Methods for Treating Lysosomal Storage Disorders - Patent application

Abstract

The present disclosure provides novel animal models for use in studying lysosomal storage diseases, and methods of treating lysosomal storage diseases by increasing the level or activity of TPP1. In some embodiments, the disease or disorder is selected from juvenile neuronal ceroid lipofuscinosis (CLN3) disease, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease, neuronal ceroid lipofuscinosis type 7 (CLN7) disease, northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease, late-onset neuronal ceroid lipofuscinosis (CLN12), and Kufuor-Rakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and osteopetrosis autosomal recessive 4 (OPTB4).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 362,574, filed April 6, 2022. The aforementioned application is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to methods of treating lysosomal storage diseases. [Background technology]

[0003] Neuronal ceroid lipofuscinosis (NCL) is a group of more than a dozen genetically distinct but similar lysosomal storage disorders. Characterized by the accumulation of autofluorescent storage material within lysosomes, NCL is a neurodegenerative and progressive disorder that presents with seizures, loss of vision, and ultimately loss of mental abilities. Onset is typically in childhood, and these disorders result in premature death. Two of the most frequently encountered NCL disorders are the late infantile and juvenile forms (LINCL and JNCL, respectively). LINCL results from mutations in the gene encoding the soluble lysosomal serine protease tripeptidyl peptidase 1 (TPP1, formerly known as CLN2). LINCL disease usually begins at about 4 years of age, with a lifespan of about 8-15 years. JNCL is caused by mutations in the gene encoding the lysosomal transmembrane protein CLN3. JNCL has a later onset and is more slow-progressing than LINCL, with early signs of disease (such as vision problems) occurring at approximately 8 years, and patients often surviving into their teens or twenties. Despite differences in disease timeline and genetic etiology, LINCL and JNCL share some similarities, including lysosomal accumulation of mitochondrial ATP synthase subunit c (SCMAS).

[0004] There is considerable focus on developing effective therapies for NCL disease, led by LINCL. Enzyme replacement therapy has been clinically approved for LINCL, and there is interest in gene therapy, with promising results in animal models and clinical studies. Both enzyme replacement therapy (ERT) and gene therapy rely on the fact that TPP1 is a soluble lysosomal protein. In ERT, exogenously administered recombinant protein can be taken up by a large number of cells by endocytosis and delivered to lysosomes, whereas in gene therapy, only a fraction of cells are transduced, which may overproduce and secrete the enzyme that is taken up by non-transduced cells. In addition, LINCL animal models with well-defined phenotypes that recapitulate the human disease are essential for testing therapeutic strategies.

[0005] Currently, there is no approved treatment for JNCL, which reflects several major obstacles. First, CLN3 is a transmembrane protein, which precludes replacement therapy using exogenously administered recombinant protein. There is interest in gene therapy for JNCL, but because CLN3 is an integral membrane protein, non-transduced cells do not express the deleted protein. If the underlying metabolic defect is cell-autonomous, cross-protection between transduced and non-transduced cells may not be possible, requiring a very high percentage of transduced cells for effective therapy. Second, there is a fundamental lack of understanding of the cellular function of the CLN3 protein. Although it has been implicated in numerous cellular activities, including lysosomal pH homeostasis, endocytosis, autophagy, apoptosis, lysosomal enzyme trafficking, and others, its exact function has yet to be conclusively established. As a result, a mechanism-based approach to the treatment of JNCL is currently not an option. Third, animal models of JNCL do not present a robust phenotype, especially with respect to survival. There are several mouse models of JNCL and their phenotypes are very similar, although the disease is highly attenuated compared to other mouse models of NCL disease.

[0006] Thus, there remains a need for animal models for use in studying lysosomal storage diseases and methods of treating lysosomal storage diseases. Summary of the Invention

[0007] In one aspect, the disclosure provides a method of treating a subject having a disease or disorder characterized by accumulation of SCMAS in the lysosomes of affected cells. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that increases the level or activity of TPP1 to reduce or eliminate symptoms caused by the disease or disorder.

[0008] In some embodiments, the disease or disorder is selected from juvenile neuronal ceroid lipofuscinosis (CLN3) disease, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease, neuronal ceroid lipofuscinosis type 7 (CLN7) disease, northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease, late-onset neuronal ceroid lipofuscinosis (CLN12), and Kufuor-Rakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and osteopetrosis autosomal recessive 4 (OPTB4).

[0009] In some embodiments, the disease or disorder is characterized by a deficiency in the function of the CLN3 protein.

[0010] In some embodiments, the diseased cells are neuronal cells, hi some embodiments, the diseased cells are in a tissue or organ, such as the liver, spleen, or brain.

[0011] In some embodiments, the agent reduces the level of SCMAS accumulation in the lysosomes of the affected cells. In some embodiments, the agent comprises a recombinant human TPP1 protein. In some embodiments, the TPP1 protein is an inactive proenzyme. In some embodiments, the TPP1 protein is mannose-6-phosphorylated.

[0012] In some embodiments, the therapeutically effective amount of TPP1 protein is such that the affected cells receive about 1.0 to about 100 nM of recombinant human TPP1 protein.

[0013] In some embodiments, the agent comprises a nucleic acid molecule comprising a nucleotide sequence encoding TPP1 or a variant thereof.

[0014] In some embodiments, the agent is administered by injection. In some embodiments, the injection is an intracranial injection. In some embodiments, the agent is delivered to the lysosomes of the diseased cells. In some embodiments, the agent is administered in a controlled release system.

[0015] In some embodiments, the subject is a mammal, such as a human.

[0016] In another aspect, the present disclosure provides animal models for studying diseases or disorders, such as lysosomal storage diseases. In some embodiments, the animals include (i) tripeptidyl peptidase 1 (Tpp1) gene heterozygous knockout (Tpp1 + / - ), and (ii) Cln3 gene homozygous knockout (Cln3 - / - ), where the mouse model has a shortened lifespan compared to wild-type animals. In some embodiments, the animal is a mouse.

[0017] In some embodiments, the disease or disorder is selected from late infantile ceroid lipofuscinosis (CLN2) disease, neuro-juvenile neuronal ceroid lipofuscinosis (CLN3) disease, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease, neuronal ceroid lipofuscinosis type 7 (CLN7) disease, northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease, late-onset neuronal ceroid lipofuscinosis (CLN12), and Kufuor-Rakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and osteopetrosis autosomal recessive 4 (OPTB4).

[0018] In some embodiments, the animal has at least a 25% reduction in lifespan compared to a wild-type animal.

[0019] In some embodiments, the Tpp1 gene or Cln3 gene comprises at least one mutation selected from a deletion, an insertion, a frameshift mutation, a rearrangement, or a substitution. In some embodiments, the mutation is constitutive. In some embodiments, the mutation is conditional.

[0020] In some embodiments, the Tpp1 gene is located at Chr 7 E3;7 55.97 cM. In some embodiments, the Tpp1 gene comprises a deletion of at least a portion of an exon in the Tpp1 gene. In some embodiments, the Tpp1 gene comprises an insertion of neo into intron 11 and an Arg446His missense mutation into exon 11 immediately upstream of the neo insertion.

[0021] In some embodiments, the Cln3 gene is located at Chr 7 F3;7 69.16 cM. In some embodiments, the Cln3 gene comprises a deletion of at least a portion of an exon in the Cln3 gene. In some embodiments, the Cln3 gene comprises a deletion of all or a portion of exons 1-6 in the Cln3 gene.

[0022] In some embodiments, the animal model has increased levels of lysosomal accumulation of mitochondrial ATP synthase subunit c (SCMAS). In some embodiments, the animal model has at least a 50% increase in the level of lysosomal accumulation of SCMAS.

[0023] In some embodiments, the animal model has increased expression levels of Niemann-Pick type C1 (NPC1) and / or Cathepsin F (CTSF). In some embodiments, the animal model has at least a 40% increase in expression levels of NPC1 and / or CTSF, or a 40% decrease in expression levels of acid sphingomyelinase (SMPD1).

[0024] In some embodiments, the animal model is characterized by a loss of locomotor activity.

[0025] Also within the scope of the disclosure are progeny of the animal models disclosed herein, and cells, tissues, or cell lines derived from the animal models disclosed herein or the progeny.

[0026] In another aspect, the present disclosure also provides a method for obtaining the animal model disclosed above. The method includes: (a) crossing an animal with a Tpp1 knockout with a second animal with a Cln3 knockout to produce a double heterozygous (Tpp1 + / - ;Cln3 + / - (b) obtaining an animal having Tpp1 - / - ;Cln3 - / - ×Tpp1 - / - ;Cln3 - / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - and crossing the animals with the double heterozygotes by mating the animals with the double heterozygotes.

[0027] In another aspect, the disclosure further provides a method of identifying an agent for use in treating a disease or disorder of a subject. In some embodiments, the method comprises administering a candidate agent to an animal model or progeny as disclosed herein and evaluating the effect of the candidate agent on the phenotype of the animal model. In some embodiments, the method comprises contacting a cell, tissue, or cell line with a candidate agent as disclosed herein and evaluating the effect of the candidate agent on the cell, tissue, or cell line.

[0028] In some embodiments, the disease or disorder is characterized by accumulation of SCMAS in lysosomes of affected cells (eg, neuronal cells).

[0029] In some embodiments, the disease or disorder is selected from late infantile ceroid lipofuscinosis (CLN2) disease, neuro-juvenile neuronal ceroid lipofuscinosis (CLN3) disease, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease, neuronal ceroid lipofuscinosis type 7 (CLN7) disease, northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease, late-onset neuronal ceroid lipofuscinosis (CLN12), and Kufuor-Rakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and osteopetrosis autosomal recessive 4 (OPTB4).

[0030] In some embodiments, the phenotype is lifespan in an animal model.

[0031] In some embodiments, the effect is characterized by an increase in lifespan in an animal model. In some embodiments, the effect is characterized by a decrease in the level of lysosomal accumulation of SCMAS. In some embodiments, the effect is characterized by a decrease in the expression levels of NPC1 and / or CTSF.

[0032] In some embodiments, the candidate agents include proteins, peptides, peptidomimetics, nucleic acids, or small molecules.

