaaccc
3601 catctctact aaaaatacaa aattagccaa gcatgctggc acatgcctgt aatcccagct
3661 actcgggagg ctgaggtacg agaatcgctt gaacctggga ggcagaggat gcagtgagcc
3721 gagatcacgc cattgcactc cagcctgggg gacaagagtg aatctgtgtc tcaccaaaaa
3781 aaaaaagaaa aagaaagatg cttaacaaag gttaccataa gccacaaatt cataaccact
3841 tatccttcca gtttcaagta gaatatattc ataacctcaa taaagttctc cctgctccca
3901 aa
SEQ ID NO: 49 Human Cathepsin B Polypeptide, variant 3
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 50 Human Cathepsin B mRNA, variant 4
1    ggggcggggc cgggagggta cttagggccg gggctggccc aggctacggc ggctgcaggg
61   ctccggcaac cgctccggca acgccaaccg ctccgctgcg cgcaggctgg gctgcaggct
121  ctcggctgca gcgctgggct ggtgtgcagt ggtgcgacca cggctcacgg cagcctcagc
181  cacccagatg taagcgatct ggttcccacc tcagcctccc gagtagtgga tctaggatcc
241  ggcttccaac atgtggcagc tctgggcctc cctctgctgc ctgctggtgt tggccaatgc
301  ccggagcagg ccctctttcc atcccctgtc ggatgagctg gtcaactatg tcaacaaacg
361  gaataccacg tggcaggccg ggcacaactt ctacaacgtg gacatgagct acttgaagag
421  gctatgtggt accttcctgg gtgggcccaa gccaccccag agagttatgt ttaccgagga
481  cctgaagctg cctgcaagct tcgatgcacg ggaacaatgg ccacagtgtc ccaccatcaa
541  agagatcaga gaccagggct cctgtggctc ctgctgggcc ttcggggctg tggaagccat
601  ctctgaccgg atctgcatcc acaccaatgc gcacgtcagc gtggaggtgt cggcggagga
661  cctgctcaca tgctgtggca gcatgtgtgg ggacggctgt aatggtggct atcctgctga
721  agcttggaac ttctggacaa gaaaaggcct ggtttctggt ggcctctatg aatcccatgt
781  agggtgcaga ccgtactcca tccctccctg tgagcaccac gtcaacggct cccggccccc
841  atgcacgggg gagggagata cccccaagtg tagcaagatc tgtgagcctg gctacagccc
901  gacctacaaa caggacaagc actacggata caattcctac agcgtctcca atagcgagaa
961  ggacatcatg gccgagatct acaaaaacgg ccccgtggag ggagctttct ctgtgtattc
1021 ggacttcctg ctctacaagt caggagtgta ccaacacgtc accggagaga tgatgggtgg
1081 ccatgccatc cgcatcctgg gctggggagt ggagaatggc acaccctact ggctggttgc
1141 caactcctgg aacactgact ggggtgacaa tggcttcttt aaaatactca gaggacagga
1201 tcactgtgga atcgaatcag aagtggtggc tggaattcca cgcaccgatc agtactggga
1261 aaagatctaa tctgccgtgg gcctgtcgtg ccagtcctgg gggcgagatc ggggtagaaa
1321 tgcattttat tctttaagtt cacgtaagat acaagtttca gacagggtct gaaggactgg
1381 attggccaaa catcagacct gtcttccaag gagaccaagt cctggctaca tcccagcctg
1441 tggttacagt gcagacaggc catgtgagcc accgctgcca gcacagagcg tccttccccc
1501 tgtagactag tgccgtaggg agtacctgct gccccagctg actgtggccc cctccgtgat
1561 ccatccatct ccagggagca agacagagac gcaggaatgg aaagcggagt tcctaacagg
1621 atgaaagttc ccccatcagt tcccccagta cctccaagca agtagctttc cacatttgtc
1681 acagaaatca gaggagagac ggtgttggga gccctttgga gaacgccagt ctcccaggcc
1741 ccctgcatct atcgagtttg caatgtcaca acctctctga tcttgtgctc agcatgattc
1801 tttaatagaa gttttatttt ttcgtgcact ctgctaatca tgtgggtgag ccagtggaac
1861 agcgggagac ctgtgctagt tttacagatt gcctccttat gacgcggctc aaaaggaaac
1921 caagtggtca ggagttgttt ctgacccact gatctctact accacaagga aaatagttta
1981 ggagaaacca gcttttactg tttttgaaaa attacagctt caccctgtca agttaacaag
2041 gaatgcctgt gccaataaaa gttttctcca acttgaagtc tactctgatg ggatctcaga
2101 tcctttgtca ctgcctatag acttgtagct gctgtctctc tttgtccctg cagagaatca
2161 cgtcctggaa ctgcatgttc ttgcgactct tgggacttca tcttaacttc tcgctgcccc
2221 agccatgttt tcaaccatgg catccctccc ccaattagtt ccctgtcatc ctcgtcaacc
2281 ttctctgtaa gtgcctggta agcttgccct tgcttaagaa ctcaaaacat agctgtgctc
2341 tatttttttg ttgttgttgt gactgacaga gtgagattcc gtctcccagg ctggagtgca
2401 gtggcgcctt ctcagctcac tgcaacctgc agcctcctag attcaagcga ttctcctgct
2461 tcagccttcc gagtagctgg gatgacaggc actcaccaat atgcctgggt aatttttgta
2521 tttttaagta catacaggat ttcaccatgt tggccaggct agtttcaaac tcccggcctc
2581 aggtggtctg cctgcctcag cctcccaaag tgttgggatt acaggcgtga gccactgggc
2641 cctgcctgta ttttttatca gccacaaatc cagcaacaag ctgaggattc agctcataaa
2701 acaggcttgg tgtcttggtg atctcacata accaagatgc taccccgtgg ggaaccacat
2761 ccccctggat gccctccagc cttggtttgg gctggagtca gggcctgtat acagtatttt
2821 gaatttgtat gccactggtt tgcattgctg gtcaggaact ctagtgcttt gcatagccct
2881 ggtttagaaa catgttatag cagttcttgg tatagagcaa actagaagaa ccagcaatca
2941 ttccactgtc ctgccaaggt acacctcagt actccccttc ccaactgaag tggtatgagg
3001 ctagctcttt ccaaaagcat tcaagtttgg cttctgatgt gactcagaat ttaggaacca
3061 gatgctagat caaataagct ctgaaaatct gaggaacatt gtaggaaagg tttgttaagc
3121 atctcttaag tgccatgatg agcataacag ccggccgtcg tggctcacgc ctgtaatccc
3181 agcactttgg gaggccaagg tgggaggatg acaaggtcag gagttcaaga ccagcctggc
3241 caacatgctg aaacctcacc tctactaaaa atacaaaaat tagctgggca tggtggcaca
3301 tgcctgtaat cccagctact tgggaggctg aggcaggaga atcgcttgaa cccgggaggc
3361 ggaggttgca gtgagccaag acagtgccag tgcactccag cctcggtgac agcgcaaggc
3421 tccgtctcaa taattaaaaa aaaaaaaaaa aaaaaaaagg ccgggcgcag tggctcaagc
3481 ctgtaatccc agcactttgg gaggctgagg cgggcagatc acctgaggtc aggagttttg
