Methods for treating lysosomal storage diseases

Abstract

The present disclosure provides novel animal models for studying lysosomal storage diseases and methods of treating lysosomal storage diseases by increasing the level or activity of TPP1.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 362,574, filed on April 6, 2022. The foregoing application is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to methods of treating lysosomal storage diseases. Background Art

[0004] Neuronal ceroid lipofuscinosis (NCL) is a group of more than twelve genetically different but similar lysosomal storage diseases. NCL is characterized by the accumulation of autofluorescent storage substances in lysosomes, is a neurodegenerative and progressive disease, manifested as epileptic seizures, loss of vision and ultimately loss of mental ability. Onset is typically in childhood, and these diseases lead to premature death. The two most common NCL diseases are late infantile and adolescent types (LINCL and JNCL, respectively). LINCL is caused by mutations in the gene encoding soluble lysosomal serine protease tripeptidyl peptidase 1 (TPP1, formerly known as CLN2). The disease of LINCL typically occurs at around 4 years old, and the patient life span is about 8 to 15 years old. JNCL is caused by mutations in the gene encoding lysosomal transmembrane protein CLN3. Compared with LINCL, JNCL onset is later and progresses more slowly, and the initial symptoms of the disease (such as vision problems) appear at around 8 years old, and patients usually survive to their teens or twenties. Despite differences in disease timelines and genetic etiologies, LINCL and JNCL share many similarities, including lysosomal storage of mitochondrial ATP synthase subunit c (SCMAS).

[0005] The development of effective NCL disease therapies has received great attention, and LINCL is at the forefront. Enzyme replacement therapy has been clinically approved for LINCL, and gene therapy has also received attention, 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 proteins can be taken up by a large number of cells through endocytosis and delivered to lysosomes, while in gene therapy, only a portion of cells are transduced, but these cells can overproduce and secrete enzymes, which can be taken up by non-transduced cells. In addition, LINCL animal models with well-defined phenotypes that reproduce human disease have become an integral part of testing treatment strategies.

[0006] There are currently no approved therapies for JNCL, and this reflects several major obstacles. First, CLN3 is a transmembrane protein, which precludes replacement therapy with exogenously administered recombinant proteins. Gene therapy for JNCL has received attention, but because CLN3 is an integral membrane protein, non-transduced cells do not express the missing protein. If the underlying metabolic defect is cell autonomous, cross-protection between transduced and non-transduced cells may be impossible, requiring a very high proportion of cells to be transduced for effective therapy. Second, there is a fundamental lack of understanding of the cellular function of the CLN3 protein. The protein is associated with many cellular activities, including lysosomal pH homeostasis, endocytosis, autophagy, apoptosis, lysosomal enzyme transport, etc., but its exact function is still unclear. Therefore, mechanism-based treatments for JNCL are currently not feasible. Third, animal models of JNCL do not present a robust phenotype, especially in terms of survival. There are several mouse models of JNCL, and their phenotypes are very similar, but the disease is highly attenuated compared to mouse models of other NCL diseases.

[0007] Therefore, there remains a need for animal models for studying lysosomal storage diseases and methods for treating lysosomal storage diseases. Summary of the invention

[0008] In one aspect, the present disclosure provides a method of treating a subject suffering from a disease or condition 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 condition.

[0009] In some embodiments, the disease or disorder is selected from juvenile neuronal ceroid lipofuscinosis (CLN3) disease, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, variant 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 Kufor-Rakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID), and autosomal recessive osteopetrosis type 4 (OPTB4).

[0010] In some embodiments, the disease or disorder is characterized by a defective function of the CLN3 protein.

[0011] In some embodiments, the affected cells are neuronal cells. In some embodiments, the affected cells are located in a tissue or organ, such as the liver, spleen, or brain.

[0012] In some embodiments, the agent reduces the accumulation level of SCMAS in the lysosomes of the affected cells. In some embodiments, the agent comprises recombinant human TPP1 protein. In some embodiments, the TPP1 protein is an inactive zymogen. In some embodiments, the TPP1 protein is mannose-6-phosphorylated.

[0013] In some embodiments, the therapeutically effective amount of the TPP1 protein is such that the affected cells receive about 1.0 to about 100 nM of the recombinant human TPP1 protein.

[0014] In some embodiments, the agent comprises a nucleic acid molecule comprising a nucleotide sequence encoding TPP1 or a variant thereof.

[0015] In some embodiments, the agent is administered by injection. In some embodiments, the injection is intracranial. In some embodiments, the agent is delivered to the lysosomes of the affected cells. In some embodiments, the agent is administered in a controlled release system.

[0016] In some embodiments, the subject is a mammal, such as a human.

[0017] In another aspect, the present disclosure provides an animal model for studying diseases or conditions such as lysosomal storage diseases. In some embodiments, the animal model comprises: (i) a heterozygous knockout of the tripeptidyl peptidase 1 (Tpp1) gene (Tpp1 + / - ), and (ii) homozygous knockout of Cln3 gene (Cln3 - / - ), wherein the life span of the mouse model is shortened compared to a wild-type animal. In some embodiments, the animal is a mouse.

[0018] In some embodiments, the disease or disorder is selected from late infantile neuronal ceroid lipofuscinosis (CLN2) disease, juvenile neuronal ceroid lipofuscinosis (CLN3) disease, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, variant 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 Kuller-Lae syndrome, Sanphilip D syndrome (mucopolysaccharidosis type IIID), and autosomal recessive osteopetrosis type 4 (OPTB4).

[0019] In some embodiments, the lifespan of the animal is shortened by at least 25% compared to a wild-type animal.

[0020] In some embodiments, the Tpp1 gene or the Cln3 gene comprises at least one mutation selected from the group consisting of 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.

[0021] In some embodiments, the Tpp1 gene is located at Chr 7E3;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 a neo insertion in intron 11 and an Arg446His missense mutation in exon 11 immediately upstream of the neo insertion.

[0022] In some embodiments, the Cln3 gene is located at Chr 7F3; 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 part of exons 1-6 in the Cln3 gene.

[0023] In some embodiments, the lysosomal accumulation level of mitochondrial ATP synthase subunit c (SCMAS) is increased in the animal model. In some embodiments, the lysosomal accumulation level of SCMAS is increased by at least 50% in the animal model.

[0024] In some embodiments, the expression level of Niemann-Pick disease type C1 (NPC1) and / or cathepsin F (CTSF) in the animal model is increased. In some embodiments, the expression level of NPC1 and / or CTSF in the animal model is increased by at least 40%, or the expression level of acid sphingomyelinase (SMPD1) is decreased by 40%.

[0025] In some embodiments, the animal model is characterized by deficits in locomotor activity.

[0026] Also within the scope of the present disclosure are descendants of the animal models disclosed herein, as well as cells, tissues or cell lines derived from the animal models or descendants as disclosed herein.

[0027] In another aspect, the present disclosure also provides a method for obtaining the animal model disclosed above. The method comprises: (a) crossing a Tpp1 knockout animal with a Cln3 knockout second animal to obtain a double heterozygous animal (Tpp1 + / - ;Cln3 + / - ); and (b) by mating Tpp1 - / -;Cln3 - / - ×Tpp1 - / - ;Cln3 - / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - , crossing animals that are doubly heterozygous.