[0033] The foregoing summary is not intended to define all aspects of the disclosure, and additional aspects are described in other sections, such as the following detailed description. It should be understood that the entire document is intended to relate as a unified disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the present specification. Other features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are given by way of example only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0034] [Figure 1] Results of survival analysis of mice in a Tpp1- / - background are shown. Data were obtained from approximately equal numbers of male and female littermates from crosses between Tpp1 and Cln3 mutants. Previous data for Tpp1- / -;Cln3+ / + are included for animals on a mixed C57BL / 6N, C57BL / 6J genetic background. Survival was not significantly different from littermates of the same genotype from crosses between Tpp1 and Cln3 mutants (Log-rank P=0.8475. Median survival and number of animals analyzed per genotype are shown in Table 1. [Diagram 2]Results of survival analysis of mice in Tpp1+ / - or Tpp1+ / + backgrounds are shown. Figure 2A shows a comparison of Tpp1Cln3 mutant mice. Data were obtained from an equal number of male and female animals. Median survival times and the number of animals analyzed per genotype are shown in Table 1. The Yuan survival data set (Yuan R, et al. (2012) Proc Natl Acad Sci USA 109:8224-9) was obtained from Jackson Laboratories (https: / / phenome.jax.org / projects / Yuan2) and consisted of male and female mice in a C57BL / 6J substrain background. Figure 2B shows a comparison of survival times of Tpp1+ / +;Cln3- / - animals from this study with historical data of wild-type and Tpp1+ / +;Cln3- / - animals. [Diagram 3] Similar levels of astrocytes in Tpp1- / -Cln3+ / + and Tpp1- / -Cln3- / - animals are shown. Immunofluorescence staining for the astrocyte marker glial fibrillary associated protein (GFAP) revealed extensive astrocytes in the primary somatosensory cortex (S1BF) and ventral posterior thalamic nucleus (VPM / VPL) of both Tpp1- / -Cln3+ / + and Tpp1- / -Cln3- / - animals, compared with relatively low levels of GFAP immunoreactivity in the gray matter of Tpp1+ / -Cln3- / - and Tpp1+ / -Cln3+ / -. This was comparable to previously published data from wild-type mice, where GFAP staining was mainly restricted to the white matter. Insets show the corresponding astrocytic hypertrophy in Tpp1- / -Cln3+ / + and Tpp1- / -Cln3- / - mice. Scale bars in insets = 100 μm and 25 μm. [Figure 4]Similar levels of microglial activation in Tpp1- / -Cln3+ / + and Tpp1- / -Cln3- / - animals are shown. Immunofluorescence staining for the microglial marker CD68 shows greater microglial activation in the primary somatosensory cortex (S1BF) and ventral posterior thalamic nucleus (VPM / VPL) of both Tpp1+ / +Cln3+ / + and Tpp1- / -Cln3- / - animals compared to relatively low levels of CD68 immunoreactivity in Tpp1+ / -Cln3- / - and Tpp1+ / -Cln3+ / -, which is comparable to previously published data in wild-type mice. Insets show corresponding microglial hypertrophy in Tpp1- / -Cln3+ / + and Tpp1- / -Cln3- / - mice. Scale bars in insets = 100 μm and 25 μm. [Diagram 5] Results of quantitative analysis of glial activation are shown. Thresholded image analysis confirms different levels of glial fibrillary associated protein (GFAP, astrocytes) and CD68 (microglia) in the primary somatosensory cortex (S1BF) and ventral posterior thalamic nucleus (VPM / VPL) of animals of different genotypes. These data confirm significantly elevated levels of both antigens in Tpp1- / -Cln3+ / + and Tpp1- / -Cln3- / - mice compared to animals of other genotypes that showed very low levels of immunoreactivity for these markers. Asterisks indicate adjusted p-values ​​<0.05 when compared to Tpp1+ / +;Cln3+ / - by one-way ANOVA with Dunnett's test for multiple comparisons. [Figure 6]Relative protein expression in Tpp1 and Cln3 mutant mice as determined by SPS-MS3 quantification of isobarically labeled peptides is shown. Q values ​​were calculated based on peptide data for each protein using the false discovery rate two-step step-up method of Benjamini, Krieger, and Yekutieli. Dashed lines indicate 1% FDR (y-axis) and an arbitrary fold change of 2 (x-axis). Axes are truncated at a Q value of 1E-10 and a ratio of 1 / 16 to 16. Red and black symbols indicate known lysosomal (based on (17)) and other proteins, respectively. Names of selected proteins of interest are shown in red (lysosomal) or black (others). [Figure 7] Correlation between significant proteomic changes in animals of different genotypes is shown. Figure 7A shows protein levels of Tpp1- / -;Cln3- / - compared to those of Tpp1- / -;-Cln3+ / +. Goodness of fit, R=0.7901. NNT is truncated (see Examples). Figure 7B shows protein levels of Tpp+ / +;Cln3- / - compared to Tpp1+ / -;Cln3- / -. Goodness of fit, R=0.5752). Compared to wild type, the proteins shown are significantly changed in at least one model (FDR1%), have a magnitude of change of 1.5-fold or greater in at least one model, and have a consistent direction of change in both models. The names of selected proteins of interest are shown in red (lysosomal) or black (others). Filled symbols indicate proteins that are significantly changed in both genotypes compared, whereas unfilled symbols indicate proteins that are significantly changed in one of the two genotypes compared. [Figure 8] Figure 1 shows TPP1 activity in mutant mice. Activity measured in each genotype was compared to wild type using Dunnett's multiple comparison test. ns: not significant; **, p<0.01; ***, P<0.001. [Figure 9]Figure 9 shows TPP1 expression in mice expressing TgTPP1+. Figure 9A shows that the transgene TgTPP1+ is inserted into ROSA26 and uses a synthetic intron to drive the expression of mouse TPP1 from the chicken actin (CAG) promoter. Figure 9B shows the expression of TPP1 activity driven by TgTPP1+. Figure 9C shows the effect of TgTPP1+ on survival time. Hemizygous mouse strains containing this transgene are called R26TgTPP1+ / 0, and mice lacking the TPP1 transgene are called R26Tg0 / 0. Double-labeled immunofluorescence images show TPP1 and the neuronal marker NeuN (Figure 9D) or the microglial marker Iba1 (Figure 9E), as well as Hoescht stained nuclei. [Figure 10] Shown is SCMAS staining in the liver. [Figure 11] Shown is SCMAS staining in the spleen. [Figure 12] Shown is SCMAS staining in the brain. [Figure 13] Quantification of SCMAS accumulation is shown. The area of ​​images occluded by SCMAS-containing inclusions was quantified in a genotype-blind analysis using ImageJ. Data were analyzed using a non-paired one-way ANOVA with the mean of each sample compared to the mean of all other samples using Tukey's test to correct for multiple comparisons. Note that although all possible comparisons were tested, only pairwise comparison bars for Tg-Cln3- / - and Tg+Cln3- / - are displayed. ****. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure provides methods for treating lysosomal storage diseases by increasing the level or activity of tripeptidyl peptidase 1 (TPP1). The present disclosure also provides novel animal models for use in studying lysosomal storage diseases. In particular, Tpp1 + / - ;Cln3 - / -Animal models with double knockouts have shorter survival times compared to wild-type animals and are useful for developing therapies for lysosomal storage diseases such as juvenile neuronal ceroid lipofuscinosis (JNCL) using survival time as an endpoint.

[0036] Methods for Treating Lysosomal Storage Disorders - Patent application In one aspect, the disclosure provides a method of treating a subject having a disease or disorder characterized by accumulation of SCMAS in lysosomes of affected cells (e.g., neuronal cells). In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that increases the level or activity of TPP1 to reduce or eliminate symptoms caused by the disease or disorder.

[0037] In some embodiments, the methods involve selecting a subject with increased levels of accumulation of SCMAS in lysosomes of diseased cells compared to a reference level.

[0038] In some embodiments, the method includes: (a) obtaining a sample containing diseased cells; (b) performing an assay on the sample to determine a level of accumulation of SCMAS in lysosomes of the diseased cells; (c) identifying the subject as likely to benefit from treatment with an agent that increases a level or activity of TPP1 if the subject has an increased level of accumulation of SCMAS in lysosomes of the diseased cells compared to a reference level; and (d) administering a therapeutically effective amount of the agent to the subject to reduce or eliminate symptoms caused by the disease or disorder.

[0039] In some embodiments, the disease or disorder is characterized by the accumulation of one or more storage products (e.g., SCMAS) in the lysosomes of affected cells, such as neurons. One way to determine the disorder is to find that the lysosomes have accumulated storage materials, which can be done by known methods such as microscopy or immunofluorescence. An example of a storage material that may be detected in lysosomes is SCMAS. In some embodiments, treatment with TPP1 protein reduces or eliminates mitochondrial ATP synthase, particularly SCMAS in the lysosomes of affected cells, such as neurons. Detecting the removal of storage materials, such as mitochondrial SCMAS, in the lysosomes of affected cells can be done by known methods such as those described above.

[0040] In some embodiments, the disease or disorder is selected from the group consisting of late infantile neuronal ceroid lipofuscinosis (CLN2) caused by mutations in the Tpp1 gene, juvenile neuronal ceroid lipofuscinosis (CLN3) caused by mutations in the Cln3 gene, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease caused by mutations in the Cln5 gene, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease caused by mutations in the Cln6 gene, neuronal ceroid lipofuscinosis type 7 (CLN7) disease caused by mutations in the MFSD8 gene, Northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease caused by mutations in the Cln8 gene, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease caused by mutations in the CTSD gene, late-onset neuronal ceroid lipofuscinosis (CLN12) and Kufour-Rakeb syndrome caused by mutations in the gene ATP13A2 I, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) caused by mutations in the GNS gene, osteopetrosis autosomal recessive 4 (OPTB4) caused by mutations in the CLCN7 gene.

[0041] In some embodiments, the disease or disorder is characterized by a deficiency in the function of the CLN3 protein. An example of such a disorder is JNCL.

[0042] In some embodiments, the diseased cells may belong to any cell or tissue type, such as neurons. In some embodiments, the diseased cells are neural cells. In some embodiments, the diseased cells are in a tissue or organ, such as the liver, spleen, or brain.

[0043] The level or activity of TPP1 can be measured by determining or estimating protein level or mRNA level. Methods for determining or estimating protein level or mRNA level are well known in the art. Such methods can include enzyme activity assay, microscopy, immunofluorescence, and nucleic acid hybridization (e.g., using proteins and nucleic acids described in US Ser. No. 08 / 931,608 and Sleat et al. (1997)). For example, the protein level (e.g., protein expression level) of TPP1 can be determined by SDS-PAGE, Western blot, or immunoassay (e.g., immunoblotting assay, immunoprecipitation assay). The mRNA level can be determined by RT-PCR.

[0044] In some embodiments, the reference level may be obtained from a subject prior to administration of an agent for increasing the level or activity of TPP1 or a composition thereof. In some embodiments, the reference level may be obtained from a control subject or a group of individuals who do not have a disease or disorder or have not been diagnosed with a disease or disorder. In some embodiments, the reference level is obtained based on, for example, the average level of TPP1 level or activity in a population not affected by a disease or disorder. In some embodiments, the reference level is obtained based on the median or median level of a set of individuals including patients with a disease or disorder.

[0045] In some embodiments, the agent reduces the accumulation level of SCMAS in the lysosomes of the diseased cells. In some embodiments, the diseased cells are in a tissue or organ, such as the liver, spleen, or brain. In some embodiments, the agent reduces the accumulation level of SCMAS in the lysosomes of the liver, spleen, and / or brain.

[0046] In some embodiments, the agent comprises a protein, peptide, peptidomimetic, nucleic acid, or small molecule.

[0047] In some embodiments, the agent comprises a recombinant human TPP1 protein. In some embodiments, the TPP1 protein is an inactive proenzyme. In some embodiments, the TPP1 protein is mannose-6-phosphorylated.

[0048] In some embodiments, the agent is a TPP1 protein or a variant thereof. In some embodiments, the TPP1 protein comprises an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 99%) sequence identity to the amino acid sequence of SEQ ID NO:1, or comprises the amino acid sequence of SEQ ID NO:1.

[0049] In some embodiments, the TPP1 protein is encoded by a nucleotide sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 99%) sequence identity to the nucleotide sequence of SEQ ID NO:2 or is encoded by the nucleotide sequence of SEQ ID NO:2. SEQ ID NO:1 MGLQACLLGLFALILSGKCSYSPEPDQRRTLPPGWVSLGRADPEEELSLTFALRQQNVERLSELVQAVSDPSSPQYGKYLTLENVADLVRPSPLTLHTVQKWLLAAGAQKCHSVITQDFLTCWLSIRQAELLLPGAEFHH YVGGPTETHVVRSPHPPYQLPQALAPHVDFVGGLHRFPPTSSLRQRPEPQVTGTVGLHLGVTPSVIRKRYNLTSQDVGSGTSNNSQACAQFLEQYFHDSDLAQFMRLFGGNFAHQASVARVVGQQGRGRAGIEASLDVQYLM SAGANISTVVYSSPGRHEGQEPFLQWLMLLSNESALPHVHTVSYGDDEDSLSSAYIQRVNTELMKAARGLTLLFASGDSGAGCWSVSGRHQFRPTFPASSPYVTTVGGTSFQEPFLITNEIVDYISGGGFSNVFPRPSYQ EEAVTKFLSSSPHLPPSSYFNASGRAYPDVAALSDGYWVVSNRVPIPWVSGTSASTPVFGGILSLINEHRILSGRPPLGFNLNPRLYQQHGAGLFDVTRGCHESCLDEEVEGQGFCSGPGWDPVTGWGTPNFPALLKTLLNP கார்க்கை ATGGGACTCCAAGCCTGCCTCCTAGGGCTCTTTGCCCTCATCCTCTCTGGGCAAATGCAGTTACAGCCCGG AGCCCGACCAGCGGAGGACGCTGCCCCCAGGCTGGGTTCCCTGGGCCGTGCGGACCCTGAGGAAGAGCT GAGTCTCACCTTTGCCCTGAGACAGCAGAATGTGGAAAGACTCTCGGAGCTGGTGCAGGCTGTGTCGGAT CCCAGCTCTCCTCAATCGGAAAATACCTGACCCTAGAGAATGTGGCTGATCTGGTGAGGCCATCCCCAC TGACCCTCCAACGGTGCAAAAATGGCTCTTGGCAGCCGGAGCCCAGAAGTGCCATTCTGTGATCACACA GGACTTTCTGACTTGCTGGCTGAGCATCCGACAAGCAGAGCTGCTGCTCCCTGGGGCTGAGTTTCATCAC TATGTGGGAGGACCTACGGAAACCCATGTTGTAAGGTCCCCACATCCCTACCAGCTTCCACAGGCCTTGG CCCCCCATGTGGACTTTGTGGGGGGACTGCACCGTTTTCCCCCAACATCATCCCTGAGGCAACGTCCTGA GCCGCAGGTGACAGGGACTGTAGGCCTGCATCTGGGGGTAACCCCCTCTGTGATCCGTAAGCGATACAAC TTGACCTCACAAGACGTGGGCTCTGGCACCAGCAATAACAGCCAAGCCTGTGCCCAGTTCCTGGAGCAGT ATTTCCATGACTCAGACCTGGCTCAGTTCATGCGCCTCTTCGGTGGCAACTTTGCACATCAGGCATCAGT AGCCCGTGTGGTTGGACAACAGGGCCGGGGCCGGGCCGGGATTGAGGCCAGTCTAGATGTGCAGTACCTG ATGAGTGCTGGTGCCAACATCTCCACCTGGGTCTACAGTAGCCCTGGCCGGCATGAGGGACAGGAGCCCT TCCTGCAGTGGCTCATGCTGCTCAGTAATGAGTCAGCCCTGCCACATGTGCATACTGTGAGCTATGGAGA TGATGAGGACTCCCTCAGCAGCGCCTACATCCAGCGGGTCAACACTGAGCTCATGAAGGCTGCCGCTCGG GGTCTCACCCTGCTCTTCGCCTCAGGTGACAGTGGGGCCGGGTGTTGGTCTGTCTCTGGAAGACACCAGT TCCGCCCTACCTTCCCTGCCTCCAGCCCCTATGTCACCACAGTGGGAGGCACATCCTTCCAGGAACCTTT CCTCATCACAAATGAAATTGTTGACTATATCAGTGGTGGGTGGCTTCAGCAATGGTTCCCACGGCCTTCA TACCAGGAGGAAGCTGTAACGAAGTTCCTGAGCTCTAGCCCCCACCTGCCACCATCCAGTTACTTCAATG CCAGTGGCCGTGCCTACCCAGATGTGGCTGCACTTTCTGATGGCTACTGGGTGGTCAGCAACAGAGTGCC CATTCCATGGGTGTCCGGAACCTCGGCCTCTACTCCAGTGTTTGGGGGGATCCTATCCTTGATCAATGAG CACAGGATCCTTAGTGGCCGCCCCCCTCTTGGCTTTCTCAACCCAAGGCTCTACCAGCAGCATGGGGCAG GACTCTTTGATGTAACCCGTGGCTGCCATGAGTCCTGTCTGGATGAAGAGGTAGAGGGCCAGGGTTTCTG CTCTGGTCCTGGCTGGGATCCTGTAACAGGCTGGGGAACACCCAACTTCCCAGCTTTGCTGAAGACTCTA CTCAACCCCTGA

[0050] As used herein, the term "variant" refers to a first molecule relative to a second molecule (also called a "parent" molecule). A variant molecule may be derived from, isolated from, based on, or homologous to a parent molecule. For example, variant forms of TPP1, including TPP1 mutants with cysteine ​​substitutions, are mutants of wild-type TPP1. The term variant can be used to describe either a polynucleotide or a polypeptide.