3541 agatcagcct tggcaacacg gtgaaacccc atctctacta aaaatacaaa attagccaag
3601 catgctggca catgcctgta atcccagcta ctcgggaggc tgaggtacga gaatcgcttg
3661 aacctgggag gcagaggatg cagtgagccg agatcacgcc attgcactcc agcctggggg
3721 acaagagtga atctgtgtct caccaaaaaa aaaaagaaaa agaaagatgc ttaacaaagg
3781 ttaccataag ccacaaattc ataaccactt atccttccag tttcaagtag aatatattca
3841 taacctcaat aaagttctcc ctgctcccaa a
SEQ ID NO: 51 Human Cathepsin B Polypeptide, variant 4
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 52 Human Cathepsin B mRNA, variant 5
1    ggggcggggc cgggagggta cttagggccg gggctggccc aggctacggc ggctgcaggg
61   ctccggcaac cgctccggca acgccaaccg ctccgctgcg cgcaggctgg gctgcaggct
121  ctcggctgca gcgctgggtg tcttcaggcc tatggagagc agcttgcgtg ggctgggcct
181  gcagtacctg gtttgcatag atgattggca ggtggatcta ggatccggct tccaacatgt
241  ggcagctctg ggcctccctc tgctgcctgc tggtgttggc caatgcccgg agcaggccct
301  ctttccatcc cctgtcggat gagctggtca actatgtcaa caaacggaat accacgtggc
361  aggccgggca caacttctac aacgtggaca tgagctactt gaagaggcta tgtggtacct
421  tcctgggtgg gcccaagcca ccccagagag ttatgtttac cgaggacctg aagctgcctg
481  caagcttcga tgcacgggaa caatggccac agtgtcccac catcaaagag atcagagacc
541  agggctcctg tggctcctgc tgggccttcg gggctgtgga agccatctct gaccggatct
601  gcatccacac caatgcgcac gtcagcgtgg aggtgtcggc ggaggacctg ctcacatgct
661  gtggcagcat gtgtggggac ggctgtaatg gtggctatcc tgctgaagct tggaacttct
721  ggacaagaaa aggcctggtt tctggtggcc tctatgaatc ccatgtaggg tgcagaccgt
781  actccatccc tccctgtgag caccacgtca acggctcccg gcccccatgc acgggggagg
841  gagatacccc caagtgtagc aagatctgtg agcctggcta cagcccgacc tacaaacagg
901  acaagcacta cggatacaat tcctacagcg tctccaatag cgagaaggac atcatggccg
961  agatctacaa aaacggcccc gtggagggag ctttctctgt gtattcggac ttcctgctct
1021 acaagtcagg agtgtaccaa cacgtcaccg gagagatgat gggtggccat gccatccgca
1081 tcctgggctg gggagtggag aatggcacac cctactggct ggttgccaac tcctggaaca
1141 ctgactgggg tgacaatggc ttctttaaaa tactcagagg acaggatcac tgtggaatcg
1201 aatcagaagt ggtggctgga attccacgca ccgatcagta ctgggaaaag atctaatctg
1261 ccgtgggcct gtcgtgccag tcctgggggc gagatcgggg tagaaatgca ttttattctt
1321 taagttcacg taagatacaa gtttcagaca gggtctgaag gactggattg gccaaacatc
1381 agacctgtct tccaaggaga ccaagtcctg gctacatccc agcctgtggt tacagtgcag
1441 acaggccatg tgagccaccg ctgccagcac agagcgtcct tccccctgta gactagtgcc
1501 gtagggagta cctgctgccc cagctgactg tggccccctc cgtgatccat ccatctccag
1561 ggagcaagac agagacgcag gaatggaaag cggagttcct aacaggatga aagttccccc
1621 atcagttccc ccagtacctc caagcaagta gctttccaca tttgtcacag aaatcagagg
1681 agagacggtg ttgggagccc tttggagaac gccagtctcc caggccccct gcatctatcg
1741 agtttgcaat gtcacaacct ctctgatctt gtgctcagca tgattcttta atagaagttt
1801 tattttttcg tgcactctgc taatcatgtg ggtgagccag tggaacagcg ggagacctgt
1861 gctagtttta cagattgcct ccttatgacg cggctcaaaa ggaaaccaag tggtcaggag
1921 ttgtttctga cccactgatc tctactacca caaggaaaat agtttaggag aaaccagctt
1981 ttactgtttt tgaaaaatta cagcttcacc ctgtcaagtt aacaaggaat gcctgtgcca
2041 ataaaagttt tctccaactt gaagtctact ctgatgggat ctcagatcct ttgtcactgc
2101 ctatagactt gtagctgctg tctctctttg tccctgcaga gaatcacgtc ctggaactgc
2161 atgttcttgc gactcttggg acttcatctt aacttctcgc tgccccagcc atgttttcaa
2221 ccatggcatc cctcccccaa ttagttccct gtcatcctcg tcaaccttct ctgtaagtgc
2281 ctggtaagct tgcccttgct taagaactca aaacatagct gtgctctatt tttttgttgt
2341 tgttgtgact gacagagtga gattccgtct cccaggctgg agtgcagtgg cgccttctca
2401 gctcactgca acctgcagcc tcctagattc aagcgattct cctgcttcag ccttccgagt
2461 agctgggatg acaggcactc accaatatgc ctgggtaatt tttgtatttt taagtacata
2521 caggatttca ccatgttggc caggctagtt tcaaactccc ggcctcaggt ggtctgcctg
2581 cctcagcctc ccaaagtgtt gggattacag gcgtgagcca ctgggccctg cctgtatttt
2641 ttatcagcca caaatccagc aacaagctga ggattcagct cataaaacag gcttggtgtc
2701 ttggtgatct cacataacca agatgctacc ccgtggggaa ccacatcccc ctggatgccc
2761 tccagccttg gtttgggctg gagtcagggc ctgtatacag tattttgaat ttgtatgcca
2821 ctggtttgca ttgctggtca ggaactctag tgctttgcat agccctggtt tagaaacatg
2881 ttatagcagt tcttggtata gagcaaacta gaagaaccag caatcattcc actgtcctgc
2941 caaggtacac ctcagtactc cccttcccaa ctgaagtggt atgaggctag ctctttccaa
3001 aagcattcaa gtttggcttc tgatgtgact cagaatttag gaaccagatg ctagatcaaa
3061 taagctctga aaatctgagg aacattgtag gaaaggtttg ttaagcatct cttaagtgcc
3121 atgatgagca taacagccgg ccgtcgtggc tcacgcctgt aatcccagca ctttgggagg
3181 ccaaggtggg aggatgacaa ggtcaggagt tcaagaccag cctggccaac atgctgaaac
3241 ctcacctcta ctaaaaatac aaaaattagc tgggcatggt ggcacatgcc tgtaatccca
3301 gctacttggg aggctgaggc aggagaatcg cttgaacccg ggaggcggag gttgcagtga
3361 gccaagacag tgccagtgca ctccagcctc ggtgacagcg caaggctccg tctcaataat
3421 taaaaaaaaa aaaaaaaaaa aaaaggccgg gcgcagtggc tcaagcctgt aatcccagca