[0028] In another aspect, the present disclosure further provides a method for identifying an agent for treating a disease or condition in 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 as disclosed herein with a candidate agent, and evaluating the effect of the candidate agent on the cell, tissue, or cell line.

[0029] In some embodiments, the disease or disorder is characterized by accumulation of SCMAS in lysosomes of affected cells (eg, neuronal cells).

[0030] In some embodiments, the disease or disorder is selected from late infantile neuronal ceroid lipofuscinosis (CLN2) disease, juvenile neuronal ceroid lipofuscinosis (CLN3) disease, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, variant 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 Kuller-Lae syndrome, Sanphilip D syndrome (mucopolysaccharidosis type IIID), and autosomal recessive osteopetrosis type 4 (OPTB4).

[0031] In some embodiments, the phenotype is lifespan in the animal model.

[0032] 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 level of NPC1 and / or CTSF.

[0033] In some embodiments, the candidate agent comprises a protein, a peptide, a peptidomimetic, a nucleic acid, or a small molecule.

[0034] The foregoing summary of the invention is not intended to limit every aspect of the present disclosure, and additional aspects are described in other parts such as the following specific embodiments. The entire document is intended to be associated as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found in the same sentence or paragraph or section of the document. Other features and advantages of the present invention will become apparent from the following specific embodiments. However, it should be understood that although the specific embodiments and specific examples indicate specific embodiments of the present disclosure, they are given only by way of illustration, because various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art based on this specific embodiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Display Tpp1 - / - Results of survival analysis of mice on the mixed C57BL / 6N, C57BL / 6J genetic background including Tpp1 - / - ;Cln3 + / + Historical data: There was no significant difference in survival compared to littermates of the same genotype produced by matings between Tpp1 and Cln3 mutants (log-rank P = 0.8475. Table 1 shows the median survival and number of animals analyzed by genotype.

[0036] Figures 2A and 2B Display Tpp1 + / - or Tpp1 + / + Results of survival analysis of mice in the C57BL / 6J background. Figure 2A shows a comparison of Tpp1Cln3 mutant mice. Data were obtained from an equal number of male and female animals. Table 1 shows the median survival and number of animals analyzed by genotype. The Yuan survival dataset (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 contains male and female mice in the C57BL / 6J substrain background. Figure 2B shows Tpp1 from the current study. + / + ;Cln3 - / - Survival of animals is similar between wild type and Tpp1 + / + ;Cln3 - / - Comparison of historical data for animals.

[0037] Figure 3Display Tpp1 - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Similar levels of astrogliosis in animals. Immunofluorescence staining of the astrocyte marker glial fibrillary associated protein (GFAP) showed that Tpp1 - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Tpp1 + / - Cln3 - / - and Tpp1 + / - Cln3 + / - The levels of GFAP immunoreactivity in the gray matter of Tpp1 were relatively low, which is comparable to previously published data from wild-type mice in which GFAP staining was primarily confined to the white matter. - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Corresponding astrocyte hypertrophy in mice. Scale bars = 100 μm and 25 μm (inset).

[0038] Figure 4 Display Tpp1 - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Similar levels of microglial activation in animals. Immunofluorescence staining of microglial marker CD68 showed that Tpp1 - / - Cln3 + / + and Tpp1 - / - Cln3 - / - In contrast, Tpp1 + / - Cln3 - / - and Tpp1 + / - Cln3 + / - The level of CD68 immunoreactivity in Tpp1 was relatively low, which is comparable to previously published data in wild-type mice. - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Corresponding microglial hypertrophy in mice. Scale bars = 100 μm and 25 μm (inset).

[0039] Figure 5Results of quantitative analysis of glial activation are shown. Threshold image analysis demonstrated different levels of glial fibrillary associated protein (GFAP, astrocytes) and CD68 (microglia) in the primary somatosensory cortex (S1BF) and ventral posterior nucleus of the thalamus (VPM / VPL) of animals of different genotypes. These data demonstrate that Tpp1 is activated in animals of different genotypes compared to animals of other genotypes. - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Levels of both antigens were significantly elevated in mice with the other genotypes, while animals of the other genotypes showed very low levels of immunoreactivity to these markers. Asterisks indicate differences in Tpp1 expression by one-way ANOVA using Dunnett's test for multiple comparisons. + / + ;Cln3 + / - Compared with the adjusted p value < 0.05.

[0040] Figure 6 Relative protein expression in Tpp1 and Cln3 mutant mice determined by SPS-MS3 quantification of isobaric labeled peptides is shown. Q values ​​were calculated based on peptide data for each protein using the false discovery rate two-stage incremental method of Benjamini, Krieger and Yekutieli. Dashed lines represent 1% FDR (y-axis) and 2-fold arbitrary multiple change (x-axis). The axes are cut off at Q values ​​of 1E-10 and ratios of 1 / 16 to 16. Red and black symbols represent known lysosomes (based on (17)) and other proteins, respectively. The names of selected related proteins are shown in red (lysosomes) or black (others).

[0041] Figures 7A and 7B Figure 7A shows the correlation between significant proteomic changes in animals of different genotypes. - / ; - Cln3 + / + Protein levels of Tpp1 - / - ;Cln3 - / - Comparison of protein levels of Tpp; goodness of fit, R = 0.7901. NNT was reviewed (see Examples). Figure 7B shows that Tpp + / + ;Cln3 - / - Protein levels of Tpp1 + / - ;Cln3 - / -Comparison of protein levels of ; goodness of fit, R = 0.5752. Compared with the wild type, the indicated proteins are significantly changed in at least one model (FDR 1%), the change amplitude is ≥ 1.5 times in at least one model, and the change direction is consistent in the two models. The names of the selected related proteins are shown in red (lysosome) or black (other). Solid symbols represent proteins that are significantly changed in the two genotypes being compared, and hollow symbols represent proteins that are significantly changed in one of the two genotypes being compared.

[0042] Figure 8 TPP1 activity in mutant mice is shown. The activity measured in each genotype was compared with the wild type using Dunnett's multiple comparison test. ns, not significant; **, p < 0.01; ***, p < 0.001.

[0043] Figures 9A, 9B, 9C, 9D and 9E Show expression of Tg TPP1+ Figure 9A shows the expression of TPP1 in transgenic Tg TPP1+ was integrated into ROSA26, and a synthetic intron was used to drive mouse TPP1 expression from the chicken actin (CAG) promoter. TPP1+ Figure 9C shows that Tg TPP1+ The mouse strain hemizygous for this transgene was designated R26Tg TPP1+ / 0 , mice lacking the TPP1 transgene were designated R26Tg 0 / 0 In the double-labeled immunofluorescence images, TPP1 and the neuronal marker NeuN ( FIG. 9D ) or the microglial marker Iba1 ( FIG. 9E ) as well as Hoescht-stained nuclei are indicated.

[0044] Fig.10 Shown is SCMAS staining in liver.

[0045] Fig.11 SCMAS staining in spleen is shown.

[0046] Fig.12 SCMAS staining in the brain is shown.

[0047] Fig.13 Quantification of SCMAS accumulation is shown. The area of ​​the image obscured by SCMAS-containing inclusions was quantified using ImageJ in a genotype-blind analysis. Data were analyzed using unpaired one-way ANOVA, comparing the mean of each sample to the mean of all other samples, with Tukey's test corrected for multiple comparisons. Note that while all possible comparisons were tested, we only show the Tg-Cln3 - / -and Tg+Cln3 - / - Paired comparison bars. ****. DETAILED DESCRIPTION

[0048] 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 studying lysosomal storage diseases. In particular, compared with wild-type animals, animals with Tpp1 + / - ;Cln3 - / - The double knockout animal model has a truncated survival, making it useful for developing therapies for lysosomal storage diseases such as juvenile neuronal ceroid lipofuscinosis (JNCL) using survival as an endpoint.