[0051] When applied to proteins, a variant polypeptide can have full amino acid sequence identity with the original parent polypeptide, or can have less than 100% amino acid identity with the parent protein. For example, an amino acid sequence variant can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in amino acid sequence compared to the original amino acid sequence. Polypeptide variants include polypeptides that include the entire parent polypeptide, and further include additional fused amino acid sequences. Polypeptide variants also include polypeptides that are portions or subsequences of the parent polypeptide. For example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques) of the polypeptides disclosed herein are also encompassed by the present invention.

[0052] In another aspect, a polypeptide variant includes a polypeptide that contains small, trivial, or insignificant changes to the parent amino acid sequence. For example, small, trivial, or insignificant changes result in a functionally identical polypeptide, including amino acid changes (including substitutions, deletions, and insertions) that have little or no effect on the biological activity of the polypeptide, and the addition of non-functional peptide sequences. In another aspect, a variant polypeptide of the invention alters the biological activity of the parent molecule. One of skill in the art will appreciate that many variants of the disclosed polypeptides are encompassed by the invention.

[0053] In some aspects, polynucleotide or polypeptide variants of the invention may include variant molecules in which only a small percentage of the nucleotide or amino acid positions are modified, added or deleted, e.g., typically less than about 10%, less than about 5%, less than 4%, less than 2%, or less than 1%.

[0054] A "functional variant" of a protein, as used herein, refers to a variant of such a protein that retains at least some of the activity of that protein. Functional variants can include mutations, including polymorphisms and the like, which can be insertion, deletion, or substitution variants. Functional variants also include fusion products of such a protein with another, usually unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants can be naturally occurring or artificial.

[0055] In some embodiments, the TPP1 variant may include one or more conservative modifications. The TPP1 variant with one or more conservative modifications may retain the desired functional properties, which can be tested using functional assays known in the art. As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and include one or more conservative mutations. Cas proteins with one or more conservative modifications may retain desired functional properties, which can be tested using functional assays known in the art. As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding properties of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0056] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). Comparison of sequences and determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm, as described in the non-limiting examples below.

[0057] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0058] Additionally or alternatively, the protein sequences of the present invention can be further used as a "query sequence" to perform searches against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

[0059] In some embodiments, the TPP1 variant may be conjugated or linked to a detectable tag or detectable marker (e.g., radionuclide, fluorescent dye). In some embodiments, the detectable tag may be an affinity tag. The term "affinity tag" as used herein refers to a moiety attached to a polypeptide that allows the polypeptide to be purified from a biochemical mixture. The affinity tag may consist of an amino acid sequence or may comprise an amino acid sequence to which a chemical group is attached by post-translational modification. Non-limiting examples of affinity tags include His-tag, CBP-tag (CBP: calmodulin binding protein), CYD-tag (CYD: covalent but dissociable NorpD peptide), Strep-tag, StrepII-tag, FLAG-tag, HPC-tag (HPC: heavy chain of protein C), GST-tag (GST: glutathione S-transferase), Avi-tag, biotinylated tag, Myc-tag, 3xFLAG-tag, SUMO-tag, and MBP-tag (MBP: maltose-binding protein). Further examples of affinity tags can be found in Kimple et al., Curr Protoc Protein Sci. 2013 Sep 24;73:Unit 9.9.

[0060] In some embodiments, the detectable tag may be conjugated or linked to the N-terminus and / or C-terminus of the TPP1 variant. The detectable tag and the affinity tag may also be separated by one or more amino acids. In some embodiments, the detectable tag may be conjugated or linked to the TPP1 variant via a cleavable element. In the context of the present invention, the term "cleavable element" relates to a peptide sequence that is susceptible to cleavage by chemical or enzymatic means such as a protease. The protease may be sequence specific (e.g. thrombin) or have limited sequence specificity (e.g. trypsin). Cleavable elements I and II may also be included in the amino acid sequence of the detection tag or polypeptide, especially when the last amino acid of the detection tag or polypeptide is K or R.

[0061] As used herein, the term "conjugate" or "conjugation" or "linked" as used herein refers to the joining of two or more entities to form one entity. Conjugates encompass both peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0062] The term "fusion polypeptide" or "fusion protein" refers to a protein produced by linking two or more polypeptide sequences together. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct that links a nucleic acid sequence encoding a first polypeptide with a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by a peptide bond or through several peptides. A fusion protein may contain a peptide linker between the two domains.

[0063] In some embodiments, the agent comprises a fusion protein comprising TPP1 or a variant thereof.

[0064] In some embodiments, the agent comprises a nucleic acid (e.g., DNA, RNA) having a polynucleotide sequence encoding a TPP1 protein or variant thereof. In some embodiments, the polynucleotide sequence is RNA. In some embodiments, the agent comprises a vector having a polynucleotide sequence encoding a TPP1 protein or variant thereof.

[0065] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and induce expression of a particular gene with which it is operatively associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as a vector integrated into the genome of a host cell into which it is introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding TPP1 or a variant thereof.

[0066] The vector may comprise a polynucleotide encoding an RNA (e.g., RNAi, ribozyme, miRNA, siRNA) that, when transcribed from the vector polynucleotide, results in the accumulation of the chimeric protein on the plasma membrane of the target cell. Vectors that may be used include, but are not limited to, lentivirus vectors, HSV vectors, and adenovirus vectors. Lentiviruses include, but are not limited to, HIV-1, HIV-2, SIV, FIV, and EIAV. Lentiviruses may be pseudotyped with envelope proteins of other viruses, including, but not limited to, VSV, rabies, Mo-MLV, baculovirus, and Ebola. Such vectors may be prepared using standard methods in the art.

[0067] In some embodiments, the vector is a recombinant AAV vector. AAV vectors are DNA viruses of relatively small size that can integrate in a stable and site-specific manner into the genome of the cells they infect. They can infect a wide range of cells without inducing any effect on cell growth, morphology or differentiation, and do not appear to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. It encompasses about 4700 bases and contains an inverted terminal repeat (ITR) region of about 145 bases at each end, which serves as the origin of viral replication. The remainder of the genome is divided into two essential regions responsible for encapsidation functions: the left part of the genome contains the rep gene, which is involved in viral replication and expression of viral genes, and the right part of the genome contains the cap gene, which codes for the viral capsid protein.

[0068] For example, the application of AAV as a vector for gene therapy has been rapidly developed in recent years. Wild-type AAV can infect dividing or non-dividing cells or tissues of mammals, including humans, with relatively high titers, and can also integrate into human cells at a specific site (on the long arm of chromosome 19) (see Kotin, RM, et al., Proc. Natl. Acad. Sci. USA 87:2211-2215, 1990) (Samulski, RJ, et al., EMBO J. 10:3941-3950, 1991, the disclosure of which is incorporated herein by reference in its entirety). AAV vectors that do not contain the rep and cap genes lose the specificity of site-specific integration but can still mediate long-term stable expression of exogenous genes. AAV vectors exist in cells in two forms: one is episomal outside the chromosome, and the other is integrated into the chromosome, with the former being the predominant form. Furthermore, AAV has not been linked to any human disease so far. No association has been found, and no changes in biological properties resulting from integration have been observed. Sixteen AAV serotypes, designated AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, and their recombinant variants, have been reported in the literature, with AAV5 originally isolated from humans (Bantel-Schaal, and H. zur Hausen 1984. Virology 134:52-63), while AAV1-4 and AAV6 are all found in the study of adenoviruses (Ursula Bantel-Schaal, Hajo Delius, and Harald zur Hausen J. Virol. 1999, 73:939-947).

[0069] AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable (see, for example, Blacklow, "Parvoviruses and Human Disease" JR Pattison, ed. (1988) pp. 165-174; Rose, Comprehensive Virology 3:1, 1974; P. Tattersall "The Evolution of Parvovirus Taxonomy" In Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski "The Genus Dependovirus" (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p15-23, Hudder Arnold, London, UK (2007). See, Arnold, London, UK (2006), the disclosures of which are incorporated herein by reference in their entirety. Methods for purifying vectors are described, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, as well as in "Methods for Generating High Titer Helper-free Preparation of Recombinant AAV The preparation of hybrid vectors can be found in WO / 1999 / 011764, entitled "AAV-Based Vectors," the disclosure of which is incorporated herein by reference in its entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors to transfer genes in vitro and in vivo has been described (see, e.g., International Patent Application Publication Nos. WO 91 / 18088 and WO 93 / 09239, U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entireties).These publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced by a gene of interest, and the use of these constructs to transfer the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). Replication-defective recombinant AAV can be prepared by co-introducing a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV packaging genes (rep and cap genes), into a cell line infected with a human helper virus (e.g., adenovirus). The resulting AAV recombinants are then purified by standard techniques.

[0070] In some embodiments, the vectors may be encapsidated into viral particles (e.g., AAV viral particles, including but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, also provided are recombinant viral particles (which contain a recombinant polynucleotide and are therefore recombinant) that comprise any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0071] In some embodiments, the viral vector comprises an AAV vector, a lentiviral vector, an adenoviral vector, or a non-viral plasmid vector. In some embodiments, the adeno-associated viral vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh74, and recombinant subtypes thereof.

[0072] In some embodiments, vectors may be derived from retroviruses, including avian retinal endotheliosis viruses (Duck infectious anemia virus, Spleen necrosis virus, Twiehaus strain reticuloendotheliosis virus, Type C retroviruses, Reticuloendotheliosis virus Hungary-2 (REV-H-2)), and feline leukemia virus (FeLV). Retroviral genomes have been modified for use as vectors (Cone & Mulligan, Proc. Natl. Acad. Sci. (USA, 81:6349-6353, (1984)). Non-limiting examples of retroviruses include lentiviruses, such as human immunodeficiency viruses (HIV-1 and HIV-2), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), Maedi / Visna virus, Caprine arthritis / encephalitis virus, equine infectious anemia virus (EIAV), and bovine immunodeficiency virus (BIV); avian type C retroviruses, such as avian leukemia virus (ALV); bovine leukemia virus (BLV), human T-cell lymphoma virus (HCV ... mammalian type B retroviruses, such as mouse mammary tumor virus (MMTV); mammalian type C retroviruses, such as murine leukemia virus (MLV), feline sarcoma virus (FeSV), murine sarcoma virus, gibbon ape leukemia virus, guinea pig type C virus, equine type C virus, woolly monkey sarcoma virus, and viper retrovirus; spumaviruses (formerly foamy viruses), such as human spumavirus (HSRV), feline syncytium-forming virus (FeSFV), human foamy virus, simian foamy virus, and equine syncytial virus, and type D retroviruses, such as Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus, and langur monkey virus.

[0073] In some embodiments, the vector comprises a retroviral vector or a lentiviral vector. In some embodiments, lentiviral and retroviral vectors may be packaged using their native envelope proteins or may be modified to be encapsulated with heterologous envelope proteins. Examples of envelope proteins include, but are not limited to, amphotropic envelopes, ecotropic envelopes, or xenotropic envelopes, or may be envelopes that include amphotropic and ecotropic portions. The protein may also be any of the retroviral and lentiviral proteins listed above. Alternatively, the env protein may be a modified env construct, a synthetic or chimeric env construct, or may be obtained from a non-retrovirus, such as vesicular stomatitis virus and HVJ virus. Specific non-limiting examples include Moloney murine leukemia virus (MMLV), Rous sarcoma virus, baculovirus, Jaagsiekte ovine retrovirus (JSRV) envelope proteins, and the envelope of feline endogenous virus RD114, gibbon leukemia virus (GALV) envelope, baboon endogenous virus (BaEV) envelope, simian sarcoma-associated virus (SSAV) envelope, amphoteric murine leukemia virus (MLV-A) envelope, human immunodeficiency virus envelope, avian leukosis virus envelope, endogenous xenotropic NZB virus envelope, and envelopes of the paramyxovirus family, such as, but not limited to, the HVJ virus envelope.

[0074] Once the DNA sequence comprising the expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can be modified or optimized based on this description depending on the specific expression vector and mammalian host cell used.

[0075] In some embodiments, the agent is a TPP1 agonist. The term "agonist" refers to a compound that causes agonism of the TPP1 pathway and induces a response in a cell. A TPP1 agonist mimics the action of an endogenous ligand (TPP1) and produces a physiological response similar to that provided by the endogenous ligand. This term includes agents (e.g., TPP1 variants / fragments, fusion proteins containing TPP1 that, when administered to a subject in need of TPP1, cause upregulation and / or increase in the activity of TPP1-mediated signaling pathways. In some embodiments, this term includes agents that, when administered to a subject, cause a decrease in the number or activity of T cells.