3481 ctttgggagg ctgaggcggg cagatcacct gaggtcagga gttttgagat cagccttggc
3541 aacacggtga aaccccatct ctactaaaaa tacaaaatta gccaagcatg ctggcacatg
3601 cctgtaatcc cagctactcg ggaggctgag gtacgagaat cgcttgaacc tgggaggcag
3661 aggatgcagt gagccgagat cacgccattg cactccagcc tgggggacaa gagtgaatct
3721 gtgtctcacc aaaaaaaaaa agaaaaagaa agatgcttaa caaaggttac cataagccac
3781 aaattcataa ccacttatcc ttccagtttc aagtagaata tattcataac ctcaataaag
3841 ttctccctgc tcccaaa
SEQ ID NO: 53 Human Cathepsin B Polypeptide, variant 5
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 54 Human Cathepsin B mRNA, variant 6
1    agggccgggg ctggcccagg ctacggcggc tgcagggctc cggcaaccgc tccggcaacg
61   ccaaccgctc cgctgcgcgc aggctgggct gcaggctctc ggctgcagcg ctgggctggt
121  gtgcagtggt gcgaccacgg ctcacggcag cctcagccac ccagatgtaa gcgatctggt
181  tcccacctca gcctcccgag tagatacttc tgaaaataga aatgatgact ctgggatgca
241  aacgttggct gtcctatgta taaggagatg gcttttcacg ctcccagtga ctgaggaagt
301  ttctcccaga tggcgctgct ctgagcctgg tgcagggtgg atctaggatc cggcttccaa
361  catgtggcag ctctgggcct ccctctgctg cctgctggtg ttggccaatg cccggagcag
421  gccctctttc catcccctgt cggatgagct ggtcaactat gtcaacaaac ggaataccac
481  gtggcaggcc gggcacaact tctacaacgt ggacatgagc tacttgaaga ggctatgtgg
541  taccttcctg ggtgggccca agccacccca gagagttatg tttaccgagg acctgaagct
601  gcctgcaagc ttcgatgcac gggaacaatg gccacagtgt cccaccatca aagagatcag
661  agaccagggc tcctgtggct cctgctgggc cttcggggct gtggaagcca tctctgaccg
721  gatctgcatc cacaccaatg cgcacgtcag cgtggaggtg tcggcggagg acctgctcac
781  atgctgtggc agcatgtgtg gggacggctg taatggtggc tatcctgctg aagcttggaa
841  cttctggaca agaaaaggcc tggtttctgg tggcctctat gaatcccatg tagggtgcag
901  accgtactcc atccctccct gtgagcacca cgtcaacggc tcccggcccc catgcacggg
961  ggagggagat acccccaagt gtagcaagat ctgtgagcct ggctacagcc cgacctacaa
1021 acaggacaag cactacggat acaattccta cagcgtctcc aatagcgaga aggacatcat
1081 ggccgagatc tacaaaaacg gccccgtgga gggagctttc tctgtgtatt cggacttcct
1141 gctctacaag tcaggagtgt accaacacgt caccggagag atgatgggtg gccatgccat
1201 ccgcatcctg ggctggggag tggagaatgg cacaccctac tggctggttg ccaactcctg
1261 gaacactgac tggggtgaca atggcttctt taaaatactc agaggacagg atcactgtgg
1321 aatcgaatca gaagtggtgg ctggaattcc acgcaccgat cagtactggg aaaagatcta
1381 atctgccgtg ggcctgtcgt gccagtcctg ggggcgagat cggggtagaa atgcatttta
1441 ttctttaagt tcacgtaaga tacaagtttc agacagggtc tgaaggactg gattggccaa
1501 acatcagacc tgtcttccaa ggagaccaag tcctggctac atcccagcct gtggttacag
1561 tgcagacagg ccatgtgagc caccgctgcc agcacagagc gtccttcccc ctgtagacta
1621 gtgccgtagg gagtacctgc tgccccagct gactgtggcc ccctccgtga tccatccatc
1681 tccagggagc aagacagaga cgcaggaatg gaaagcggag ttcctaacag gatgaaagtt
1741 cccccatcag ttcccccagt acctccaagc aagtagcttt ccacatttgt cacagaaatc
1801 agaggagaga cggtgttggg agccctttgg agaacgccag tctcccaggc cccctgcatc
1861 tatcgagttt gcaatgtcac aacctctctg atcttgtgct cagcatgatt ctttaataga
1921 agttttattt tttcgtgcac tctgctaatc atgtgggtga gccagtggaa cagcgggaga
1981 cctgtgctag ttttacagat tgcctcctta tgacgcggct caaaaggaaa ccaagtggtc
2041 aggagttgtt tctgacccac tgatctctac taccacaagg aaaatagttt aggagaaacc
2101 agcttttact gtttttgaaa aattacagct tcaccctgtc aagttaacaa ggaatgcctg
2161 tgccaataaa agttttctcc aacttgaagt ctactctgat gggatctcag atcctttgtc
2221 actgcctata gacttgtagc tgctgtctct ctttgtccct gcagagaatc acgtcctgga
2281 actgcatgtt cttgcgactc ttgggacttc atcttaactt ctcgctgccc cagccatgtt
2341 ttcaaccatg gcatccctcc cccaattagt tccctgtcat cctcgtcaac cttctctgta
2401 agtgcctggt aagcttgccc ttgcttaaga actcaaaaca tagctgtgct ctattttttt
2461 gttgttgttg tgactgacag agtgagattc cgtctcccag gctggagtgc agtggcgcct
2521 tctcagctca ctgcaacctg cagcctccta gattcaagcg attctcctgc ttcagccttc
2581 cgagtagctg ggatgacagg cactcaccaa tatgcctggg taatttttgt atttttaagt
2641 acatacagga tttcaccatg ttggccaggc tagtttcaaa ctcccggcct caggtggtct
2701 gcctgcctca gcctcccaaa gtgttgggat tacaggcgtg agccactggg ccctgcctgt
2761 attttttatc agccacaaat ccagcaacaa gctgaggatt cagctcataa aacaggcttg
2821 gtgtcttggt gatctcacat aaccaagatg ctaccccgtg gggaaccaca tccccctgga
2881 tgccctccag ccttggtttg ggctggagtc agggcctgta tacagtattt tgaatttgta
2941 tgccactggt ttgcattgct ggtcaggaac tctagtgctt tgcatagccc tggtttagaa
3001 acatgttata gcagttcttg gtatagagca aactagaaga accagcaatc attccactgt
3061 cctgccaagg tacacctcag tactcccctt cccaactgaa gtggtatgag gctagctctt
3121 tccaaaagca ttcaagtttg gcttctgatg tgactcagaa tttaggaacc agatgctaga
3181 tcaaataagc tctgaaaatc tgaggaacat tgtaggaaag gtttgttaag catctcttaa
3241 gtgccatgat gagcataaca gccggccgtc gtggctcacg cctgtaatcc cagcactttg
3301 ggaggccaag gtgggaggat gacaaggtca ggagttcaag accagcctgg ccaacatgct
3361 gaaacctcac ctctactaaa aatacaaaaa ttagctgggc atggtggcac atgcctgtaa