[0049] Methods of treating lysosomal storage diseases

[0050] In one aspect, the present disclosure provides a method of treating a subject having a disease or condition 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 the symptoms caused by the disease or condition.

[0051] In some embodiments, the method comprises selecting a subject having elevated levels of accumulation of SCMAS in lysosomes of affected cells compared to a reference level.

[0052] In some embodiments, the method comprises: (a) obtaining a sample containing affected cells; (b) analyzing the sample and determining the accumulation level of SCMAS in the lysosomes of the affected cells; (c) if the accumulation level of SCMAS in the lysosomes of the affected cells of the subject is increased compared to a reference level, identifying the subject as being likely to benefit from treatment with an agent that increases the level or activity of TPP1; and (d) administering to the subject a therapeutically effective amount of the agent to reduce or eliminate the symptoms caused by the disease or condition.

[0053] In some embodiments, the disease or condition is characterized by the accumulation of one or more storage products (e.g., SCMAS) in the lysosomes of affected cells (e.g., neurons). One way to determine the condition is to find that the lysosomes have accumulated storage materials, which can be accomplished by known methods (e.g., microscopy or immunofluorescence). An example of a storage material that will be detected in the lysosome is SCMAS. In some embodiments, treatment with TPP1 protein will reduce or eliminate mitochondrial ATP synthase, especially SCMAS, in the lysosomes of affected cells (e.g., neurons). Detection of the elimination of storage materials (e.g., SCMAS) in the lysosomes of affected cells can be performed by known methods as described above.

[0054] In some embodiments, the disease or condition is selected from 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, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease caused by mutations in the Cln5 gene, variant late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease caused by mutations in the Cln6 gene, neuronal ceroid lipofuscinosis type 7 (CLN8) disease caused by mutations in the MFSD8 gene. 7) diseases, northern epileptic 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 Kuller-Lae syndrome caused by mutations in the gene ATP13A2, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) caused by mutations in the GNS gene, and autosomal recessive osteopetrosis type 4 (OPTB4) caused by mutations in the CLCN7 gene.

[0055] In some embodiments, the disease or disorder is characterized by a functional defect in the CLN3 protein. An example of such a disorder is JNCL.

[0056] In some embodiments, the affected cells can be of any cell or tissue type, such as neurons. In some embodiments, the affected cells are neuronal cells. In some embodiments, the affected cells are located in tissues or organs, such as the liver, spleen, or brain.

[0057] The level or activity of TPP1 can be measured by determining or estimating protein levels or mRNA levels. Methods for determining or estimating protein levels or mRNA levels are well known in the art. Such methods may include enzyme activity analysis, microscopy, immunofluorescence and nucleic acid hybridization (e.g., using proteins and nucleic acids described in U.S. Serial 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., immunoblot analysis, immunoprecipitation analysis). The mRNA level can be determined by RT-PCR.

[0058] In some embodiments, the reference level can be obtained from a subject before administering an agent or composition thereof for increasing the level or activity of TPP1. In some embodiments, the reference level can be obtained from a control subject or a group of individuals who do not suffer from a disease or condition or have not yet been diagnosed with a disease or condition. In some embodiments, the reference level is based on the average level of the level or activity of TPP1 in a group that does not suffer from a disease or condition, for example. In some embodiments, the reference level is based on the median or median level of a group of individuals including patients with a disease or condition.

[0059] In some embodiments, the agent reduces the accumulation level of SCMAS in the lysosomes of the affected cells. In some embodiments, the affected cells are located in tissues or organs 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.

[0060] In some embodiments, the agent comprises a protein, a peptide, a peptidomimetic, a nucleic acid, or a small molecule.

[0061] In some embodiments, the agent comprises a recombinant human TPP1 protein. In some embodiments, the TPP1 protein is an inactive zymogen. In some embodiments, the TPP1 protein is mannose-6-phosphorylated.

[0062] 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 with the amino acid sequence of SEQ ID NO: 1, or comprises an amino acid sequence of SEQ ID NO: 1.

[0063] 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.

[0064] SEQ ID NO:1

[0065]

[0066] SEQ ID NO:2

[0067]

[0068] As used herein, the term "variant" refers to a first molecule that is related to a second molecule (also referred to as a "parent" molecule). The variant molecule can be derived from, separated from, based on, or homologous to a parent molecule. For example, a mutant form of TPP1 (including a TPP1 mutant with a cysteine ​​substitution) is a variant of wild-type TPP1. The term variant can be used to describe a polynucleotide or a polypeptide.

[0069] When applied to proteins, variant polypeptides may have complete amino acid sequence identity with the original parent polypeptide, or may have less than 100% amino acid identity with the parent protein. For example, the variant of an amino acid sequence may 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 fusion amino acid sequences. Polypeptide variants also include polypeptides that are part or subsequences of a parent polypeptide. For example, the present invention also encompasses unique subsequences of polypeptides disclosed herein (e.g., as determined by standard sequence comparison and alignment techniques).

[0070] On the other hand, polypeptide variants include polypeptides containing small, insignificant or insignificant changes to the parent amino acid sequence. For example, small, insignificant or insignificant changes include amino acid changes (including substitutions, deletions and insertions) that have little or no effect on the biological activity of the polypeptide, and produce functionally identical polypeptides, including the addition of non-functional peptide sequences. In other aspects, variant polypeptides of the present invention change the biological activity of the parent molecule. It will be appreciated by the technician that the present invention encompasses many variants of the disclosed polypeptides.

[0071] In some aspects, polynucleotide or polypeptide variants of the invention may include variant molecules in which a small percentage of nucleotide or amino acid positions are altered, added or deleted, such as typically less than about 10%, less than about 5%, less than 4%, less than 2% or less than 1%.

[0072] As used herein, a "functional variant" of a protein refers to a variant of such a protein that at least partially retains the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs, etc. Functional variants also include fusion products of such a protein with another generally unrelated nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be artificial.

[0073] In certain embodiments, TPP1 variants may include one or more conservative modifications. TPP1 variants with one or more conservative modifications may retain desired functional properties, which may 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 a protein containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which an amino acid residue is substituted by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), including one or more conservative modifications. Cas proteins with one or more conservative modifications can 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 amino acid modifications that do not significantly affect or change the binding properties of proteins containing amino acid sequences. 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 amino acid substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0074] As used herein, the homology percentage between two amino acid sequences is equivalent to the identity percentage between the two sequences. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences (i.e., homology %=number of identical positions / total number of positions × 100), which takes into account the number of spaces that need to be introduced for the best comparison of the two sequences and the length of each space. The comparison of the sequence between the two sequences and the determination of the identity percentage can be completed using a mathematical algorithm as described in the following non-limiting examples.