[0076] The TPP1 protein may be in an inactive proenzyme (or prodrug) form or in a shorter active form. Either of these may be naturally isolated or recombinant. For example, when produced in Chinese Hamster Ovary (CHO) cells, the TPP1 protein is obtained in its proenzyme form. This form is converted to the active form after acidification. Thus, the proenzyme is a very suitable prodrug that remains inactive until delivered to the lysosome where the acidic environment activates it. Obtaining the TPP1 protein in either of these forms is described in detail below and in US Ser. No. 08 / 931,608 and Sleat et al. (1997). Briefly, the TPP1 protein can be isolated using known methods from human brain samples by purifying mannose-6-phosphate containing glycoproteins from normal JNCL or LINCL brain samples and isolating the protein bands that are normally present but not present in the JNCL or LINCL specimens. Once the protein is obtained, the corresponding gene and cDNA are also isolated using known methods. Recombinant proteins are then produced from the cDNA using known methods. Any of the above forms of TPP1 protein may or may not be mannose-6-phosphorylated.

[0077] In some embodiments, the agent may include an agonist of peroxisome proliferator-activated receptor alpha (PPARα) or a stereoisomer thereof, a derivative thereof, an analog thereof, a prodrug thereof, or a pharma- ceutically acceptable salt thereof.

[0078] As used herein, "PPARα agonist" refers to a compound or composition that, when combined directly or indirectly with PPARα (preferably by direct binding to PPARα), stimulates or increases an in vivo or in vitro response typical of the receptor, e.g., transcriptional regulatory activity, as measured by assays known to those of skill in the art, including those described in U.S. Pat. Nos. 4,981,784, 5,071,773, 5,298,429, 5,506,102, W089 / 05355, W091 / 06677, W092 / 05447, W093 / 11235, W093 / 23431, W094 / 23068, W095 / 18380, CA2,034,220, and Lehmann et al. These include, but are not limited to, the "cotransfection" or "cis-trans" assays described in W. et al., J. Biol. Chm. 270:12953-12956 (1995). PPARα agonists can also be identified according to the assays described in U.S. Patent No. 6,008,239.

[0079] In some embodiments, the PPARα agonist can include a fibrate compound, including but not limited to gemfibrozil, fenofibrate, bezafibrate, clofibrate, ciprofibrate, and analogues, derivatives, and pharmaceutically acceptable salts thereof. The PPARα compounds disclosed in Tontonez et al., Cell 79:1147-1156 (1994), Lehmann et al., J. Biol. Chem. 270(22):1-4, 1995, Amri et al., J. Lipid Res. 32:14491456 (1991), Kliewer et al., Proc. Natl. Acad. Sci. USA 94:4318-4323 (1997), Amri et al., J. Lipid Res. 32:1457-1463, (1991) and Grimaldi et al., Proc. Natl. Acad. Sci. USA 89:10930-10934 (1992) are incorporated herein by reference. The PPARα agonist compounds described in U.S. Patent No. 6,008,239, WO97 / 27847, WO97 / 27857, WO97 / 28115, WO97 / 28137 and WO97 / 28149 are further incorporated herein by reference. Certain fibrate compounds described in WO92 / 10468 and WO01 / 80852 are also incorporated herein by reference.

[0080] "Isomers" are different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the way the atoms are arranged in space, i.e., have different stereochemical configurations. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. The term "(±)" is used to designate racemic mixtures when appropriate. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is specified according to the Cahn-Ingold-Prelog RS system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by either (R) or (S). Resolved compounds of unknown absolute configuration can be specified as (+) or (-) depending on the direction (right-handed or left-handed) they rotate plane-polarized light at the wavelength of the sodium D line. Certain compounds of the compounds described herein contain one or more asymmetric centers and therefore may give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R) or (S). The chemical moieties, pharmaceutical compositions, and methods of the present invention are meant to include all such possible isomers, including racemic mixtures, optically pure forms, and intermediate mixtures. Optically active (R) and (S) isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0081] Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer" means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms. "R" and "S" represent the configuration of the substituents around one or more chiral carbon atoms. Thus, "R*" and "S*" indicate the relative configuration of the substituents around one or more chiral carbon atoms. The symbol "*" in a structural formula represents the presence of a chiral carbon center.

[0082] A "racemate" or "racemic mixture" means a compound of equimolar amounts of two enantiomers; such a mixture exhibits no optical activity, i.e., does not rotate the plane of polarized light.

[0083] "Geometric isomer" means an isomer that differs in the orientation of substituent atoms in relationship to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. The atoms (other than H) on each side of the carbon-carbon double bond can be in the E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side) configuration. "R", "S", "St", "R*", "E", "Z", "cis", and "trans" denote configurations relative to the core molecule.

[0084] As used herein, a "derivative" refers to a chemical that is structurally related to another, i.e., "original" substance, which may be referred to as a "parent" compound. A "derivative" may be made in one or more steps from a structurally related parent compound. The phrase "closely related derivative" refers to a derivative whose molecular weight does not exceed 50% of the weight of the parent compound. The general physical and chemical properties of a closely related derivative are also similar to the parent compound. A "pharmacologically active derivative" refers to any compound that is capable of providing, directly or indirectly, an activity disclosed herein when administered to a recipient.

[0085] "Analog" refers to a small organic compound, nucleotide, protein, or polypeptide that has a similar or identical activity or function to a compound, nucleotide, protein, or polypeptide having a desired activity of the disclosure, but may not necessarily contain a similar or identical sequence or structure to the sequence or structure of the preferred embodiments.

[0086] "Prodrug" refers to a compound that can be converted under physiological conditions or by solvolysis into a biologically active compound as described herein. Thus, the term "prodrug" refers to a pharma- ceutically acceptable precursor of a biologically active compound. A prodrug may be inactive when administered to a subject, but is converted to an active compound in vivo, for example, by hydrolysis. Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (see, for example, Bundgaard, H., Design of Prodrugs (1985) (Elsevier, Amsterdam)). The term "prodrug" also refers to any covalently bonded carrier that releases an active compound in vivo when administered to a subject. Prodrugs of the active compounds described herein can be prepared by modifying functional groups present in the active compound such that the modifications are cleaved, either by routine manipulation or in vivo, to obtain the active parent compound. Prodrugs include compounds in which, for example, a hydroxy, amino, or mercapto group of an active compound is bonded to any group that cleaves to form a free hydroxy, amino, or mercapto group, respectively, when the prodrug of the active compound is administered to a mammalian subject. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols, various ester derivatives of carboxylic acids, or acetamide, formamide, and benzamide derivatives of amine functional groups in the active compound.Various forms of prodrugs are well known in the art and are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31, (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs 113-191 (Harwood Academic Publishers, 1991), and (d) Hydrolysis in Drug and Prodrug Metabolism, Bernard Testa and Joachim M. Mayer, (Wiley-VCH, 2003).

[0087] In some embodiments, the agent for increasing the level or activity of TPP1 may be provided as a composition, e.g., a pharmaceutical composition. The composition may include one or a combination of TPP1 proteins or variants thereof, formulated with a pharma- ceutically acceptable carrier. In some embodiments, such a composition may include one or a combination (e.g., two or more different) of TPP1 variants. For example, the composition may include a combination of TPP1 variants with different genetic modifications.

[0088] Thus, the present disclosure also provides a pharmaceutical composition for treating a disease or disorder in a subject. The pharmaceutical composition comprises (a) an agent capable of increasing the level or activity of TPP1 in a subject, and (b) optionally a pharma- ceutical acceptable carrier. In some embodiments, the agent comprises a fusion protein comprising a TPP1 protein or a variant / fragment thereof, or a fusion protein comprising a TPP1 protein or a variant thereof.

[0089] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one component useful within the present invention with other components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. A pharmaceutical composition facilitates administration of one or more components of the present invention to an organism.

[0090] A pharmaceutical composition or therapeutic formulation of an agent for increasing the level or activity of TPP1 (e.g., a TPP1 protein or variant thereof, a TPP1 agonist) can be prepared by mixing the agent having the desired purity in the form of a lyophilized formulation or an aqueous solution with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) proteins, e.g., serum albumin, zeolite, sorbitol, sorbitol derivatives, and the like). hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN®, PLURONIC®, or polyethylene glycol (PEG).

[0091] The formulation may also contain two or more active ingredients, preferably those with complementary activities that do not adversely affect each other, as necessary for the particular indication being treated. For example, the formulation may further include another anti-inflammatory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0092] The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or by interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0093] Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing agents for increasing the level or activity of TPP1 (e.g., TPP1 protein or its variants), where the matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate), and poly-d(-)-3-hydroxybutyric acid (PHB). Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. Once encapsulated, the drug remains in the body for a long period of time. It may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational strategies for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular SS bond formation through thiol-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solution, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0094] The preparations used for in vivo administration must be sterile, and can be easily achieved by filtration through a sterile filtration membrane. Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the ingredients listed above as necessary, and carrying out sterile filtration. In general, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which obtains a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0095] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the subject being treated and the specific mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent of the active ingredient combined with a pharma- ceutically acceptable carrier, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent.

[0096] The agent for increasing the level or activity of TPP1 (e.g., TPP1 protein or its variant) can be administered as a single dose, or more commonly, multiple doses. The interval between single doses can be, for example, weekly, monthly, every three months, or yearly. The interval can be irregular, as indicated by measuring the blood level of TPP1 protein or its variant in the patient.

[0097] The agent or pharmaceutical composition thereof can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. For example, administration of the TPP1 protein or variants thereof may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein refers to forms of administration other than enteral administration and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraarticular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the TPP1 protein or variant thereof can be administered via a non-parenteral route, such as a topical, epithelial, or mucosal administration route, such as an intranasal, oral, vaginal, rectal, sublingual, or topical administration route.

[0098] In some embodiments, the TPP1 protein or a composition comprising the TPP1 protein may be introduced parenterally, transmucosally, for example orally, nasally, or rectally, or transdermally. Preferably, administration is by injection, particularly parenterally, for example, intravenous injection, including but not limited to intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.

[0099] In some embodiments, the agent is administered by injection. In some embodiments, the injection is an intracranial injection. In some embodiments, the agent is delivered to the lysosomes of the diseased cells.

[0100] The TPP1 protein or composition can be delivered in vesicles, in particular liposomes (Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). This may be the preferred method for introducing TPP1 in order to reduce its systemic side effects and increase cellular penetration.

[0101] The TPP1 protein or composition can be delivered in a controlled release system. For example, it can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can 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 (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design, and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in the vicinity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, the controlled release device is introduced into the subject in the vicinity of the tissue site affected by LINCL. Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

[0102] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions into unit dosage forms for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as units for the subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are dictated by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active ingredients for the treatment of individual susceptibility.

[0103] The amount of TPP1 protein effective to reduce or eliminate symptoms caused by a deficiency of TPP1 protein can be easily determined by one skilled in the art. As discussed above, alleviation (i.e., reduction or elimination) of symptoms can be determined based on the patient's physical condition, e.g., cessation of seizures or reduction in the amount or intensity of seizures. Alternatively, measurements can be made on cell samples, e.g., brain neurons, by determining the amount of storage products present in lysosomes and comparing with normal control cells to confirm alleviation of the condition. Thus, dosages can be determined by one skilled in the art according to the age, size, and condition of the patient. Alternatively, the amount of TPP1 protein administered can be such that the normal level+ of TPP1 protein in the cells is restored, e.g., as determined by comparison with normal cells. In a preferred treatment, the effective amount of TPP1 protein is such that the affected cells receive about 1.0 nM to about 100 nM of TPP1 protein. The dosage used to ensure that affected cells receive about 1.0-100 nM of TPP1 protein can be determined by one of skill in the art, for example, by biopsy and analysis of treated cells after administration by known methods to determine the amount of injected or oral or inhaled TPP1 needed to provide the desired cellular level. When administered with an uptake inhibitor, the uptake inhibitor should be at a concentration that inhibits immediate clearance of TPP1 protein near the site of administration. Such dosages can be determined by one of skill in the art. When the uptake inhibitor is mannose-6-phosphate, 5 mM is a preferred dosage.

[0104] In some embodiments, the TPP1 protein may be administered according to one of the following dosing schedules: (i) six doses every four weeks, then every three months, (ii) every three weeks, and (iii) one dose at 3 mg / kg body weight followed by one dose at 1 mg / kg body weight every three weeks.

[0105] Alternatively, the drug can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the drug in the patient. Dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages may be required at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete improvement of the symptoms of the disease. The patient can then be administered a preventive regime.