3421 tcccagctac ttgggaggct gaggcaggag aatcgcttga acccgggagg cggaggttgc
3481 agtgagccaa gacagtgcca gtgcactcca gcctcggtga cagcgcaagg ctccgtctca
3541 ataattaaaa aaaaaaaaaa aaaaaaaaag gccgggcgca gtggctcaag cctgtaatcc
3601 cagcactttg ggaggctgag gcgggcagat cacctgaggt caggagtttt gagatcagcc
3661 ttggcaacac ggtgaaaccc catctctact aaaaatacaa aattagccaa gcatgctggc
3721 acatgcctgt aatcccagct actcgggagg ctgaggtacg agaatcgctt gaacctggga
3781 ggcagaggat gcagtgagcc gagatcacgc cattgcactc cagcctgggg gacaagagtg
3841 aatctgtgtc tcaccaaaaa aaaaaagaaa aagaaagatg cttaacaaag gttaccataa
3901 gccacaaatt cataaccact tatccttcca gtttcaagta gaatatattc ataacctcaa
3961 taaagttctc cctgctccca aa
SEQ ID NO: 55 Human Cathepsin B Polypeptide, variant 6
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 56 Human Cathepsin B mRNA, variant 7
1    caggaccgcc gagggaggcg cctgcgagga agagctcggc cgggtccgga gactgctgcc
61   tgggaccgcg ctcccagcgc ctgggcctcg gtgtctccgg gccaaactgc cgacataatc
121  gcatctgccg gcatctattt tcggtttatt tccccctcat tgcgaaggat ttgcctggcc
181  aactttctgc gcaagatccc acgcaattcc tgggacccca gaagacaggt cctgttgaag
241  aacaggaatc tggcactggg tgggctgggg aggaagccgc acggtgttaa atccataaac
301  aggaagagaa accagacagc gaaaccaaga ggcgaatggg cgattggatg ccggtgggga
361  gaaggccggg ggcgcaccct gctcctggac tccagtaaag ggaggccggg cagagtccct
421  ggggcgccac ctccccctcg gtggatctag gatccggctt ccaacatgtg gcagctctgg
481  gcctccctct gctgcctgct ggtgttggcc aatgcccgga gcaggccctc tttccatccc
541  ctgtcggatg agctggtcaa ctatgtcaac aaacggaata ccacgtggca ggccgggcac
601  aacttctaca acgtggacat gagctacttg aagaggctat gtggtacctt cctgggtggg
661  cccaagccac cccagagagt tatgtttacc gaggacctga agctgcctgc aagcttcgat
721  gcacgggaac aatggccaca gtgtcccacc atcaaagaga tcagagacca gggctcctgt
781  ggctcctgct gggccttcgg ggctgtggaa gccatctctg accggatctg catccacacc
841  aatgcgcacg tcagcgtgga ggtgtcggcg gaggacctgc tcacatgctg tggcagcatg
901  tgtggggacg gctgtaatgg tggctatcct gctgaagctt ggaacttctg gacaagaaaa
961  ggcctggttt ctggtggcct ctatgaatcc catgtagggt gcagaccgta ctccatccct
1021 ccctgtgagc accacgtcaa cggctcccgg cccccatgca cgggggaggg agataccccc
1081 aagtgtagca agatctgtga gcctggctac agcccgacct acaaacagga caagcactac
1141 ggatacaatt cctacagcgt ctccaatagc gagaaggaca tcatggccga gatctacaaa
1201 aacggccccg tggagggagc tttctctgtg tattcggact tcctgctcta caagtcagga
1261 gtgtaccaac acgtcaccgg agagatgatg ggtggccatg ccatccgcat cctgggctgg
1321 ggagtggaga atggcacacc ctactggctg gttgccaact cctggaacac tgactggggt
1381 gacaatggct tctttaaaat actcagagga caggatcact gtggaatcga atcagaagtg
1441 gtggctggaa ttccacgcac cgatcagtac tgggaaaaga tctaatctgc cgtgggcctg
1501 tcgtgccagt cctgggggcg agatcggggt agaaatgcat tttattcttt aagttcacgt
1561 aagatacaag tttcagacag ggtctgaagg actggattgg ccaaacatca gacctgtctt
1621 ccaaggagac caagtcctgg ctacatccca gcctgtggtt acagtgcaga caggccatgt
1681 gagccaccgc tgccagcaca gagcgtcctt ccccctgtag actagtgccg tagggagtac
1741 ctgctgcccc agctgactgt ggccccctcc gtgatccatc catctccagg gagcaagaca
1801 gagacgcagg aatggaaagc ggagttccta acaggatgaa agttccccca tcagttcccc
1861 cagtacctcc aagcaagtag ctttccacat ttgtcacaga aatcagagga gagacggtgt
1921 tgggagccct ttggagaacg ccagtctccc aggccccctg catctatcga gtttgcaatg
1981 tcacaacctc tctgatcttg tgctcagcat gattctttaa tagaagtttt attttttcgt
2041 gcactctgct aatcatgtgg gtgagccagt ggaacagcgg gagacctgtg ctagttttac
2101 agattgcctc cttatgacgc ggctcaaaag gaaaccaagt ggtcaggagt tgtttctgac
2161 ccactgatct ctactaccac aaggaaaata gtttaggaga aaccagcttt tactgttttt
2221 gaaaaattac agcttcaccc tgtcaagtta acaaggaatg cctgtgccaa taaaagtttt
2281 ctccaacttg aagtctactc tgatgggatc tcagatcctt tgtcactgcc tatagacttg
2341 tagctgctgt ctctctttgt ccctgcagag aatcacgtcc tggaactgca tgttcttgcg
2401 actcttggga cttcatctta acttctcgct gccccagcca tgttttcaac catggcatcc
2461 ctcccccaat tagttccctg tcatcctcgt caaccttctc tgtaagtgcc tggtaagctt
2521 gcccttgctt aagaactcaa aacatagctg tgctctattt ttttgttgtt gttgtgactg
2581 acagagtgag attccgtctc ccaggctgga gtgcagtggc gccttctcag ctcactgcaa
2641 cctgcagcct cctagattca agcgattctc ctgcttcagc cttccgagta gctgggatga
2701 caggcactca ccaatatgcc tgggtaattt ttgtattttt aagtacatac aggatttcac
2761 catgttggcc aggctagttt caaactcccg gcctcaggtg gtctgcctgc ctcagcctcc
2821 caaagtgttg ggattacagg cgtgagccac tgggccctgc ctgtattttt tatcagccac
2881 aaatccagca acaagctgag gattcagctc ataaaacagg cttggtgtct tggtgatctc
2941 acataaccaa gatgctaccc cgtggggaac cacatccccc tggatgccct ccagccttgg
3001 tttgggctgg agtcagggcc tgtatacagt attttgaatt tgtatgccac tggtttgcat
3061 tgctggtcag gaactctagt gctttgcata gccctggttt agaaacatgt tatagcagtt
3121 cttggtatag agcaaactag aagaaccagc aatcattcca ctgtcctgcc aaggtacacc
3181 tcagtactcc ccttcccaac tgaagtggta tgaggctagc tctttccaaa agcattcaag
3241 tttggcttct gatgtgactc agaatttagg aaccagatgc tagatcaaat aagctctgaa