[0075] The percent identity between two amino acid sequences can be determined using the E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) algorithm, which has been 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 Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0076] Additionally or alternatively, the protein sequences of the invention can be further used as "query sequences" to search public databases, e.g., 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, word length = 3) to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be employed as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST programs and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

[0077] In some embodiments, the TPP1 variant can be conjugated or connected to a detectable label or a detectable marker (e.g., a radionuclide, a fluorescent dye). In some embodiments, the detectable label can be an affinity tag. As used herein, the term "affinity tag" refers to a portion connected to a polypeptide that allows the polypeptide to be purified from a biochemical mixture. The affinity tag can be composed of an amino acid sequence, or can include an amino acid sequence to which a chemical group is connected by post-translational modification. Non-limiting examples of affinity tags include His tags, CBP tags (CBP: calmodulin binding protein), CYD tags (CYD: covalent but dissociable NorpD peptide), Strep tags, StrepII tags, FLAG tags, HPC tags (HPC: heavy chain of protein C), GST tags (GST: glutathione S transferase), Avi tags, biotinylation tags, Myc tags, 3×FLAG tags, SUMO tags, and MBP tags (MBP: maltose binding protein). Additional examples of affinity tags can be found in Kimple et al., Curr Protoc Protein Sci. 2013 Sep 24;73:Unit 9.9.

[0078] In certain embodiments, the detectable label can be conjugated or connected to the N-terminal and / or C-terminal of TPP1 variant. Detectable label and affinity tag can also be separated by one or more amino acids. In certain embodiments, the detectable label can be conjugated or connected to TPP1 variant by cleavable element. In the context of the present invention, the term "cleavable element" relates to a peptide sequence that is easily cracked by chemical reagents or enzyme means (such as protease). Protease can be sequence-specific (for example, thrombin) or can have limited sequence specificity (for example, trypsin). Cleavable elements I and II can also be included in the amino acid sequence of detection label or polypeptide, particularly when the last amino acid of detection label or polypeptide is K or R.

[0079] As used herein, the term "conjugate" or "conjugation" or "linking" as used herein refers to the joining of two or more entities to form one entity. Conjugates encompass peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0080] The term "fusion polypeptide" or "fusion protein" means a protein produced by joining two or more polypeptide sequences together. Fusion polypeptides encompassed in the present invention include the translation product of a chimeric gene construct that joins a nucleic acid sequence encoding a first polypeptide to 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 connected by a peptide bond or by several peptides. The fusion protein may also include a peptide linker between the two domains.

[0081] In some embodiments, the agent comprises a fusion protein comprising TPP1 or a variant thereof.

[0082] In some embodiments, the agent comprises a nucleic acid (e.g., DNA, RNA) having a polynucleotide sequence encoding a TPP1 protein or a 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 a variant thereof.

[0083] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene operably associated therewith in a target cell and direct the expression of the gene. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows transcription of a large amount of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or the protein encoded by the gene is produced by the cell 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.

[0084] The vector may comprise a polynucleotide encoding RNA (e.g., RNAi, ribozymes, miRNA, siRNA) that, when transcribed from the polynucleotide of the vector, will cause the chimeric protein to accumulate on the plasma membrane of the target cell. Useful vectors include, but are not limited to, lentiviral vectors, HSV vectors, and adenoviral vectors. Slow viruses include, but are not limited to, HIV-1, HIV-2, SIV, FIV, and EIAV. Slow viruses 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.

[0085] In certain embodiments, the vector is a recombinant AAV vector. The AAV vector is a relatively small DNA virus that can be integrated into the genome of the cell infected by it in a stable and site-specific manner. The AAV vector can infect a wide spectrum of cells without inducing any effects on cell growth, morphology or differentiation, and it does not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. It covers about 4700 bases and contains about 145 bases of reverse terminal repeat (ITR) regions at each end, and the ITR region serves as the replication origin of the virus. The rest of the genome is divided into two basic regions carrying encapsidation function: the left-hand part of the genome, which contains the rep gene involved in viral replication and viral gene expression; and the right-hand part of the genome, which contains the cap gene of the capsid protein encoding the virus.

[0086] In recent years, the application of AAV as a vector for gene therapy has been rapidly developed. Wild-type AAV can infect the division or non-division cells or tissues of mammals (including humans) with relatively high titers, and can also be integrated into human cells at specific sites (on the long arm of chromosome 19) (Kotin, RM et al., Proceedings of the National Academy of Sciences of the United States of America 87:2211-2215, 1990) (Samulski, RJ et al., Journal of European Molecular Biology (EMBO J.) 10:3941-3950, 1991, the disclosure of which is hereby incorporated herein by reference in its entirety). AAV vectors without 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 of which is free outside the chromosome; the other is integrated into the chromosome, of which the former is the main form. In addition, AAV has not been found to be associated with any human disease to date, and no changes in biological properties caused by integration have been observed. Sixteen AAV serotypes have been reported in the literature, namely AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16 and recombinant variants thereof, among which AAV5 was originally isolated from humans (Bantel-Schaal and H. zur Hausen. 1984. Virology 134: 52-63), while AAV1-4 and AAV6 were both discovered in adenovirus research (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Virol. 1999, 73: 939-947).

[0087] AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable (see, e.g., 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, ed. JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, pp. 5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski, "The Genus Dependovirus," in JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, eds., pp. 15-23, Hudder Arnold, London, UK). Arnold (2006), the disclosure of which is hereby incorporated by reference in its entirety. Methods for purifying vectors can be found, for example, in U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006, and WO / 1999 / 011764, entitled "Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors," the disclosure of which is hereby incorporated by reference in its entirety. Preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosure of which is hereby incorporated 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., Intl. Pat. Appl. Pub. Nos. 91 / 18088 and WO 93 / 09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941; and EP 0 488 528, all of which are incorporated herein by reference in their entireties).These publications describe various AAV-derived constructs in which rep and / or cap genes are deleted and replaced by relevant genes, and the use of these constructs for transferring relevant genes in vitro (into cultured cells) or in vivo (directly into an organism). Replication-defective recombinant AAV can be prepared by co-transfecting a plasmid containing relevant nucleic acid sequences flanking two AAV inverted terminal repeat (ITR) regions and a plasmid carrying AAV encapsidation genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The resulting AAV recombinant is then purified by standard techniques.

[0088] In some embodiments, the vector can be encapsidated into a viral particle (e.g., an AAV viral particle, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, a recombinant viral particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein is also provided. Methods for producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0089] 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, AAV 12, AAV13, AAV rh74 and recombinant subtypes thereof.

[0090] In some embodiments, the vector may be derived from a retrovirus, including avian reticuloendothelioma virus (duck infectious anemia virus, spleen necrosis virus, Twiehaus-strain reticuloendothelioma virus, type C retrovirus, reticuloendothelioma virus Hungary-2 (REV-H-2) and feline leukemia virus (FeLV). Retroviral genomes have been modified for use as vectors (Cone and Mulligan, Proceedings of the National Academy of Sciences, 81: 6349-6353, (1984)). Non-limiting examples of retroviruses include lentiviruses, such as human immunodeficiency virus (HIV-1 and HIV-2), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), Maedi / Visnavirus, caprine arthritis / encephalitis virus, equine infectious anemia virus (EIAV), and bovine immunodeficiency virus (BIV); avian type C retroviruses, such as avian leukemia virus (ALV); HTLV-BLV retroviruses, such as bovine leukemia virus (ALV); retroviruses, such as human spumavirus (HSRV), feline syncytial virus (FeSFV), human spumavirus, simian spumavirus, and bovine spumavirus; and type D retroviruses, such as Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus, and leaf monkey virus.