[0106] The actual dosage level of the active ingredient in the composition may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of excretion of the particular active ingredient used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0107] The term "effective amount", "effective dose", or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug / agent is an amount of drug that inhibits the onset or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering a recurrence of a disease. The ability of a therapeutic or prophylactic agent to promote disease reversion or inhibit the onset or recurrence of a disease can be evaluated using a variety of methods known to those skilled in the art, for example, by assaying the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0108] Pharmaceutical compositions can be administered with medical devices known in the art. For example, therapeutic compositions of the present invention can be administered with needleless hypodermic injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556. Examples of well-known implants and modules useful in the present invention include those described in U.S. Patent Nos. 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and 4,475,196. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0109] In some embodiments, the method of treating a disease or disorder in a subject as described above further comprises administering an additional therapeutic agent or therapy to the subject. In some embodiments, the TPP1 protein may be used alone or with other active ingredients. It may be conjugated to a polyalkylene glycol moiety by known methods or may be used as part of a chimeric protein, for example, linked to an antibody or portion thereof, transferrin, hormone, or growth factor. The TPP1 protein may be provided in the form of a prodrug, i.e., a stable inactive form that becomes active when administered (e.g., as described above). The present invention provides for conjugating targeting molecules to TPP1, DNA vectors (including viruses) encoding TPP1, and carriers (i.e., liposomes) for targeting a desired cell or tissue, e.g., the brain. As used herein, "targeting molecule" refers to a molecule that localizes to a desired location when administered in vivo. In various embodiments, the targeting molecule may be a peptide or protein, an antibody, a lectin, a carbohydrate, or a steroid. In one embodiment, the targeting molecule is a protein or peptide ligand of an internalizing receptor on a target cell. In certain embodiments, the targeting molecule is a peptide containing the well-known RGD sequence, or a variant thereof, that binds to an RGD receptor on the surface of cancer cells, such as cells such as human ova that have a receptor that recognizes the RGD sequence. Other ligands include, but are not limited to, transferrin, insulin, amylin, and the like. To facilitate intracellular delivery of the TPP1 protein, receptor internalization is preferred. In another embodiment, the targeting molecule is an antibody. Preferably, the targeting molecule is a monoclonal antibody. In one embodiment, the antibody can be reduced to two heavy and light chain heterodimers, or F(ab) to facilitate crosslinking. 2The fragments can be reduced and cross-linked to TPP1 via the reduced sulfhydryls. Antibodies for use as targeting molecules are specific for cell surface antigens. In one embodiment, the antigen is a receptor. For example, antibodies specific for receptors on cancer cells, such as melanoma cells, can be used. The present invention further provides for the use of other targeting molecules, such as lectins, carbohydrates, proteins, and steroids.

[0110] The composition of the present invention preferably includes an uptake inhibitor that reduces the local clearance of the TPP1 protein by cell surface receptors. This helps ensure that the TPP1 protein is evenly administered so that more cells get some TPP1 protein, rather than a few cells close to the administration site that get most of it. Clearance mechanisms include endocytosis by cell surface receptors such as the mannose receptor, the asialoglycoprotein receptor, and the mannose-6-phosphate receptor. Thus, the preferred uptake inhibitor is mannose-6-phosphate. The uptake inhibitor can be administered in a composition with the TPP1 protein, or can be administered separately and simultaneously, or the two can be administered at different times, as long as the uptake inhibitor can have the desired effect.

[0111] As used herein, "treatment" or "treat" or "alleviate" or "ameliorate" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement or effect in one or more diseases (e.g., inflammatory diseases), conditions, or symptoms being treated. For prophylactic benefit, the agent or composition thereof may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who reports one or more of the physiological symptoms of the disease, even if the disease, condition, or symptom has not yet manifested.

[0112] As used herein, the term "co-administration" or "co-administered" refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is simultaneous. In other embodiments, a first agent / therapy is administered before a second agent / therapy. One of skill in the art will appreciate that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0113] In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment. As used herein, the terms "subject" and "subject(s)" may refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus monkeys and chimpanzees), and humans). The subject may be human or non-human. In a more exemplary aspect, the mammal is a human. As used herein, the phrase "subject in need thereof" or "patient in need thereof" refers to a human or non-human mammal that exhibits one or more symptoms or signs of a disease or disorder and / or has been diagnosed as having a disease or disorder. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0114] "Sample", "test sample", and "patient sample" may be used interchangeably herein. A sample may be a serum, urine plasma, amniotic fluid, cerebrospinal fluid, cell, or tissue sample. Such samples may be used directly as obtained from a patient, or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the characteristics of the sample in some way, as discussed herein or known in the art. As used herein, the terms "sample" and "biological sample" generally refer to biological material suspected of containing an analyte being tested and / or of interest, such as an antibody. A sample may be any tissue sample from a subject. A sample may include proteins from a subject.

[0115] The terms "increased," "increase," "elevate," "promote," or "activate" are all used herein to mean an increase that is generally a statically significant amount, and for the avoidance of doubt, the terms "increased," "increase," or "promote," or "activate" mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% compared to a reference level, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0116] Animal models for studying lysosomal storage disorders In one aspect, the disclosure provides an animal model for studying a disease or disorder. In some embodiments, the animal model is (i) a Tpp1 gene heterozygous knockout (Tpp1 + / - ), and (ii) Cln3 gene homozygous knockout (Cln3 - / -), and mouse models have a shorter lifespan compared to wild-type animals.

[0117] "Gene" refers to a DNA sequence that encodes a protein and may or may not include regulatory sequences, such as introns, exons, promoter or enhancer sequences, and 5' untranslated regions. A "transcript," as referred to herein, is an RNA molecule derived by transcription of a coding gene or nucleic acid.

[0118] The term "knockout" in relation to a gene refers to an alteration of the wild-type sequence of the gene such that no functional gene product is produced. The term "gene product" encompasses the transcript of the gene as well as the protein translated from the transcript. For example, mutation, insertion or deletion of nucleotides in the wild-type gene sequence can result in complete silencing of gene expression or expression of a gene sequence that is altered such that only non-functional transcripts are produced. Non-functional transcripts cannot be translated into functional proteins. If only one allele of a gene is altered, the knocked-out gene is referred to as a "heterozygous knockout." If both alleles are altered, the knockout is referred to as a "homozygous knockout." In accordance with the conventions of the field, heterozygous gene knockouts are designated "het" or "+ / -" and homozygous gene knockouts are designated "horn" or "- / -." If both alleles of a gene remain unchanged (i.e., have wild-type gene sequences), it is designated by "wt" or "++."

[0119] The term "heterozygosity" refers to the genetic condition that exists when different alleles reside at corresponding loci on homologous chromosomes. The term "homozygosity" refers to the genetic condition that exists when identical alleles reside at corresponding loci on homologous chromosomes.

[0120] In some embodiments, the animal may be a mammal, including, but not limited to, for example, a mouse, rat, sheep, dog, cow, horse, non-human primate, pig, cat, rabbit, goat, ferret, guinea pig, gerbil, or hamster. The animal may also be a bird, including, but not limited to, a chicken, duck, or quail. In some embodiments, the animal is a member of the mouse family. In further embodiments, the animal is a mouse.

[0121] In some embodiments, the disease or disorder is selected from the group consisting of late infantile neuronal ceroid lipofuscinosis (CLN2) caused by mutations in the Tpp1 gene, juvenile neuronal ceroid lipofuscinosis (CLN3) caused by mutations in the Cln3 gene, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease caused by mutations in the Cln5 gene, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease caused by mutations in the Cln6 gene, neuronal ceroid lipofuscinosis type 7 (CLN7) disease caused by mutations in the MFSD8 gene, Northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease caused by mutations in the Cln8 gene, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease caused by mutations in the CTSD gene, late-onset neuronal ceroid lipofuscinosis (CLN12) and Kufour-Rakeb syndrome caused by mutations in the gene ATP13A2 I, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) caused by mutations in the GNS gene, osteopetrosis autosomal recessive 4 (OPTB4) caused by mutations in the CLCN7 gene.

[0122] In some embodiments, the animals have a reduction in lifespan of at least 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%) compared to wild-type animals.

[0123] In some embodiments, the Tpp1 gene or Cln3 gene comprises at least one mutation selected from a deletion, an insertion, a frameshift mutation, a rearrangement, or a substitution. In some embodiments, the mutation is constitutive. In some embodiments, the mutation is conditional.

[0124] In some embodiments, the Tpp1 gene is located at Chr 7 E3;7 55.97 cM. In some embodiments, the Tpp1 gene comprises a deletion of at least a portion of an exon in the Tpp1 gene. In some embodiments, the Tpp1 gene comprises an insertion of neo into intron 11 and an Arg446His missense mutation into exon 11 immediately upstream of the neo insertion.

[0125] In some embodiments, the Cln3 gene is located at Chr 7 F3;7 69.16 cM. In some embodiments, the Cln3 gene comprises a deletion of at least a portion of an exon in the Cln3 gene. In some embodiments, the Cln3 gene comprises a deletion of all or a portion of exons 1-6 in the Cln3 gene.

[0126] In some embodiments, the animal model has increased levels of lysosomal accumulation of mitochondrial ATP synthase subunit c (SCMAS). In some embodiments, the animal model has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200% or more increased levels of lysosomal accumulation of SCMAS. In some embodiments, the animal model has at least 50% increased levels of lysosomal accumulation of SCMAS.

[0127] In some embodiments, the animal model has increased expression levels of NPC1 and / or CTSF. In some embodiments, the animal model has at least 40% or more increased expression levels of NPC1 and / or CTSF, or at least 40% or more decreased expression levels of SMPD1. In some embodiments, the animal model has at least 40% increased expression levels of NPC1 and / or CTSF, or at least 40% decreased expression levels of SMPD1.

[0128] In some embodiments, the animal model is characterized by a loss of locomotor activity.

[0129] Also within the scope of the disclosure are progeny of the animal models disclosed herein, and cells, tissues, or cell lines derived from the animal models disclosed herein or the progeny.

[0130] The term "progeny" refers to the descendant of a particular mating. Typically, progeny results from the breeding of two individuals. Progeny can be, for example, an F1, an F2, or any subsequent generation.

[0131] In another aspect, the present disclosure also provides a method for obtaining the animal model disclosed above. The method includes: (a) crossing an animal with a Tpp1 knockout with a second animal with a Cln3 knockout to produce a double heterozygote (Tpp1 + / - ;Cln3 + / - (b) obtaining an animal having Tpp1 - / - ;Cln3 - / - ×Tpp1 - / - ;Cln3 - / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - and crossing the animals with the double heterozygotes by mating the animals with the double heterozygotes.

[0132] Methods for identifying agents for treating lysosomal storage disorders In another aspect, the disclosure further provides a method of identifying an agent for use in treating a disease or disorder of a subject, in some embodiments, the method comprises administering a candidate agent to an animal model or progeny as disclosed herein and evaluating the effect of the candidate agent on the phenotype of the animal model.

[0133] In some embodiments, the methods include contacting a cell, tissue, or cell line with a candidate agent as disclosed herein and evaluating the effect of the candidate agent on the cell, tissue, or cell line.

[0134] In some embodiments, the phenotype is lifespan in an animal model.

[0135] In some embodiments, the effect is characterized by an increase in lifespan in an animal model. In some embodiments, the effect is characterized by a decrease in the level of lysosomal accumulation of SCMAS. In some embodiments, the effect is characterized by a decrease in the expression levels of NPC1 and / or CTSF.

[0136] In some embodiments, the candidate agents include proteins, peptides, peptidomimetics, nucleic acids, or small molecules.

[0137] In yet another aspect, the present disclosure further provides a method for identifying an agent having TPP1 agonist activity. In some embodiments, the method includes: (a) administering an amount of a candidate agent to a subject, such as an animal model disclosed herein; (b) performing an assay on a sample obtained from the subject to determine the level of lysosomal accumulation of SCMAS and / or the activity / level of TPP1 in the sample; and (c) identifying an agent having TPP1 agonist activity when the subject has a reduced level of lysosomal accumulation of SCMAS and / or an increased activity / level of TPP1 compared to a reference level.

[0138] In some embodiments, the method includes: (a) contacting a candidate agent as disclosed herein with a sample comprising a cell, tissue, or cell line; (b) performing an assay on the sample to determine lysosomal accumulation levels of SCMAS and / or activity / levels of TPP1 in the sample; and (c) identifying an agent having TPP1 agonist activity if the sample exhibits a reduced lysosomal accumulation level of SCMAS and / or an increased activity / level of TPP1 compared to a reference level.

[0139] In some embodiments, the method may include culturing or expanding the test cells and / or the control cells. The terms "culturing" or "expansion" refer to maintaining or culturing cells under conditions that allow the cells to proliferate and avoid senescence. For example, the cells may be cultured in a medium that optionally contains one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include a neo-antigen peptide. A stable cell line may be established to allow for continued proliferation of the cells.

[0140] In some embodiments, the reference level may be obtained from a subject prior to administration of an agent or from a cell prior to contact with an agent to increase the level or activity of TPP1 or a composition thereof. In some embodiments, the reference level may be obtained from a control subject or population that does not have or has not been diagnosed with a disease or disorder, or from a control cell of a subject or an unaffected cell. In some embodiments, the reference level is obtained based on, for example, the average level of TPP1 level or activity in a population that is not affected by a disease or disorder. In some embodiments, the reference level is obtained based on the median or median level of a set of individuals that includes patients with a disease or disorder.

[0141] In some embodiments, the disease or disorder is characterized by the accumulation of one or more storage products (e.g., SCMAS) in the lysosomes of affected cells, such as neurons. One way to determine the disorder is to find that the lysosomes have accumulated storage materials, which can be done by known methods such as microscopy or immunofluorescence. An example of a storage material that may be detected in lysosomes is SCMAS. In some embodiments, treatment with TPP1 protein reduces or eliminates mitochondrial ATP synthase, particularly SCMAS in the lysosomes of affected cells, such as neurons. Detecting the removal of storage materials, such as mitochondrial SCMAS, in the lysosomes of affected cells can be done by known methods such as those described above.