3301 aatctgagga acattgtagg aaaggtttgt taagcatctc ttaagtgcca tgatgagcat
3361 aacagccggc cgtcgtggct cacgcctgta atcccagcac tttgggaggc caaggtggga
3421 ggatgacaag gtcaggagtt caagaccagc ctggccaaca tgctgaaacc tcacctctac
3481 taaaaataca aaaattagct gggcatggtg gcacatgcct gtaatcccag ctacttggga
3541 ggctgaggca ggagaatcgc ttgaacccgg gaggcggagg ttgcagtgag ccaagacagt
3601 gccagtgcac tccagcctcg gtgacagcgc aaggctccgt ctcaataatt aaaaaaaaaa
3661 aaaaaaaaaa aaaggccggg cgcagtggct caagcctgta atcccagcac tttgggaggc
3721 tgaggcgggc agatcacctg aggtcaggag ttttgagatc agccttggca acacggtgaa
3781 accccatctc tactaaaaat acaaaattag ccaagcatgc tggcacatgc ctgtaatccc
3841 agctactcgg gaggctgagg tacgagaatc gcttgaacct gggaggcaga ggatgcagtg
3901 agccgagatc acgccattgc actccagcct gggggacaag agtgaatctg tgtctcacca
3961 aaaaaaaaaa gaaaaagaaa gatgcttaac aaaggttacc ataagccaca aattcataac
4021 cacttatcct tccagtttca agtagaatat attcataacc tcaataaagt tctccctgct
4081 cccaaa
SEQ ID NO: 57 Human Cathepsin B Polypeptide, variant 7
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAG
HNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQ
GSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLICCGSMCGDGCNGGYPAEAWN
FWIRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPT
YKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMG
GHAIRILGWGVENGTPYWLVANSWNIDWGDNGFFKILRGQDHCGIESEVVAGIPRIDQ
YWEKI
SEQ ID NO: 58 Human Cathepsin L mRNA, variant 2
1    ggcggtgccg gccgaaccca gacccgaggt tttagaagca gagtcaggcg aagctgggcc
61   agaaccgcga cctccgcaac cttgagcggc atccgtggag tgcgcctgcg cagctacgac
121  cgcagcagga aagcgccgcc ggccaggccc agctgtggcc ggacagggac tggaagagag
181  gacgcggtcg agtaggtttt aaaacatgaa tcctacactc atccttgctg ccttttgcct
241  gggaattgcc tcagctactc taacatttga tcacagttta gaggcacagt ggaccaagtg
301  gaaggcgatg cacaacagat tatacggcat gaatgaagaa ggatggagga gagcagtgtg
361  ggagaagaac atgaagatga ttgaactgca caatcaggaa tacagggaag ggaaacacag
421  cttcacaatg gccatgaacg cctttggaga catgaccagt gaagaattca ggcaggtgat
481  gaatggcttt caaaaccgta agcccaggaa ggggaaagtg ttccaggaac ctctgtttta
541  tgaggccccc agatctgtgg attggagaga gaaaggctac gtgactcctg tgaagaatca
601  gggtcagtgt ggttcttgtt gggcttttag tgctactggt gctcttgaag gacagatgtt
661  ccggaaaact gggaggctta tctcactgag tgagcagaat ctggtagact gctctgggcc
721  tcaaggcaat gaaggctgca atggtggcct aatggattat gctttccagt atgttcagga
781  taatggaggc ctggactctg aggaatccta tccatatgag gcaacagaag aatcctgtaa
841  gtacaatccc aagtattctg ttgctaatga caccggcttt gtggacatcc ctaagcagga
901  gaaggccctg atgaaggcag ttgcaactgt ggggcccatt tctgttgcta ttgatgcagg
961  tcatgagtcc ttcctgttct ataaagaagg catttatttt gagccagact gtagcagtga
1021 agacatggat catggtgtgc tggtggttgg ctacggattt gaaagcacag aatcagataa
1081 caataaatat tggctggtga agaacagctg gggtgaagaa tggggcatgg gtggctacgt
1141 aaagatggcc aaagaccgga gaaaccattg tggaattgcc tcagcagcca gctaccccac
1201 tgtgtgagct ggtggacggt gatgaggaag gacttgactg gggatggcgc atgcatggga
1261 ggaattcatc ttcagtctac cagcccccgc tgtgtcggat acacactcga atcattgaag
1321 atccgagtgt gatttgaatt ctgtgatatt ttcacactgg taaatgttac ctctatttta
1381 attactgcta taaataggtt tatattattg attcacttac tgactttgca ttttcgtttt
1441 taaaaggatg tataaatttt tacctgttta aataaaattt aatttcaaat gtagtggtgg
1501 ggcttctttc tatttttgat gcactgaatt tttgtgtaat aaagaacata attgggctct
1561 aagccataaa aaaaaaaaaa aaaaaaa
SEQ ID NO: 59 Human Cathepsin L Polypeptide, variant 2
MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNE
EGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRK
GKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLIS
LSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYS
VANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDH
GVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
SEQ ID NO: 60 Human Cathepsin L mRNA, variant 3
1    ggcggtgccg gccgaaccca gacccgaggt tttagaagca gagtcaggcg aagctgggcc
61   agaaccgcga cctccgcaac cttgagcggc atccgtggag tgcgcctgcg cagctacgac
121  cgcagcagga aagcgccgcc ggccaggccc agctgtggcc ggacagggac tggaagagag
181  gacgcggtcg agtaggtgtg caccagccct ggcaacgaga gcgtctaccc cgaactctgc
241  tggccttgag gttttaaaac atgaatccta cactcatcct tgctgccttt tgcctgggaa
301  ttgcctcagc tactctaaca tttgatcaca gtttagaggc acagtggacc aagtggaagg
361  cgatgcacaa cagattatac ggcatgaatg aagaaggatg gaggagagca gtgtgggaga
421  agaacatgaa gatgattgaa ctgcacaatc aggaatacag ggaagggaaa cacagcttca
481  caatggccat gaacgccttt ggagacatga ccagtgaaga attcaggcag gtgatgaatg
541  gctttcaaaa ccgtaagccc aggaagggga aagtgttcca ggaacctctg ttttatgagg
601  cccccagatc tgtggattgg agagagaaag gctacgtgac tcctgtgaag aatcagggtc
661  agtgtggttc ttgttgggct tttagtgcta ctggtgctct tgaaggacag atgttccgga
721  aaactgggag gcttatctca ctgagtgagc agaatctggt agactgctct gggcctcaag