[0091] In some embodiments, the vector comprises a retroviral vector or a lentiviral vector. In some embodiments, lentiviral and retroviral vectors can be packaged using their native envelope proteins, or can be modified to be encapsulated with heterologous envelope proteins. Examples of envelope proteins include, but are not limited to, amphophilic envelopes, monophilic envelopes, or heterophilic envelopes, or can be envelopes comprising amphophilic portions and monophilic portions. The protein can also be a protein of any of the above-mentioned retroviruses and lentiviruses. Alternatively, the env protein can be a modified, synthetic, or chimeric env construct, or can be obtained from non-retroviruses such as vesicular stomatitis virus and HVJ virus. Specific non-limiting examples include the envelope of Moloney Murine Leukemia Virus (MMLV), Rous Sarcoma Virus, baculovirus, Jaagsiekte Sheep Retrovirus (JSRV) envelope protein, and feline endogenous virus RD114; the gibbon ape leukemia virus (GALV) envelope; the baboon endogenous virus (BaEV) envelope; the simian sarcoma-associated virus (SSAV) envelope; the amphotropic murine leukemia virus (MLV-A) envelope; the human immunodeficiency virus envelope; the avian leukemia virus envelope; the endogenous heterotropic NZB virus envelope; and the envelope of the paramyxoviridiae family, such as, but not limited to, the HVJ virus envelope.

[0092] Once the expression vector or DNA sequence containing the construct has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, 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 varied or optimized based on the present specification according to the specific expression vector and mammalian host cell used.

[0093] In some embodiments, the agent is a TPP1 agonist. The term "agonist" refers to a compound that causes the excitation of the TPP1 pathway and causes a cellular response. TPP1 agonists mimic the effects of endogenous ligands (TPP1) and produce physiological responses similar to those provided by endogenous ligands. The term includes agents that cause upregulation and / or increase in the activity of TPP1-mediated signaling pathways when administered to subjects in need (e.g., TPP1 variants / fragments, fusion proteins comprising TPP1. In some embodiments, the term includes agents that cause a decrease in the number or activity of T cells when administered to a subject.

[0094] TPP1 protein can be in the form of an inactive zymogen (or prodrug), or in a shorter active form. Any of these can be naturally isolated or recombinant. When produced, for example, in Chinese hamster ovary (CHO) cells, TPP1 protein in the form of a zymogen is obtained. This form is converted into an active form after acidification. Therefore, the zymogen is a highly suitable prodrug that remains inactive until delivered to the lysosome, and the acidic environment of the lysosome will activate the prodrug. Obtaining TPP1 protein in any of these forms is described in detail below and in U.S. Serial No. 08 / 931,608 and Sleat et al. (1997). In short, known methods can be used to separate TPP1 protein from human brain samples by purifying glycoproteins containing mannose-6-phosphate from normal, JNCL brain samples or LINCL brain samples and separating protein bands present in normal samples but not present in JNCL or LINCL samples. Once the protein is obtained, the corresponding gene and cDNA are also separated using known methods. Recombinant protein is then produced from cDNA using known methods. Any of the above forms of TPP1 protein may or may not be mannose-6-phosphorylated.

[0095] In some embodiments, the pharmaceutical 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 pharmaceutically acceptable salt thereof.

[0096] As used herein, "PPARα agonist" refers to a compound or composition that, when combined directly or indirectly with PPARα (preferably directly binds to PPARα), stimulates or increases a receptor-typical in vivo or in vitro response (e.g., transcriptional regulatory activity) as measured by assays known to those skilled in the art, including but not limited to U.S. Pat. Nos. 4,981,784, 5,071,773, 5,298,429, 5,506,102, WO89 / 05355, WO91 / 06677, WO92 / 05447, WO93 / 11235, WO93 / 23431, WO94 / 23068, WO95 / 18380, CA 2,034,220 and Lehmann et al., J. Biol. Chem. 270:12953-12956 (1995), which are incorporated herein by reference. PPARα agonists can also be identified according to the assay described in U.S. Pat. No. 6,008,239.

[0097] In some embodiments, the PPARα agonist may include a fibrate compound, including but not limited to gemfibrozil, fenofibrate, bezafibrate, clofibrate, ciprofibrate, and analogs, 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: 1449-1456 (1991), Kliewer et al., PNAS 94: 4318-4323 (1997), Amri et al., PNAS 32: 1457-1463, (1991), and Grimaldi et al., PNAS 89: 10930-10934 (1992) are incorporated herein by reference. PPARα agonist compounds described in U.S. Pat. No. 6,008,239, WO 97 / 27847, WO 97 / 27857, WO 97 / 28115, WO 97 / 28137 and WO 97 / 28149 are further incorporated herein by reference. Certain fibrates as described in WO92 / 10468 and WO01 / 80852 are also incorporated herein by reference.

[0098] "Isomers" are different compounds with the same molecular formula. "Stereoisomers" are isomers with different stereochemical configurations, i.e., only the atoms are arranged differently in space. "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 represent a racemic mixture where appropriate. "Diastereomers" 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 (R) or (S). The resolved compound with unknown absolute configuration can be specified as (+) or (-) according to the direction (right-handed or left-handed) of rotating plane polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R) or (S) based on absolute stereochemistry. The chemical entities, pharmaceutical compositions, and methods of the present invention are intended to include all such possible isomers, including racemic mixtures, optically pure forms, and mixtures of intermediates. 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 centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers.

[0099] Stereoisomers are compounds that differ only in spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are non-superimposable, most often because they contain asymmetrically substituted carbon atoms that serve as chiral centers. "Enantiomers" means one of a pair of molecules that are mirror images of each other and non-superimposable. Diastereomers are stereoisomers that are independent of the mirror image, most often 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. Therefore, "R*" and "S*" represent the relative configuration of the substituents around one or more chiral carbon atoms. The symbol "*" in the structural formula represents the presence of a chiral carbon center.

[0100] "Racemate" or "racemic mixture" means a compound of equimolar amounts of two enantiomers, wherein such mixture exhibits no optical activity, ie, it does not rotate the plane of polarized light.

[0101] "Geometric isomers" means isomers that differ in the orientation of substituent atoms in relation to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. The atoms on each side of the carbon-carbon double bond (except H) 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" indicate configurations relative to the core molecule.

[0102] As used herein, "derivative" refers to a chemical substance that is structurally related to another chemical substance (i.e., the "original" substance, which may be referred to as the "parent" compound). A "derivative" may be prepared from a structurally related parent compound in one or more steps. The phrase "closely related derivative" means a derivative whose molecular weight does not exceed 50% of the weight of the parent compound. The general physical and chemical properties of closely related derivatives are also similar to those of the parent compound. A "pharmaceutically active derivative" refers to any compound that is capable of directly or indirectly providing an activity disclosed herein when administered to a recipient.

[0103] "Analog" refers to a small organic compound, nucleotide, protein or polypeptide that has an activity or function similar or identical to a compound, nucleotide, protein or polypeptide or compound having the desired activity of the present disclosure, but may not necessarily include a sequence or structure similar or identical to that of the preferred embodiments.