[0142] In some embodiments, the disease or disorder is selected from the group consisting of late infantile neuronal ceroid lipofuscinosis (CLN2) caused by mutations in the Tpp1 gene, juvenile neuronal ceroid lipofuscinosis (CLN3) caused by mutations in the Cln3 gene, atypical late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease caused by mutations in the Cln5 gene, atypical late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease caused by mutations in the Cln6 gene, neuronal ceroid lipofuscinosis type 7 (CLN7) disease caused by mutations in the MFSD8 gene, Northern epilepsy neuronal ceroid lipofuscinosis type 8 (CLN8) disease caused by mutations in the Cln8 gene, congenital neuronal ceroid lipofuscinosis type 10 (CLN10) disease caused by mutations in the CTSD gene, late-onset neuronal ceroid lipofuscinosis (CLN12) and Kufour-Rakeb syndrome caused by mutations in the gene ATP13A2 I, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) caused by mutations in the GNS gene, osteopetrosis autosomal recessive 4 (OPTB4) caused by mutations in the CLCN7 gene.

[0143] In some embodiments, the disease or disorder is characterized by a deficiency in the function of the CLN3 protein. An example of such a disorder is JNCL.

[0144] In some embodiments, the diseased cells may belong to any cell or tissue type, such as neurons, hi some embodiments, the diseased cells are neural cells.

[0145] The level or activity of TPP1 can be measured by determining or estimating protein level or mRNA level. Methods for determining or estimating protein level or mRNA level are well known in the art. Such methods can include enzyme activity assay, microscopy, immunofluorescence, and nucleic acid hybridization (e.g., using proteins and nucleic acids described in US Ser. No. 08 / 931,608 and Sleat et al. (1997)). For example, the protein level (e.g., protein expression level) of TPP1 can be determined by SDS-PAGE, Western blot, or immunoassay (e.g., immunoblotting assay, immunoprecipitation assay). The mRNA level can be determined by RT-PCR.

[0146] Measurement of the levels of TPP1 can be performed by assaying the proteins themselves (by Western blotting, ELISA, RIA, and other techniques known to those of skill in the art), by assaying the mRNAs encoding these proteins (such as quantitative PCR, Northern blotting, RNAse protection assay, RNA dot blotting, and other techniques known to those of skill in the art), or by assaying the activity of regulatory elements of genes for TPP1. For example, the activity of regulatory elements can be assessed by reporter constructs consisting of DNA segments from promoters, enhancers, and / or intronic elements (such as luciferase, beta-galactosidase, green fluorescent protein, or other reporter genes that can be easily assayed) linked to a cDNA encoding a reporter. These reporter constructs can be transfected into cells either stably or transiently.

[0147] Additional definitions To aid in understanding the detailed description of the compositions and methods according to the present disclosure, some explicit definitions are provided to facilitate clear disclosure of the various aspects of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0148] The term "agent" is used herein to denote a chemical substance, a mixture of chemical substances, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide, etc.), or an extract made from biological material such as a bacterial, plant, fungus, or animal (especially mammalian) cell or tissue. The activity of such agents may qualify as "therapeutic agents," which are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a subject.

[0149] The terms "therapeutic agent," "therapeutic agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that provides some beneficial effect when administered to a subject. Beneficial effects include enabling a diagnostic determination, ameliorating a disease, symptom, disorder, or pathological condition, reducing or preventing the onset of a disease, symptom, disorder, or condition, and generally preventing a disease, symptom, disorder, or pathological condition.

[0150] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified by any other manipulation, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or conjugation with a labeling moiety. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0151] "Nucleic acid" or "polynucleotide" refers to a DNA molecule (e.g., but not limited to, cDNA or genomic DNA) or an RNA molecule (e.g., but not limited to, mRNA), including DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. DNA or RNA molecules can contain non-naturally occurring moieties, such as modified bases, modified backbones, deoxyribonucleotides in RNA, etc. Nucleic acid molecules can be single-stranded or double-stranded.

[0152] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence (resulting in expression of the latter). For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, join two protein coding regions in the same reading frame.

[0153] The term "linker" refers to any means, entity, or moiety used to connect two or more entities. A linker may be a covalent linker or a non-covalent linker. Examples of covalent linkers include a covalent bond or linker moiety covalently attached to one or more of the proteins or domains to be connected. A linker may also be a non-covalent bond, e.g., an organometallic bond through a metal center, such as a platinum atom. For covalent attachment, various functional groups can be used, such as carbonic acid derivatives, ethers, esters including organic and inorganic esters, amide groups including amino, urethane, urea, etc. To provide attachment, the domains may be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide sites for coupling. Methods for conjugation are well known by those of skill in the art and are encompassed for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or, for example, peptide linker moieties (linker sequences).

[0154] An "isolated" nucleic acid molecule or polynucleotide is intended as a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a therapeutic polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule that is normally contained in a cell that contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, as well as positive and negative stranded forms, and double stranded forms. Isolated polynucleotides or nucleic acids may further include such molecules that are synthetically produced. Additionally, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, a ribosome binding site, or a transcription terminator.

[0155] The term "substantial identity" or "substantial identity," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is at least about 90% nucleotide sequence identity of the nucleotide bases, more preferably at least about 95%, 96%, 97%, 98% or 99%, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. In certain cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0156] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0157] The term "disease" as used herein is intended to be generally synonymous with, and used interchangeably with, the terms "disorder" and "condition" (as in medical conditions), all of which reflect an abnormal state (e.g., disease or disorder) of the human or animal body or a part thereof that impairs normal functioning, typically manifested by distinguishing signs and symptoms, and resulting in a reduced duration or quality of life for the human or animal.

[0158] As used herein, the term "modulate" is meant to refer to any change in a biological state, ie, an increase, a decrease, and the like.

[0159] As used herein, the term "pharmaceutical acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the composition and is relatively non-toxic, i.e., that may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0160] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, that are involved in carrying or transporting a compound of the present invention in or to a subject so that it can perform its intended function. Typically, such compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials which may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; diluents; grinding agents; lubricants; binders; disintegrating agents; wetting agents. agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutical acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, etc., that are compatible with the activity of one or more components of the present invention and are physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0161] As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., a test tube or reaction vessel, cell culture, etc.

[0162] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0163] It is noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0164] The terms "including," "comprising," "containing," or "having," and variations thereof, unless otherwise expressly stated, are intended to encompass the items listed thereafter and equivalents thereof, as well as additional subject matter.

[0165] The phrases "in one embodiment," "in various embodiments," "in some embodiments," and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may, unless context dictates otherwise.

[0166] The term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0167] The word "substantially" does not exclude "completely", e.g. a composition that is "substantially" free of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definition of the invention.

[0168] As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise specified or clear from the context (except where such number exceeds 100% of the possible values). Unless otherwise indicated herein, the term "about" is intended to include values ​​close to the recited range, e.g., weight percent, that are equivalent with respect to the functionality of the individual components, compositions, or embodiments.

[0169] When values ​​and ranges are provided herein, it should be understood that all values ​​and ranges subsumed within those values ​​and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the application.

[0170] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where explicit disclosure or context clearly dictates otherwise.

[0171] The use of any and all examples provided herein, or exemplary language (e.g., "etc."), is intended merely to better clarify the invention and does not impose limitations on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the invention. As used herein, the term "exemplary" is intended to mean "by way of example" and is not intended to indicate that any particular exemplary item is preferred or required.

[0172] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. For any of the methods provided, the method steps may occur simultaneously or sequentially. When method steps are performed sequentially, the steps may be performed in any order unless otherwise specified.

[0173] Where the method includes a combination of steps, unless otherwise stated herein, any combination or subcombination of steps is encompassed within the scope of the disclosure.

[0174] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety unless it contradicts this disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0175] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof may be suggested to one skilled in the art and are to be included within the spirit and scope of this application and the appended claims. EXAMPLES

[0176] Example 1 This example describes the materials and methods used in the subsequent examples below.

[0177] animal Mice were maintained and used according to a protocol approved by the Rutgers University and Robert Wood Johnson Medical School Institutional Animal Care and Use Committee ("Preclinical Evaluation of Therapies in Animal Models at LINCL", protocol I09-0274-4). - / - and Cln3 - / - Mice were on a C57BL / 6 genetic background and genotyping was performed as described (Sleat DE, et al. J Neurosci 24:9117-26 (2004); Greene ND, et al. Mol Genet Metab 66:309-13 (1999)). Experimental cohorts contained equal numbers of male and female animals and were analyzed at approximately 120 days of age. For biochemical analysis, animals were deeply anesthetized with sodium pentobarbital / phenytoin (1:4 dilution of Euthasol; Delmarva Laboratories, Midlothian, VA) and euthanized by transcardial perfusion with 0.9% saline. Brains were dissected and frozen on dry ice. For histopathology, mice were anesthetized, perfused with saline, and then perfusion-fixed with 4% paraformaldehyde in PBS. Brains were excised and fixed in 4% paraformaldehyde in PBS for 48 hours, then transferred to 30% sucrose / PBS at 4° C. until they sunk.

[0178] quantitative mass spectrometry Sample preparation and quantitative mass spectrometry of whole brain extracts were performed as previously described (Sleat DE, et al. Mol Cell Proteomics 18:2244-2261 (2019)). Briefly, detergent-solubilized extracts were prepared, proteins were sequentially digested in solution with trypsin (specificity, carboxyl side of K and R) and endoprotease LysC (specificity, carboxyl side of K), and the resulting peptides were labeled with TMT11-plex isobaric reagents (ThermoFisher Scientific). Labeled samples were pooled and analyzed by synchronous precursor selection MS3 on a Lumos Tribrid instrument (ThermoFisher Scientific). Peak lists were generated using Proteome Discoverer 2.2 (ThermoFisher Scientific) and the data were searched using a local implementation of the Global Proteome Machine (GPM) (Craig R, Cortens JP and Beavis RC (2004) J Proteome Res 3:1234-42; Beavis RC (2006) Methods Mol Biol 328:217-28). Reporter ion intensities were extracted using an in-house script (https: / / github.com / cgermain / IDEAA). Mass spectrometry files (mgf and raw files, GPM search files, and Excel files showing protein assignments, peptide-spectrum matches, and corresponding reporter ion intensities) will be archived in the MassIVE (http.massive.ucsd.edu) and ProteomeXchange (http. / / www.proteomexchange.org / ) repositories under submission MSV000087613.

[0179] Mass spectrometry data normalization and statistical analysis TMT-16 reporter ion intensities were normalized and analyzed as previously described (Sleat DE, et al. (2017) J Proteome Res 16:3787-3804). Briefly, reporter ion intensity data were extracted from peak list files using custom in-house scripts (https: / / github.com / cgermain / IDEAA), and spectra were then normalized to the total reporter ion intensity per channel to correct for differences in labeling efficiency and / or amount of labeled protein. Peptides were first filtered for complete trypsin cleavage, no false cleavage sites, and complete iTRAQ labeling of lysines and amino termini. Peptides were then filtered to remove those containing post-translational modifications that may increase data variability (i.e., asparagine or glutamine deamidation, methionine dioxide, tryptophan mono- and di-oxidation, and isobaric labeling of tyrosine at positions other than the amino terminus). For selected comparisons, ratios of expression and q values ​​corrected for multiple comparisons using the Benjamini-Hochberg procedure were generated using a nested procedure that accounts for variability at both the peptide and protein levels (YB and YH (1995) Journal of the Royal Statistical Society: series B (Statistical Methodology) 57:289-300).

[0180] immunostaining For immunohistochemistry, one of six series of coronal brain sections from each mouse was stained using a modified immunofluorescence protocol (Nelvagal HR, et al. Neuropathol Appl Neurobiol 47:251-267) for the astrocyte marker glial fibrillary associated protein (GFAP, rabbit anti-GFAP, 1:1000, DAKO), and the microglial marker CD68 (rat anti-mouse CD68, 1:400, Bio-Rad). Briefly, 40 μm coronal sections were mounted on Superfrost Plus slides (Fisher Scientific) and air-dried for 30 minutes. Slides were then blocked for 1 hour in 15% serum solution in 2% TBS-T (Tris-buffered saline 1×, pH 7.6 with 2% Triton®-X100, Fisher Scientific). Slides were then incubated in primary antibodies in 10% serum solution in 2% TBS-T for 2 hours, washed three times in 1× TBS, and incubated in fluorescent Alexa-Fluor-labeled IgG secondary antibodies (Alexa-Fluor goat anti-rabbit 488, goat anti-rat 546, Invitrogen) in 10% serum solution in 2% TBS-T for 2 hours. Slides were washed three times in 1× TBS and incubated in a 1× solution of TrueBlack lipofuscin autofluorescence quencher (Biotium, Fremont, CA) in 70% ethanol for 2 minutes, followed by rinsing in 1× TBS. Slides were coverslipped in Fluoromount-G mounting medium with DAPI (Southern Biotech, Birmingham, AL).

[0181] Thresholding Image Analysis To analyze the extent of glial activation in the gray matter (GFAP-positive astrocytes + CD68-positive microglia), a semi-automated thresholding image analysis method was used with Image-Pro Premier software (Media Cybernetics). Briefly, stained sections were scanned at 10x magnification for each of six serial sections per animal using a Zeiss AxioScan Z1 slide scanner at the Washington University Center for Cellular Imaging (WUCCI), followed by demarcation of all areas of interest. Images were then analyzed using Image-Pro Premier (Media Cybernetics) using an appropriate threshold that selected foreground immunoreactivity over background. This threshold was then applied as a constant to all subsequent images analyzed per animal and batch of reagents used to determine the specific areas of immunoreactivity for each antigen. Histological processing measurements were performed blinded to genotype, and statistical analysis was performed using GraphPad Prism version 8.0.0 for MacOS. Data were analyzed using two-way ANOVA with post-hoc Bonferroni correction, and p values ​​of 0.05 or less were considered significant.