781  gcaatgaagg ctgcaatggt ggcctaatgg attatgcttt ccagtatgtt caggataatg
841  gaggcctgga ctctgaggaa tcctatccat atgaggcaac agaagaatcc tgtaagtaca
901  atcccaagta ttctgttgct aatgacaccg gctttgtgga catccctaag caggagaagg
961  ccctgatgaa ggcagttgca actgtggggc ccatttctgt tgctattgat gcaggtcatg
1021 agtccttcct gttctataaa gaaggcattt attttgagcc agactgtagc agtgaagaca
1081 tggatcatgg tgtgctggtg gttggctacg gatttgaaag cacagaatca gataacaata
1141 aatattggct ggtgaagaac agctggggtg aagaatgggg catgggtggc tacgtaaaga
1201 tggccaaaga ccggagaaac cattgtggaa ttgcctcagc agccagctac cccactgtgt
1261 gagctggtgg acggtgatga ggaaggactt gactggggat ggcgcatgca tgggaggaat
1321 tcatcttcag tctaccagcc cccgctgtgt cggatacaca ctcgaatcat tgaagatccg
1381 agtgtgattt gaattctgtg atattttcac actggtaaat gttacctcta ttttaattac
1441 tgctataaat aggtttatat tattgattca cttactgact ttgcattttc gtttttaaaa
1501 ggatgtataa atttttacct gtttaaataa aatttaattt caaatgtagt ggtggggctt
1561 ctttctattt ttgatgcact gaatttttgt gtaataaaga acataattgg gctctaagcc
1621 ataaaa
SEQ ID NO: 61 Human Cathepsin L Polypeptide, variant 3
MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNE
EGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRK
GKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLIS
LSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYS
VANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDH
GVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
SEQ ID NO: 62 Human Cathepsin L mRNA, variant 4
1    ggcggtgccg gccgaaccca gacccgaggt tttagaagca gagtcaggcg aagctgggcc
61   agaaccgcga cctccgcaac cttgagcggc atccgtggag tgcgcctgcg cagctacgac
121  cgcagcagga aagcgccgcc ggccaggccc agctgtggcc ggacagggac tggaagagag
181  gacgcggtcg agttttaaaa catgaatcct acactcatcc ttgctgcctt ttgcctggga
241  attgcctcag ctactctaac atttgatcac agtttagagg cacagtggac caagtggaag
301  gcgatgcaca acagattata cggcatgaat gaagaaggat ggaggagagc agtgtgggag
361  aagaacatga agatgattga actgcacaat caggaataca gggaagggaa acacagcttc
421  acaatggcca tgaacgcctt tggagacatg accagtgaag aattcaggca ggtgatgaat
481  ggctttcaaa accgtaagcc caggaagggg aaagtgttcc aggaacctct gttttatgag
541  gcccccagat ctgtggattg gagagagaaa ggctacgtga ctcctgtgaa gaatcagggt
601  cagtgtggtt cttgttgggc ttttagtgct actggtgctc ttgaaggaca gatgttccgg
661  aaaactggga ggcttatctc actgagtgag cagaatctgg tagactgctc tgggcctcaa
721  ggcaatgaag gctgcaatgg tggcctaatg gattatgctt tccagtatgt tcaggataat
781  ggaggcctgg actctgagga atcctatcca tatgaggcaa cagaagaatc ctgtaagtac
841  aatcccaagt attctgttgc taatgacacc ggctttgtgg acatccctaa gcaggagaag
901  gccctgatga aggcagttgc aactgtgggg cccatttctg ttgctattga tgcaggtcat
961  gagtccttcc tgttctataa agaaggcatt tattttgagc cagactgtag cagtgaagac
1021 atggatcatg gtgtgctggt ggttggctac ggatttgaaa gcacagaatc agataacaat
1081 aaatattggc tggtgaagaa cagctggggt gaagaatggg gcatgggtgg ctacgtaaag
1141 atggccaaag accggagaaa ccattgtgga attgcctcag cagccagcta ccccactgtg
1201 tgagctggtg gacggtgatg aggaaggact tgactgggga tggcgcatgc atgggaggaa
1261 ttcatcttca gtctaccagc ccccgctgtg tcggatacac actcgaatca ttgaagatcc
1321 gagtgtgatt tgaattctgt gatattttca cactggtaaa tgttacctct attttaatta
1381 ctgctataaa taggtttata ttattgattc acttactgac tttgcatttt cgtttttaaa
1441 aggatgtata aatttttacc tgtttaaata aaatttaatt tcaaatgtag tggtggggct
1501 tctttctatt tttgatgcac tgaatttttg tgtaataaag aacataattg ggctctaagc
1561 cataaaa
SEQ ID NO: 63 Human Cathepsin L Polypeptide, variant 4
MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNE
EGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRK
GKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLIS
LSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYS
VANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDH
GVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
SEQ ID NO: 64 Human Cathepsin L mRNA, variant 5
1    ggcggtgccg gccgaaccca gacccgaggt tttagaagca gagtcaggcg aagctgggcc
61   agaaccgcga cctccgcaac cttgagcggc atccgtggag tgcgcctgcg cagctacgac
121  cgcagcagga aagcgccgcc ggccaggccc agctgtggcc ggacagggac tggaagagag
181  gacgcggtcg agtaggtttt aaaacatgaa tcctacactc atccttgctg ccttttgcct
241  gggaattgcc tcagctactc taacatttga tcacagttta gaggcacagt ggaccaagtg
301  gaaggctgca atggtggcct aatggattat gctttccagt atgttcagga taatggaggc
361  ctggactctg aggaatccta tccatatgag gcaacagaag aatcctgtaa gtacaatccc
421  aagtattctg ttgctaatga caccggcttt gtggacatcc ctaagcagga gaaggccctg
481  atgaaggcag ttgcaactgt ggggcccatt tctgttgcta ttgatgcagg tcatgagtcc
541  ttcctgttct ataaagaagg catttatttt gagccagact gtagcagtga agacatggat
601  catggtgtgc tggtggttgg ctacggattt gaaagcacag aatcagataa caataaatat
661  tggctggtga agaacagctg gggtgaagaa tggggcatgg gtggctacgt aaagatggcc
721  aaagaccgga gaaaccattg tggaattgcc tcagcagcca gctaccccac tgtgtgagct