[0104] "Prodrug" refers to a compound that can be converted to a biologically active compound described herein under physiological conditions or by solvolysis. Thus, the term "prodrug" refers to a pharmaceutically 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 generally provide advantages of solubility, tissue compatibility or delayed release in mammalian organisms (see, e.g., Bundgaard, H., Design of Prodrugs (1985) (Elsevier, Amsterdam). The term "prodrug" also refers to any covalently bonded carrier that releases the active compound in vivo when administered to a subject. Prodrugs of active compounds as described herein can be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved in routine manipulation or in vivo to produce the active parent compound. Prodrugs include compounds such as hydroxyl, amino or sulfhydryl groups bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, is cleaved to form a free hydroxyl, free amino or free sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohols in the active compound, various ester derivatives of carboxylic acids, or acetamide, formamide and benzamide derivatives of amine functional groups. Various forms of prodrugs are well known in the art and are described in: (a) The Practice of Medicinal Chemistry. of Medicinal Chemistry, Camille G. Wermuth et al., Chapter 31, (Academic Press, 1996); (b) Prodrug Design, H. Bundgaard, ed., (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., Chapter 5, pp. 113-191 (Harwood Academic Publishers, 1991); and (d) Hydrolysis in Drug and Prodrug Metabolism, Bernard Testa and Joachim M. Mayer, (Wiley-VCH, 2003).

[0105] In some embodiments, the agent for increasing the level or activity of TPP1 can 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 pharmaceutically acceptable carrier. In some embodiments, such compositions may include one or a combination of (e.g., two or more different) TPP1 variants. For example, the composition may include a combination of TPP1 variants with different genetic modifications.

[0106] Therefore, the present disclosure also provides a pharmaceutical composition for treating a disease or condition in a subject. The pharmaceutical composition comprises: (a) an agent capable of increasing the level or activity of TPP1 in a subject and (b) an optional pharmaceutically acceptable carrier. In some embodiments, the agent comprises a fusion protein containing a TPP1 protein or a variant / fragment thereof or a fusion protein containing a TPP1 protein or a variant thereof.

[0107] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one component useful in the present invention and other components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. Pharmaceutical compositions facilitate administration of one or more components of the present invention to an organism.

[0108] Pharmaceutical compositions or therapeutic formulations of agents for increasing the level or activity of TPP1 (e.g., TPP1 protein or variants thereof, TPP1 agonists) can be prepared by mixing the agent having the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride, benzethonium chloride, chloride); phenol, butyl alcohol 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 such as serum albumin, gelatin or immunoglobulins; 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 nonionic surfactants such as TWEEN, PLURONIC or polyethylene glycol (PEG).

[0109] The formulation may also contain more than one active ingredient necessary for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. For example, the formulation may further comprise another anti-inflammatory agent. These molecules are suitably present in combination in an amount effective for the intended purpose.

[0110] The active ingredient can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in 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).

[0111] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing agents (e.g., TPP1 proteins or variants thereof) for increasing the level or activity of TPP1, and the matrix is ​​in the form of a shaped article (e.g., a film or microcapsule). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT (injectable microspheres consisting of lactic acid-glycolic acid copolymers and leuprolide acetate) and poly-d(-)-3-hydroxybutyric acid (PHB). Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter time. When encapsulated, the agent is retained in the body for a long time. It may denature or aggregate due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, a rational strategy can be designed to achieve stabilization. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiol-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0112] The formulation for in vivo administration must be sterile, which can be easily achieved by filtration with a sterile filtration membrane. Sterile injectable solutions can be prepared by incorporating the required amount of active compound and one or a combination of the above-listed components into an appropriate solvent as required, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other components required from those components listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produces a powder of the active ingredient plus any additional required components from its previous sterile filtered solution.

[0113] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Typically, out of one hundred percent, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0114] The agent for increasing the level or activity of TPP1 (e.g., TPP1 protein or variant thereof) can be administered as a single dose, or more commonly multiple doses. The intervals between single doses can be, for example, weekly, monthly, every three months, or annually. The intervals can also be irregular, as indicated by measuring the patient's blood level of TPP1 protein or variant thereof.

[0115] The medicament or its pharmaceutical composition can be administered by one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode will vary according to the desired result. For example, the administration of TPP1 protein or its variants may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means an administration mode usually performed by injection in addition to enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, TPP1 protein or its variants may be administered by non-parenteral routes, such as topical, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical.

[0116] In some embodiments, the TPP1 protein or a composition comprising the TPP1 protein can be introduced parenterally, transmucosally (e.g., orally, nasally or rectally) or transdermally. Preferably, administration is by injection, especially parenterally, such as by intravenous injection, and also includes but is not limited to intra-arteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intracerebroventricular and intracranial administration.

[0117] In some embodiments, the agent is administered by injection. In some embodiments, the injection is intracranial. In some embodiments, the agent is delivered to the lysosomes of the affected cells.

[0118] TPP1 protein or composition can be delivered in the form of vesicles, particularly liposomes (see Langer, Science 249: 1527-1533 (1990); Treat et al., 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 ibid. generally). This may be a preferred method of introducing TPP1 in order to reduce systemic side effects and increase cell penetration.

[0119] The TPP1 protein or composition can be delivered in a controlled release system. For example, it can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, 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); and Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, the controlled release system can be placed near the therapeutic target, so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Preferably, the controlled release device is introduced near the tissue site affected by LINCL in the subject. Other controlled release systems are discussed in Langer's review (Science 249:1527-1533 (1990)).

[0120] The dosage regimen is adjusted to provide the optimal desired response (e.g., a 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 in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are subject to and directly dependent on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art caused by individual sensitivity to such active ingredients compounded for treatment.

[0121] Skilled practitioners easily determine the amount of TPP1 protein that effectively alleviates or eliminates the symptoms caused by TPP1 protein deficiency. As discussed above, the relief of symptoms (i.e., relief or elimination) can be determined based on the patient's physical condition, such as epileptic seizure cessation or epileptic seizure volume reduction or intensity reduction. Or the cell sample (e.g., brain neurons) can be measured by determining the amount of storage products present in lysosomes and comparing with normal control cells to confirm the relief of the symptom. Therefore, the dosage can be determined by a skilled practitioner according to the patient's age, body shape and symptom. Alternatively, the amount of TPP1 protein administered can restore the normal level of TPP1 protein in the cell+, such as determined by comparing with normal cells. In a preferred treatment, the effective amount of TPP1 protein causes the affected cells to receive about 1.0nM to about 100nM of TPP1 protein. The dosage for ensuring that the affected cells receive about 1.0 to 100 nM of TPP1 protein can be determined by a skilled practitioner, for example by performing a biopsy after administration and analyzing the treated cells by known methods to determine how much injected or oral or inhaled TPP1 is needed to provide the desired cellular levels. When administered with an uptake inhibitor, the concentration of the uptake inhibitor should inhibit the immediate clearance of TPP1 protein near the site of administration. Such dosages can be determined by a skilled practitioner. When the uptake inhibitor is mannose-6-phosphate, 5 mM is a preferred dosage.

[0122] In some embodiments, TPP1 protein can be administered according to one of the following dosing schedules: (i) once every four weeks for six doses, then once every three months; (ii) once every three weeks; and (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight once every three weeks.

[0123] Alternatively, the medicament can be used as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the medicament in the patient. The dosage and frequency of administration can vary according to whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses 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 doses are sometimes required at relatively short intervals until disease progression is reduced or terminated, and preferably until the patient shows that symptoms of the disease are partially improved or completely improved. Thereafter, a preventive regimen can be administered to the patient.