[0182] survival statistics Survival curves were compared using the log-rank test in GraphPad Prism 9.1 (GraphPad Software, San Diego, California USA, www.graphpad.com) using the Bonferroni method to correct for multiple comparisons.

[0183] Example 2 Breeding strategies Initially, double hybrid matings were performed using animals that were double heterozygous for both the Tpp1 and Cln3 mutations to generate all the genotypes required for the litter. Analysis of transmission ratios showed that offspring were not generated in the expected ratios (data not shown), and the numbers of both double mutant and double wild-type animals were significantly reduced. This reflects genetic linkage with both Tpp1 and Cln3, which are located on mouse chromosome 7 (Tpp1 location: Chr 7 E3; 7 55.97 cM, Cln3 location: Chr 7 F3; 7 69.16 cM). Subsequently, Tpp1 - / - ;Cln3 - / - ×Tpp1 - / - ;Cln3 - / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - Double knockout animals were generated by breeding the following:

[0184] Survival time Survival curves and statistical comparisons are shown in Figures 1 and 2 and Table 1. There was a substantial difference in survival time, suggesting that Tpp1 - / - The background animals are shown in Figure 1, and Tpp1 + / - or Tpp1 + / + Background animals are shown in FIG.

[0185] Tpp1 - / - ;Cln3 - / - , Tpp1 - / - ;Cln3 +-- , and Tpp1 - / - ;Cln3 + / + Litters generated by double mutant matings had median survival times between 120 and 124 days (Table 1) and were not significantly different from each other (after correction for multiple comparisons). - / - ;Cln3 - / - The survival curves of mice were - / - ;Cln3 + / + It is slightly rectangular compared to the mouse. This is because Tpp1 - / -This could be an effect of the CLN3 mutation on the phenotype, or alternatively, could reflect subtle differences in the genetic background of the animals. The Tpp1 mutant alleles used in these studies were backcrossed to C57BL / 6 (Sleat DE, et al. (2004) J Neurosci 24:9117-26), as was the Cln3 allele (Hersrud SL, et al. Biochim Biophys Acta 1862:1324-36). However, the strain backgrounds of the two mutants are similar to those of the Tpp1 - / - Mouse is Nnt + / + whereas Cln3 + / + Mouse is Nnt - / - (Sleat DE, et al. Mol Cell Proteomics 18:2244-2261). Mutations in Nnt differ in different C57BL / 6 substrains. - / - The genotype was generated at Jackson Laboratories https: / / ncbi.nlm.nih.gov / 19448337 / and designated as C57BL / 6J, whereas Nnt + / + The strain is designated as C57BL / 6N. The double mutant animals are therefore mixed C57BL / 6N × C57BL / 6J substrains. / Litters generated from Cln3 mutant matings: Tpp1 - / - ;Cln3 + / + Mice and littermates generated by mating with a single Tpp1 mutant Tpp1 - / - ;Cln3 + / + There was no significant difference in survival time between the Tpp1- / -Cln3- / - double mutant and the Tpp1 - / - Cln3 + / + There was no significant difference in the survival phenotype of the single mutant animals.

[0186] Tpp1 + / - or Tpp1 + / +The survival time of mutant animals in this background is shown in Figure 2. Data from C57BL / 6J animals from another study ("Yuan dataset") (Yuan R, et al. (2012) Proc Natl Acad Sci US A109:8224-9) are also included for analysis. The survival time of Tpp1 heterozygous animals (median survival time 843 days) was not significantly different from wild-type animals (median survival time 901 days) in the Yuan dataset (Figure 2A). Given that Tpp1 heterozygosity did not affect survival time, no significant difference was expected in the survival time of Cln3 heterozygotes when compared to wild type. Tpp1 + / - ;Cln3 + / - The survival time of double heterozygous mice (748 days in culture) was significantly longer than that of single Cln3 mice. - / - The mean age of Tpp1 in Tpp1 mice was 1.2 years, similar to that in Tpp1 mice, and was also significantly shorter than that in wild-type animals. + / + ;Cln3 - / - The survival time of animals was significantly shorter than that of CLN3 - / - This resulted in a decreased survival time for the animals (median 584 days). Overall, these data suggest that heterozygosity for Tpp1 reduces the survival of Cln3 + / - Animals and Cln3 - / - These results show that treatment with 1-amino-2-propanediol resulted in a decrease in survival time of both animals.

[0187] Our Tpp1 + / + ;Cln3 - / - The survival time of the mice (median 719 days) was not significantly different from previous data from a different but similar JNCL knockout mouse model (742 days) (Figure 2B). However, survival time of both Cln3 mutants was significantly reduced compared to wild-type mice.

[0188] pathology Animals were euthanized at approximately 120 days to investigate the effect of Tpp1 and Cln3 genotype on brain pathology. Sections were analyzed by immunofluorescence for simultaneous detection of glial activation markers (CD68 in microglia and GFAP in astrocytes) in two brain regions where prominent glial activation is consistently observed in multiple NCL mouse models: the somatosensory barrel field (S1BF) cortex of the thalamus and the medial and lateral ventral posterior thalamic (VPM / VPL) regions. As expected, immunostaining for either GFAP or CD68 was significantly higher in Tpp1, regardless of Cln3 genotype. + / - or Tpp1 + / + In animals with the Tpp1 genotype, there were very low levels of GFAP-positive astrocytes (Figure 3) or CD68-positive microglia (Figure 4) in these animals. Marked activation of both astrocytes (Figure 3) and microglia (Figure 4) was observed in the presence of Tpp1 - / - Cln3 + / + Animal S1BF and VPM / The double mutant Tpp1 was detected in VPL and showed a marked upregulation of these markers and corresponding changes in cell morphology. - / - Cln3 - / - Similar levels of prominent glial activation were detected in the S1BF and VPM / VPL of animals, and Tpp1 - / - Cln3 + / + There was a similar degree of cellular hypertrophy of both astrocytes and microglia to that seen in Tpp1 mice compared with other genotypes. - / - Cln3 + / + and Tpp1 - / - Cln3 - / - We confirmed these qualitative observations with a significant increase in GFAP and CD68 immunoreactivity in both brain regions of animals (Figure 5). - / - Cln3 + / + and Tpp1 - / - Cln3 - / -Although there was some variation between animals, there were no significant differences for either antigen in any brain region between animals of these genotypes. - / - Cln3 + / + Animals and Tpp1 - / - Cln3 - / - This broad similarity between the animals is consistent with the similarities observed in the survival phenotypes of these mutants.

[0189] Proteomic analysis We performed proteomic analyses on brain samples from Tpp1 and Cln3 mutant mice with the aim of identifying changes that may correlate with disease survival and shed light on the cellular functions of each of these proteins. Furthermore, previous proteomic studies have shown that Cln3 - / - Given the failure to identify beneficial brain expression changes in mice, it was hypothesized that Cln3 mutations may exacerbate the changes associated with the Cln3 mutation and therefore be easier to detect in a Tpp1 mutant background. Proteins in brain extracts from animals at approximately 105 days were identified and quantified using isobaric labeling mass spectrometry. In Figure 6, the various genotypes of interest were compared using a volcano plot (a scatter plot that allows the comparison of effect sizes with the probability of significant differences). Q values ​​(i.e., p values ​​adjusted for multiple comparisons found using the Benjamini-Hochberg false discovery rate method) were calculated by comparing the reporter ion intensities of all individual spectra assigned to a given protein from all biological replicates of each genotype.

[0190] Tpp1 - / - Several significant changes in protein expression were detected when comparing animals to wild type, including increases in various lysosomal proteins, including GPMB, LYZ2, SERPINA3N, and several cathepsins (Table 2). - / - ;Cln3 - / - When comparing animals with wild type, Tpp1 - / - ;Cln3 + / + and Tpp1 - / -;Cln3 - / - For the most part, highly similar changes in protein expression were identified, as exemplified by the high degree of correlation between fold changes in expression in Tpp1 compared to wild type animals (Figure 7A). - / + or Tpp1 + / + Cln3 in the background - / - Relatively few changes were detected in mutant animals, with NPC1 and CTSF consistently elevated, and NEU4 and SMPD1 decreased (Fig. 7B ).

[0191] TPP1 activity in mutant mice. As shown in Fig. 8, TPP1 activity in Cln3 mutants was approximately twofold higher than that measured in wild-type animals, but Tpp1 was not observed regardless of Cln3 genotype. - / - It was found that no activity was detectable in animals. + / - As expected, TPP1 activity in animals was approximately 50% of that of wild type, whereas Tpp + / - Cln3 - / - The animals are approximately 100% wild type in TPP1 activity, again indicating that loss of CLN3 results in an approximately 2-fold increase in TPP1 activity.

[0192] The overall goal of this study is to determine the phenotype of double Tpp1 and Cln3 mutant mouse models, which may provide useful information regarding the cellular role of CLN3 and potential functional interactions between TPP1 and CLN3. In most cases, the phenotype of these double knockout mice is similar or identical to the single Tpp1 mutants, the median survival time of both models is within 5 days, and they show a similar degree of characteristic NCL pathology in the form of astrocytosis and microglial activation. In addition, the proteomic changes in the brain of the double knockouts are very similar to those in the single Tpp1 knockouts (Figure 7A). Consistent with these observations, one possibility is that the effects of the disease in JNCL may result, at least in part, from a secondary effect of CLN3 on TPP1. Although loss of CLN3 does not appear to directly affect TPP1 activity measured in vitro, it is possible that loss of CLN3 affects the access of TPP1 to its physiological substrates, which may include SCMAS. There are two observations that support this possibility. First, SCMAS accumulation is present in both LINCL and JNCL (but also in other lysosomal storage diseases). Second, in this study, we observed a ≈2-fold increase in TPP1 activity in the absence of CLN3 (Figure 8), indicating a compensatory response.

[0193] The life span of the JNCL mouse model (median 719 days) was shortened compared with the wild type (median 901 days), which is consistent with previous analysis of a different Cln3 knockout mouse model (Cln3 - / - , median 721 days, and wild-type median 861 days). However, Tpp1 heterozygosity was not associated with Cln3 - / - This was associated with a shortened survival time in mice, further shortening their median lifespan to 584 days. + / - The survival time of Cln3+ / - mice was also significantly reduced compared to wild type mice. Heterozygosity for Tpp1 inhibited the Cln3 - / -Although it is unclear why CLN3 deficiency exacerbates the animal phenotype or creates a survival phenotype in Cln3 heterozygotes, it is consistent with the possibility that CLN3 deficiency has downstream effects on TPP1 function. An alternative possibility is that the compensatory increase in TPP1 activity detected in the absence of CLN3 may actually provide a neuroprotective function. Thus, heterozygosity for Tpp1 may lessen such a protective role, and Cln3 may play a role in neuroprotection. - / - This results in a shorter life span for the mice.

[0194] Glial activation is a consistent pathological feature in NCL diseases, including both LINCL and JNCL. Typically, localized glial activation precedes the onset of neuronal loss and helps predict where subsequent neurodegeneration will occur. Indeed, it has been suggested that glial dysfunction may contribute to neuronal loss in multiple NCLs. Tpp1 - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Quantitative analysis of the mice revealed very similar degrees of astrocytosis and microglial activation between mice of these genotypes.

[0195] Despite its short life span, Tpp1 + / - Cln3 - / -The reduced survival of mutants may be useful for testing therapeutic strategies for JNCL. In developing any therapeutic strategy, survival provides a clear and objective endpoint: for example, in evaluating several different strategies for LINCL, survival studies in mouse models highlighted promising approaches (e.g., gene therapy, subarachnoid cerebrospinal fluid (CSF)-mediated ERT, blood flow-mediated ERT), while survival studies in dog models (Katz ML, et al. (2015) Sci Transl Med 7:313ra180) helped pave the way for the approval of enzyme replacement therapy. LINCL animal models have a significantly shortened lifespan, and therefore proof of principle of treatment in terms of survival is easily achievable. In contrast, survival of Cln3 mutant mice approaches that of wild-type, complicating survival as an endpoint. As a result, behavioral models have been widely used to analyze disease progression and the effects of potential treatments in Cln3 mutant mice. While many studies have characterized the locomotor defects in Cln3 mouse mutants, the behavioral phenotypes are subtle and mouse strain- and sex-dependent.

[0196] Cln3 - / - ;Tpp1 + / - Evaluation of potential therapeutics for JNCL in mouse mutants would provide a relatively robust survival phenotype to measure efficacy. Furthermore, behavioral phenotypes may worsen at younger ages. Effective therapies addressing CLN3 loss (e.g., brain-wide widespread gene therapy) are predicted to increase survival of this mouse mutant to resemble Tpp1 heterozygotes indistinguishable from wild type. A similar approach has been performed using mutant Cln3 mice expressing human amyloid precursor protein (APP) with familial Alzheimer's disease mutations (Centa JL, et al. (2020) Nat Med 26:1444-1451). However, mutant APP confers a severe survival phenotype even in the absence of CLN3 defects. Thus, positive treatments for CLN3 deficiency would essentially ameliorate the severe phenotype rather than restoring wild type survival.