781  ggtggacggt gatgaggaag gacttgactg gggatggcgc atgcatggga ggaattcatc
841  ttcagtctac cagcccccgc tgtgtcggat acacactcga atcattgaag atccgagtgt
901  gatttgaatt ctgtgatatt ttcacactgg taaatgttac ctctatttta attactgcta
961  taaataggtt tatattattg attcacttac tgactttgca ttttcgtttt taaaaggatg
1021 tataaatttt tacctgttta aataaaattt aatttcaaat gtagtggtgg ggcttctttc
1081 tatttttgat gcactgaatt tttgtgtaat aaagaacata attgggctct aagccataaa
1141 a
SEQ ID NO: 65 Human Cathepsin L Polypeptide, variant 5
MDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFV
DIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYG
FESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
SEQ ID NO: 66 Human Cathepsin L mRNA, variant 6
1    acagctctgg acaggctgct tttcattttg gtgagtccat ccagtacctc cacgtgccct
61   gtttttctcc aggcacatcc ttggcctctt ccacagtcct tgggttttaa aacatgaatc
121  ctacactcat ccttgctgcc ttttgcctgg gaattgcctc agctactcta acatttgatc
181  acagtttaga ggcacagtgg accaagtgga aggcgatgca caacagatta tacggcatga
241  atgaagaagg atggaggaga gcagtgtggg agaagaacat gaagatgatt gaactgcaca
301  atcaggaata cagggaaggg aaacacagct tcacaatggc catgaacgcc tttggagaca
361  tgaccagtga agaattcagg caggtgatga atggctttca aaaccgtaag cccaggaagg
421  ggaaagtgtt ccaggaacct ctgttttatg aggcccccag atctgtggat tggagagaga
481  aaggctacgt gactcctgtg aagaatcagg gtcagtgtgg ttcttgttgg gcttttagtg
541  ctactggtgc tcttgaagga cagatgttcc ggaaaactgg gaggcttatc tcactgagtg
601  agcagaatct ggtagactgc tctgggcctc aaggcaatga aggctgcaat ggtggcctaa
661  tggattatgc tttccagtat gttcaggata atggaggcct ggactctgag gaatcctatc
721  catatgaggc aacagaagaa tcctgtaagt acaatcccaa gtattctgtt gctaatgaca
781  ccggctttgt ggacatccct aagcaggaga aggccctgat gaaggcagtt gcaactgtgg
841  ggcccatttc tgttgctatt gatgcaggtc atgagtcctt cctgttctat aaagaaggca
901  tttattttga gccagactgt agcagtgaag acatggatca tggtgtgctg gtggttggct
961  acggatttga aagcacagaa tcagataaca ataaatattg gctggtgaag aacagctggg
1021 gtgaagaatg gggcatgggt ggctacgtaa agatggccaa agaccggaga aaccattgtg
1081 gaattgcctc agcagccagc taccccactg tgtgagctgg tggacggtga tgaggaagga
1141 cttgactggg gatggcgcat gcatgggagg aattcatctt cagtctacca gcccccgctg
1201 tgtcggatac acactcgaat cattgaagat ccgagtgtga tttgaattct gtgatatttt
1261 cacactggta aatgttacct ctattttaat tactgctata aataggttta tattattgat
1321 tcacttactg actttgcatt ttcgttttta aaaggatgta taaattttta cctgtttaaa
1381 taaaatttaa tttcaaatgt a
SEQ ID NO: 67 Human Cathepsin L Polypeptide, variant 6
MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNE
EGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRK
GKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLIS
LSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYS
VANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESELFYKEGIYFEPDCSSEDMDH
GVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
SEQ ID NO: 68 Human Cathepsin D Polypeptide
MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVP
AVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWIH
HKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFG
EATKQPGITFIAAKEDGILGMAYPRISVNNVLPVEDNLMQQKLVDQNIFSFYLSRDPDAQ
PGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSL
MVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQ
AGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL
SEQ ID NO: 69 Human Cathepsin E Polypeptide, Isoform 3
MKTLLLLLLVLLELGEAQGSLHRVPLRRHPSLKKKLRARSQLSEFWKSHNLDMIQFTESC
SMDQSAKEPLINYLDMEYFGTISIGSPPQNFTVIFDTGSSNLWVPSVYCTSPACKTHSRF
QPSQSSTYSQPGQSFSIQYGTGSLSGIIGADQVSAFATQVEGLTVVGQQFGESVTEPGQT
FVDAEFDGILGLGYPSLAVGGVTPVFDNMMAQNLVDLPMFSVYMSSNPEGGAGSELIFGG
YDHSHFSGSLNWVPVTKQAYWQIALDNIQVGGTVMFCSEGCQAIVDTGTSLITGPSDKIK
QLQNAIGAAPVDGEYAVECANLNVMPDVTFTINGVPYTLSPTAYTLLDFVDGMQFCSSGF
QGLDIHPPAGPLWILGDVFIRQFYSVFDRGNNRVGLAPAVP
SEQ ID NO: 70 Human Cathepsin E Polypeptide, Isoform 1
MKTLLLLLLVLLELGEAQGSLHRVPLRRHPSLKKKLRARSQLSEFWKSHNLDMIQFTESC
SMDQSAKEPLINYLDMEYFGTISIGSPPQNFTVIFDTGSSNLWVPSVYCTSPACKTHSRF
QPSQSSTYSQPGQSFSIQYGTGSLSGIIGADQVSVEGLTVVGQQFGESVTEPGQTFVDAE
FDGILGLGYPSLAVGGVTPVFDNMMAQNLVDLPMFSVYMSSNPEGGAGSELIFGGYDHSH
FSGSLNWVPVTKQAYWQIALDNIQVGGTVMFCSEGCQAIVDTGTSLITGPSDKIKQLQNA
IGAAPVDGEYAVECANLNVMPDVTFTINGVPYTLSPTAYTLLDFVDGMQFCSSGFQGLDI
HPPAGPLWILGDVFIRQFYSVFDRGNNRVGLAPAVP
SEQ ID NO: 71 Human Cathepsin E Polypeptide, Isoform 2
MKTLLLLLLVLLELGEAQGSLHRVPLRRHPSLKKKLRARSQLSEFWKSHNLDMIQFTESC
SMDQSAKEPLINYLDMEYFGTISIGSPPQNFTVIFDTGSSNLWVPSVYCTSPACKTHSRF
QPSQSSTYSQPGQSFSIQYGTGSLSGIIGADQVSVEGLTVVGQQFGESVTEPGQTFVDAE
FDGILGLGYPSLAVGGVTPVFDNMMAQNLVDLPMFSVYMSSNPEGGAGSELIFGGYDHSH
FSGSLNWVPVTKQAYWQIALDNMLWSVPTLTSCRMSPSPLTESPIPSAQLPTPYWTSWME
CSSAAVAFKDLTSTLQLGPSGSWGMSSFDSFTQSLTVGITVWDWPQQSPKEGPCVCACLS
DRP
SEQ ID NO: 72 cell permeable peptide, L803-mts
GKEAPPAPPQSP