[0124] The actual dosage level of the active ingredient in the composition can 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 a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed; the route of administration; the time of administration; the rate of excretion of the particular active ingredient employed; the duration of treatment; other drugs, compounds, and / or materials used in combination with the particular composition employed; the age, sex, weight, condition, general health, and previous medical history of the patient being treated; and similar factors well known in the medical field.

[0125] The terms "effective amount," "effective dose," or "effective dosage" are defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the 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 symptom-free periods, or prevention of impairment or disability resulting from disease affliction. A "prophylactically effective amount" or "prophylactically effective dose" of a drug / agent is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering a recurrence of the disease, inhibits the development or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease development or recurrence can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by analyzing the activity of the agent in in vitro assays.

[0126] Pharmaceutical composition can be used with medical device as known in the art.For example, therapeutic composition of the present invention can be used with needleless subcutaneous injection device, described device is as U.S. Patent No. 5399163, No. 5383851, No. 5312335, No. 5064413, No. 4941880, No. 4790824 and No. 4596556 disclosed device.The example of well-known implant and module that can be used for the present invention comprises the implant and module described in U.S. Patent No. 4487603, No. 4486194, No. 4447233, No. 4447224, No. 4439196 and No. 4475196.These patents are incorporated herein by reference.Many other such implants, delivery systems and modules are known to those skilled in the art.

[0127] In some embodiments, the method for treating a disease or condition of a subject as described above further comprises administering an additional therapeutic agent or therapy to the subject. In some embodiments, the TPP1 protein can be used alone or together with other active ingredients. It can be conjugated with a polyalkylene glycol moiety by known methods, or can be used as a part of a chimeric protein, such as being connected to an antibody or part thereof, transferrin, hormone, or growth factor. The TPP1 protein can be provided in the form of a prodrug, i.e., a stable inactive form (e.g., as described above) that becomes active once administered. The present invention provides a targeting molecule conjugated to TPP1, a DNA vector (including a virus) encoding TPP1, and a vector (i.e., a liposome) to target a desired cell or tissue (e.g., brain). As used herein, "targeting molecule" should mean a molecule that is positioned to a desired position when administered in vivo. In various embodiments, the targeting molecule can 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 internalization receptor on a target cell. In a specific embodiment, the targeting molecule is a peptide or a variant thereof comprising the well-known RGD sequence, which binds to an RGD receptor on the surface of a cell (such as a cancer cell, such as a human egg cell) having a receptor that recognizes the RGD sequence. Other ligands include, but are not limited to, transferrin, insulin, amylin, and the like. Receptor internalization is preferred for promoting intracellular delivery of TPP1 protein. In another embodiment, the targeting molecule is an antibody. Preferably, the targeting molecule is a monoclonal antibody. In one embodiment, in order to promote cross-linking, the antibody can be reduced to two heavy chain and light chain heterodimers, or the F(ab)2 fragment can be reduced and cross-linked with TPP1 through the reduced sulfhydryl group. The antibody used as a targeting molecule is specific for a cell surface antigen. In one embodiment, the antigen is a receptor. For example, an antibody specific for a receptor on a cancer cell such as a melanoma cell can be used. The present invention further provides the use of other targeting molecules such as lectins, carbohydrates, proteins, and steroids.

[0128] The compositions of the present invention preferably include an uptake inhibitor that reduces local clearance of TPP1 protein by cell surface receptors. This helps to ensure uniform application of TPP1 protein so that more cells obtain some TPP1 protein, rather than a few cells near the site of application obtaining most of the TPP1 protein. Clearance mechanisms include endocytosis of cell surface receptors such as mannose receptors, asialoglycoprotein receptors, and mannose-6-phosphate receptors. Therefore, a preferred uptake inhibitor is mannose-6-phosphate. The uptake inhibitor can be administered in the form of a composition together with the TPP1 protein, or can be administered separately but simultaneously, or both can be administered at different times, as long as the uptake inhibitor can have the desired effect.

[0129] As used herein, "treatment" or "treating" or "alleviate" or "improve" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit means any treatment-related improvement or effect on one or more diseases (e.g., inflammatory diseases), conditions, or symptoms being treated. For prophylactic benefit, an agent or composition thereof can be administered to a subject at risk for developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifested.

[0130] 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 performed simultaneously. In other embodiments, a first agent / therapy is administered before a second agent / therapy. It will be appreciated by those skilled in the art that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0131] In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms "subject" and "subjects" can refer to any vertebrate, including but not limited to mammals (e.g., cattle, 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, chimpanzees, etc.) and people). The subject can be a person or a non-human. In more exemplary aspects, the mammal is a person. As used herein, the expression "subject in need" or "patient in need" means a person or non-human mammal that exhibits one or more symptoms or indications of a disease or illness and / or has been diagnosed with a disease or illness. In some embodiments, the subject is a mammal. In some embodiments, the subject is a person.

[0132] "Sample", "test sample" and "patient sample" are used interchangeably herein. The sample can be a sample of serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells or tissues. Such samples can be used directly after being obtained from the patient, or can be pre-treated such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to change the characteristics of the sample in a certain manner as discussed herein or in other ways as known in the art. The terms "sample" and "biological sample" as used herein generally refer to test-related analytes (such as antibodies) and / or biological materials suspected of containing the analytes. The sample can be any tissue sample from a subject. The sample can contain proteins from a subject.

[0133] The terms "increased", "increase", "elevate", "enhance" or "activate" are generally used herein to refer to an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, such as 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 and including an increase of 100% or any increase between 10-100%, 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 compared to a reference level, or any increase between 2-fold and 10-fold or greater.

[0134] Animal Models for Studying Lysosomal Storage Diseases

[0135] In one aspect, the present disclosure provides an animal model for studying a disease or condition. In some embodiments, the animal model comprises: (i) a heterozygous knockout of the Tpp1 gene (Tpp1 + / -), and (ii) homozygous knockout of Cln3 gene (Cln3 - / - ), where the mouse model had a shortened lifespan compared to wild-type animals.

[0136] "Gene" refers to a DNA sequence that encodes a protein and may or may not include introns, exons, regulatory sequences (such as promoter or enhancer sequences) and a 5' untranslated region. As referred to herein, a "transcript" is an RNA molecule derived by transcription of an encoding gene or nucleic acid.

[0137] The term "knockout" related to a gene refers to changing the wild-type sequence of a gene so that a functional gene product is not produced. The term "gene product" encompasses the transcript of a gene and the protein translated from the transcript. For example, a mutation, insertion or deletion of the nucleotides of a wild-type gene sequence may result in the complete silencing of gene expression, or result in the expression of the gene sequence of the change, so that only a non-functional transcript is produced. A non-functional transcript can not be translated into a functional protein. If only one allele of a gene is changed, the gene knocked out is referred to as "heterozygous knockout". If both alleles are changed, knockout is referred to as "homozygous knockout". According to the convention in the art, heterozygous gene knockout is indicated by "het" or "+ / -", and homozygous knockout is indicated by "horn" or "- / -". If the two alleles of a gene remain unchanged (i.e., have a wild-type gene sequence), it is indicated by "wt" or "++".

[0138] The term "heterozygous" means a genetic condition that exists when different alleles are located at corresponding loci on homologous chromosomes. The term "homozygous" means a genetic condition that exists when the same alleles are located at corresponding loci on homologous chromosomes.