[0197] Finally, one aim of this study was to determine whether double Tpp1 and Cln3 knockout could highlight proteomic changes in the brain that could potentially provide a platform for clinically useful biomarkers in either or both LINCL and JNCL. Neurofilament light chain (NEFL in mouse) has been proposed as a treatment response biomarker based on studies of plasma from TPP1 patients and dog models, and levels of this and other neurofilament proteins (NEFH and NEFM) were found to be elevated in mouse CSF. However, brain levels of NEFL, NEFH, and NEFM were unchanged or reduced in TPP1 and CLN3 mouse models. This is consistent with the finding that brain NEFL is unchanged in mouse models of other neurodegenerative diseases that show elevated NEFL plasma and CSF levels. Also, several lysosomal proteins (CTSF, SMPD1, and NPC1) were found to be elevated in Cln3. - / - It was also found that CLN3 expression was significantly altered in aged mice and that although the changes in expression were moderate, they may be more pronounced in aged mice (animals were analyzed at about 120 days in this study). These changes may be a compensatory response to the loss of CLN3 and could potentially provide useful information regarding its biological function. In addition, soluble lysosomal proteins SMPD1 and CTSF would also require further investigation as potential biomarkers in JNCL. [Table 1] [Table 2-1] [Table 2-2]

[0198] Example 3 One of the aims of this study was to determine whether overexpression of TPP1 could reduce the accumulation of subunit C mitochondrial ATP synthase (SCMAS) in JNCL mice. TPP1+ / 0 A mouse transgenic model constitutively overexpressing ) was previously developed (Nemtsova, Y., et al., PLOS One, 2018.13(2):p.e0192286). The inventors were able to test this hypothesis with a genetic approach.

[0199] The transgenic lineage was a CAG promoter-driven transgene integrated into the ROSA26 locus (Tg TPP1+ We overexpress mouse TPP1 from mouse TPP1-producing mice (Figure 9A). The mice express TPP1 at levels at least 10-fold higher than normal in the brain (Figure 9B). TPP1 overexpression is constitutive and ubiquitous throughout the brain, including the cortex and hippocampus (Figure 9B). TPP1 overexpression has no obvious deleterious effects on the animals (Figure 9C). Transgenic TPP1 is expressed in both neurons and microglia and colocalizes with the respective markers NeuN (Figure 9D) and Iba1 (Figure 9E).

[0200] The approach is to transform our TPP1-overexpressing transgenic Cln3 - / - The aim of this study was to determine whether elevated TPP1 levels had any positive effect on disease progression in crosses with the JNCL mouse model. Four experimental cohorts of littermate mice were established: 1) Tg-Cln3 + / + ;2) Tg-Cln3 - / - ;3) Tg+Cln3 + / + and 4) Tg+Cln3 - / - We examined SCMAS accumulation in the liver and spleen of 6-month-old JNCL mice, as well as in the brain at this time point.

[0201] method Animals. Animals were anesthetized with Euthasol (sodium pentobarbital and sodium phenytoin) and then euthanized by exsanguination / transcardial perfusion with PBS and dissected. Half of the brain was snap frozen for biochemical analysis (see below) and the other half was drop-fixed in 4% paraformaldehyde (PFA) for immunohistochemistry.

[0202] Histological embedding, sectioning, and staining Immunohistochemistry was performed by NeuroScience Associates (Knoxville, TN). Tissue samples (brain, liver, spleen) were treated overnight with 20% glycerol and 2% dimethyl sulfoxide. Samples were then embedded in gelatin matrix using MultiBrain® / MultiCord® Technology. After hardening by immersion in crushed dry ice-cooled 2-methylbutane, blocks were rapidly frozen and mounted on the freezing stage of an AO 860 slide microtome, where blocks were sectioned. All sections were cut over the entire length of the sample segments and collected sequentially in a series of 24 containers containing antigen preservation solution (50% PBS pH 7.0, 50% ethylene glycol, 1% polyvinylpyrrolidone).

[0203] immunohistochemistry Lysosomal storage of SCMAS was visualized using an affinity purified rabbit anti-polyclonal antibody (PAC3601 / 3602; Pacific Immunology, Ramona, CA) raised against the SCMAS amino-terminal peptide (Xu, S., et al., Mol Ther, 2011.19(10):p.1842-8). Free-floating sections were stained with the desired stain. All incubation solutions from the primary antibody were used in Tris-buffered saline (TBS) with Triton® X-100 as vehicle; all rinses were with TBS. After hydrogen peroxide treatment, free-floating sections were immunostained overnight at room temperature with anti-SCMAS antibody at a dilution of 1:500. The vehicle solution contained Triton® X-100 for permeabilization. After rinsing, biotinylated secondary antibody (anti-rabbit IgG, made in goat from Vector Labs) was applied at a dilution of 1:1000. After further rinsing, Vector Lab's ABC solution (avidin-biotin-HRP complex; VECTASTAIN® Elite ABC, Vector, Burlingame, CA, instructions details) was applied. Sections were rinsed again and then treated with diaminobenzidine tetrahydrochloride (DAB) with nickel and hydrogen peroxide to make the reaction product visible. After further rinsing, sections were mounted on gelatin-coated glass slides and air-dried. Slides were dehydrated in alcohol, cleared in xylene, and coverslipped.

[0204] Imaging Slides were scanned using a Huron Digital Pathology TissueScope LE system. Entire slides were scanned at 20x resolution (0.4um / pixel). SCMAS accumulation was quantified using ImageJ (Schneider, CA, et al. Nat Methods, 2012.9(7):p.671-5.).

[0205] result Animals were euthanized at 6 months of age and SCMAS accumulation was assessed in the spleen, liver and brain.

[0206] Hepatic SCMAS staining was not detected in liver sections from control animals (Tg - Cln3 + / + and Tg+ Cln3 + / + ) (Figure 10). Cln3 lacking the TPP1 transgene - / - Mouse (Tg - Cln3 - / - ) showed widespread accumulation of SCMAS in the liver. - / - ) expressing Cln3 - / - No SCMAS accumulation was detected in mice.

[0207] Splenic SCMAS staining was not detected in spleen sections from control animals (Tg - Cln3 + / + and Tg+ Cln3 + / + ) (Figure 11). Cln3 lacking the TPP1 transgene - / - Mouse (Tg - Cln3 - / - ), widespread accumulation of SCMAS was detected in the spleen. - / - ) expressing Cln3 - / - No SCMAS accumulation was detected in mice.

[0208] Brain SCMAS accumulation was analyzed in four different regions in the brain: the CA3 region of the hippocampus, the cerebellum, the thalamus, and the cortex. No SCMAS staining was detected in brain section sections from control animals (Tg - Cln3 + / + and Tg+ Cln3 + / + ) (Figure 12). Cln3 lacking the TPP1 transgene - / - In mice (Tg- Cln3- / -), widespread accumulation of SCMAS was detected specifically in the CA3 region of the hippocampus and in Purkinje cells of the cerebellum. Widespread accumulation was observed in the thalamus and cortex. - / -In JNCL mice expressing ), SCMAS accumulation was not detected in any of these regions.

[0209] SCMAS accumulation in the liver, cortex and thalamus was quantified in terms of the area of ​​the image occluded by the inclusion (FIG. 13). The effect of transgene expression on SCMAS accumulation is clear.

[0210] TPP1 transgene (Tg+ Cln3 - / - JNCL mice expressing TPP1 showed no detectable accumulation of SCMAS in the liver, spleen, or four brain regions at 6 months of age. These results indicate that increasing TPP1 activity prevents the appearance of accumulated material in JNCL and provide support for enhancing TPP1 as a potential therapeutic approach in JNCL and other diseases that may accumulate SCMAS.

[0211] The present disclosure should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. A pharmaceutical composition for use in a method of treating a subject having a disease or disorder characterized by the accumulation of SCMAS in the lysosomes of affected cells, wherein the pharmaceutical composition comprises an agent that increases the level or activity of TPP1, wherein administration of the agent reduces or eliminates symptoms caused by the disease or disorder, and optionally the disease or disorder is characterized by a deficiency in the function of the CLN3 protein.

2. The pharmaceutical composition according to claim 1, wherein the disease or disorder is selected from juvenile neuronal ceroid lipofuscin (CLN3) disease, atypical late infant neuronal ceroid lipofuscin type 5 (CLN5) disease, atypical late infant neuronal ceroid lipofuscin type 6 (CLN6) disease, neuronal ceroid lipofuscin type 7 (CLN7) disease, northern epileptic neuronal ceroid lipofuscin type 8 (CLN8) disease, congenital neuronal ceroid lipofuscin type 10 (CLN10) disease, late-onset neuronal ceroid lipofuscinosis (CLN12), as well as Cuffor-Lakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and autosomal recessive osteopetrosis 4 (OPTB4).

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein the disease or disorder is characterized by a deficiency in the function of the CLN3 protein.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the affected cells are nerve cells.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the drug comprises a nucleic acid molecule containing recombinant human TPP1 protein or a nucleotide sequence encoding TPP1 or a variant thereof, wherein the TPP1 protein is optionally an inactive proenzyme, and the TPP1 protein is optionally mannose-6-phosphorylated.

6. The pharmaceutical composition according to claim 5, wherein the affected cells receive about 1.0 to about 100 nM of recombinant human TPP1 protein.

7. The pharmaceutical composition according to any one of claims 5 or 6, characterized in that the drug is administered by injection, and optionally the injection is an intracranial injection.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the subject is a human.

9. An animal model for studying disease or disorder, wherein (i) tripeptidyl peptidase 1 (Tpp1) gene heterozygous knockout (Tpp1 ＋／－ (ii) Cln3 gene homozygous knockout (Cln3 －／－ Animal models comprising the above, wherein the mouse model has a shorter lifespan compared to wild-type animals, and the disease or disorder is optionally selected from late infant neuronal ceroid lipofuscin (CLN2) disease, juvenile neuronal ceroid lipofuscin (CLN3) disease, atypical late infant neuronal ceroid lipofuscin type 5 (CLN5) disease, atypical late infant neuronal ceroid lipofuscin type 6 (CLN6) disease, neuronal ceroid lipofuscin type 7 (CLN7) disease, northern epileptic neuronal ceroid lipofuscin type 8 (CLN8) disease, congenital neuronal ceroid lipofuscin type 10 (CLN10) disease, late-onset neuronal ceroid lipofuscinosis (CLN12), as well as Cuffor-Lakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and autosomal recessive osteopetrosis 4 (OPTB4).

10. The animal model according to claim 9, wherein the Tpp1 gene or the Cln3 gene comprises at least one mutation selected from deletion, insertion, frameshift mutation, rearrangement, or substitution, and the mutation is optionally constitutive or conditional.

11. The animal model according to any one of claims 9 or 10, wherein the Tpp1 gene comprises i) a deletion of at least a portion of an exon within the Tpp1 gene, ii) an insertion of neo into an intron 11 and an Arg446His missense mutation in exon 11 immediately upstream of the neo insertion, or iii) located at Chr 7 E3; 7 55.97 cM.

12. The animal model according to any one of claims 9 to 11, wherein the Cln3 gene comprises i) a deletion of at least a portion of an exon in the Cln3 gene, ii) a deletion of all or part of exons 1 to 6 in the Cln3 gene, or iii) located at Chr 7 F3; 7 69.16 cM.

13. A descendant of the animal model according to any one of claims 9 to 12.

14. Cells, tissues, or cell lines derived from the animal model according to any one of claims 9 to 12 or from the offspring according to claim 13.

15. A method for obtaining an animal model according to any one of claims 9 to 12, comprising: (a) crossing an animal having a Tpp1 knockout with a second animal having a Cln3 knockout to obtain an animal having a double heterozygote (Tpp1 ＋／－ ; Cln3 ＋／－ ), and (b) crossing the animal having the double heterozygote by mating Tpp1 －／－ ; Cln3 －／－ ×Tpp1 －／－ ; Cln3 －／－ , or Tpp1 －／－ ; Cln3 －／＋ ×Tpp1 ＋／－ ; Cln3 －／－ .

16. A method for identifying drugs for use in the treatment of a disease or disorder in a subject, (i) administering the candidate drug to an animal model according to any one of claims 9 to 12 or to an offspring according to claim 13, and evaluating the effect of the candidate drug on the phenotype of the animal model, or (ii) A method comprising contacting the cells, tissue, or cell line described in claim 14 with a candidate drug to evaluate the effect of the candidate drug on the cells, tissue, or cell line, wherein the candidate drug optionally comprises a protein, peptide, peptide mimetic, nucleic acid, or small molecule.

17. The disease or disorder is characterized by the accumulation of SCMAS in the lysosomes of affected cells in the animal model, and optionally, the disease or disorder is characterized by late infant neuronal ceroid lipofuscin (CLN2) disease, juvenile neuronal ceroid lipofuscin (CLN3) disease, atypical late infant neuronal ceroid lipofuscin type 5 (CLN5) disease, atypical late infant neuronal ceroid lipofuscin type 6 (CLN6) disease, neuronal ceroid The method according to claim 16, selected from lipofuscin type 7 (CLN7) disease, northern epileptic neuroceroid lipofuscin type 8 (CLN8) disease, congenital neuroceroid lipofuscin type 10 (CLN10) disease, late-onset neuroceroid lipofuscinosis (CLN12), and Cuffor-Lakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and autosomal recessive osteopetrosis 4 (OPTB4).

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