Claims

1. A method of treating or preventing amyloidosis in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of at least one catabolic enzyme or a biologically active fragment thereof.

2. The method of claim 1, wherein the catabolic enzyme is selected from protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L.

3. The method of claim 2, wherein the catabolic enzyme is PPCA, or a biologically active fragment thereof.

4. The method of claim 3, wherein the PPCA polypeptide comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 2, 43, or 45, or a biologically active fragment thereof.

5. The method of claim 4, wherein administration of the PPCA polypeptide comprises administration of a viral vector comprising a nucleotide sequence having at least 85% identity to SEQ ID NO: 1, 42, or 44.

6.-13. (canceled)

14. The method of claim 1, wherein at least two catabolic enzymes are administered.

15. The method of claim 14, wherein the catabolic enzymes are selected from protective protein/cathepsin A (PPCA), neuraminidase 1 (NEU1), tripeptidyl peptidase 1 (TPP1), cathepsin B, cathepsin D, cathepsin E, cathepsin K, and cathepsin L.

16. The method of claim 15, wherein the catabolic enzymes are PPCA and NEU1.

17. (canceled)

18. The method of claim 1, wherein the catabolic enzyme acts to prevent the formation of and/or degrade amyloid within the lysosome.

19. The method of claim 1, wherein the catabolic enzyme is targeted to the cell lysosome.

20. The method of claim 1, wherein the catabolic enzyme acts to prevent the accumulation of and/or degrade amyloid outside the cell.

21.-24. (canceled)

25. The method of claim 1, wherein the subject is a human.

26-27. (canceled)

28. The method of claim 1, wherein the amyloidosis is light-chain (AL) amyloidosis.

29. The method of claim 28, wherein the AL amyloidosis involves one or more organs selected from the heart, the kidneys, the nervous system, and the gastrointestinal tract.

30. The method of claim 1, wherein the amyloidosis is amyloid-beta (Aβ) amyloidosis.

31. The method of claim 30, wherein the Aβ amyloidosis is associated one or more diseases selected from Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia, and inclusion body myositis.

32. The method of claim 1, further comprising the administration of one or more additional drugs for treating or preventing amyloidosis.

33. The method of claim 32, wherein the one or more additional drugs is selected from melphalan, dexamethasone, prednisone, bortezomib, lenalidomide, vincristine, doxorubicin, and cyclophosphamide.

34. The method of claim 1, further comprising the administration of one or more drugs that acidifies the lysosome.

35. The method of claim 34, wherein the drug that acidifies the lysosome is selected from an acidic nanoparticle, a catecholamine, a β-adrenergic receptor agonist, an adenosine receptor agonist, a dopamine receptor agonist, an activator of the cystic fibrosis transmembrane conductance regulator (CFTR), cyclic adenosine monophosphate (cAMP), a cAMP analog, and an inhibitor of glycogen synthase kinase-3 (GSK-3).

36.-48. (canceled)