[0139] In some embodiments, the animal can be, for example, a mammal, including but not limited to mice, rats, sheep, dogs, cows, horses, non-human primates, pigs, cats, rabbits, goats, ferrets, guinea pigs, gerbils, or hamsters. The animal can also be a bird, including but not limited to chickens, ducks, or quail. In some embodiments, the animal is a member of the family Muridae. In another embodiment, the animal is a mouse.

[0140] In some embodiments, the disease or condition is selected from 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, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease caused by mutations in the Cln5 gene, variant late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease caused by mutations in the Cln6 gene, neuronal ceroid lipofuscinosis type 7 (CLN8) disease caused by mutations in the MFSD8 gene. 7) diseases, northern epileptic 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 Kuller-Lae syndrome caused by mutations in the gene ATP13A2, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) caused by mutations in the GNS gene, and autosomal recessive osteopetrosis type 4 (OPTB4) caused by mutations in the CLCN7 gene.

[0141] In some embodiments, the lifespan of the animal is reduced by at least 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%) compared to a wild-type animal.

[0142] In some embodiments, the Tpp1 gene or the Cln3 gene comprises at least one mutation selected from the group consisting of 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.

[0143] In some embodiments, the Tpp1 gene is located at Chr 7E3;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 a neo insertion in intron 11 and an Arg446His missense mutation in exon 11 immediately upstream of the neo insertion.

[0144] In some embodiments, the Cln3 gene is located at Chr 7F3; 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 part of exons 1-6 in the Cln3 gene.

[0145] In some embodiments, the lysosomal accumulation level of mitochondrial ATP synthase subunit c (SCMAS) of the animal model is increased. In some embodiments, the lysosomal accumulation level of SCMAS of the animal model is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200% or more. In some embodiments, the lysosomal accumulation level of SCMAS of the animal model is increased by at least 50%.

[0146] In some embodiments, the expression level of NPC1 and / or CTSF in the animal model is increased. In some embodiments, the expression level of NPC1 and / or CTSF in the animal model is increased by at least 40% or more, or the expression level of SMPD1 is decreased by at least 40% or more. In some embodiments, the expression level of NPC1 and / or CTSF in the animal model is increased by at least 40%, or the expression level of SMPD1 is decreased by 40%.

[0147] In some embodiments, the animal model is characterized by deficits in locomotor activity.

[0148] Also within the scope of the present disclosure are descendants of the animal models disclosed herein, as well as cells, tissues or cell lines derived from the animal models or descendants as disclosed herein.

[0149] The term "offspring" refers to the progeny of a particular cross. Typically, the progeny is produced by breeding two individuals. The progeny can be, for example, F1, F2, or any subsequent generation.

[0150] In another aspect, the present disclosure also provides a method for obtaining the animal model disclosed above. The method comprises: (a) crossing a Tpp1 knockout animal with a Cln3 knockout second animal to obtain a double heterozygous animal (Tpp1 + / - ;Cln3 + / - ); and (b) by mating Tpp1 - / - ;Cln3 - / - ×Tpp1 - / - ;Cln3 - / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - , crossing animals that are doubly heterozygous.

[0151] Methods for identifying agents for treating lysosomal storage diseases

[0152] In another aspect, the present disclosure further provides a method of identifying an agent for treating a disease or condition in 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.

[0153] In some embodiments, the method comprises contacting a cell, tissue, or cell line as disclosed herein with a candidate agent, and evaluating the effect of the candidate agent on the cell, tissue, or cell line.

[0154] In some embodiments, the phenotype is lifespan in the animal model.

[0155] 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 level of NPC1 and / or CTSF.

[0156] In some embodiments, the candidate agent comprises a protein, a peptide, a peptidomimetic, a nucleic acid, or a small molecule.

[0157] In yet another aspect, the present disclosure further provides a method for identifying an agent having TPP1 agonist activity. In some embodiments, the method comprises: (a) administering a certain amount of a candidate agent to a subject (e.g., an animal model disclosed herein); (b) analyzing a sample obtained from the subject and determining the lysosomal accumulation level of SCMAS and / or the activity / level of TPP1 in the sample; and (c) if the lysosomal accumulation level of SCMAS of the subject is reduced and / or the activity / level of TPP1 is increased compared to a reference level, then identifying the agent as having TPP1 agonist activity.

[0158] In some embodiments, the method comprises: (a) contacting a candidate agent with a sample comprising a cell, tissue or cell line as disclosed herein; (b) analyzing the sample and determining the level of lysosomal accumulation of SCMAS and / or the activity / level of TPP1 in the sample; and (c) if the sample exhibits a decreased level of lysosomal accumulation of SCMAS and / or an increased activity / level of TPP1 as compared to a reference level, then identifying the agent as having TPP1 agonist activity.

[0159] In some embodiments, the method may include culturing or expanding test cells and / or control cells. The term "culture" or "expansion" refers to maintaining or culturing cells under conditions where cells can proliferate and avoid aging. For example, cells can be cultured in a culture medium that optionally contains one or more growth factors (i.e., a mixture of growth factors). In some embodiments, the cell culture medium is a cell culture medium with defined components. The cell culture medium may include a new antigenic peptide. A stable cell line may be established to allow cells to continue to proliferate.

[0160] In some embodiments, the reference level can be obtained from a subject before administering an agent or composition thereof for increasing the level or activity of TPP1, or from a cell before contacting the agent or composition thereof. In some embodiments, the reference level can be obtained from a control subject or a group of individuals who do not suffer from a disease or condition or have not yet been diagnosed with a disease or condition, or from a control cell or an unaffected cell of the subject. In some embodiments, the reference level is obtained based on the average level of the level or activity of TPP1 in a group of patients who do not suffer from a disease or condition, for example. In some embodiments, the reference level is obtained based on the median or median level of a group of individuals including patients with a disease or condition.

[0161] In some embodiments, the disease or condition is characterized by the accumulation of one or more storage products (e.g., SCMAS) in the lysosomes of affected cells (e.g., neurons). One way to determine the condition is to find that the lysosomes have accumulated storage materials, which can be accomplished by known methods (e.g., microscopy or immunofluorescence). An example of a storage material that will be detected in the lysosome is SCMAS. In some embodiments, treatment with TPP1 protein will reduce or eliminate mitochondrial ATP synthase, especially SCMAS, in the lysosomes of affected cells (e.g., neurons). Detection of the elimination of storage materials (e.g., SCMAS) in the lysosomes of affected cells can be performed by known methods as described above.

[0162] In some embodiments, the disease or condition is selected from 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, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease caused by mutations in the Cln5 gene, variant late infantile neuronal ceroid lipofuscinosis type 6 (CLN6) disease caused by mutations in the Cln6 gene, neuronal ceroid lipofuscinosis type 7 (CLN8) disease caused by mutations in the MFSD8 gene. 7) diseases, northern epileptic 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 Kuller-Lae syndrome caused by mutations in the gene ATP13A2, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) caused by mutations in the GNS gene, and autosomal recessive osteopetrosis type 4 (OPTB4) caused by mutations in the CLCN7 gene.

[0163] In some embodiments, the disease or disorder is characterized by a functional defect in the CLN3 protein. An example of such a disorder is JNCL.

[0164] In some embodiments, the affected cells can be of any cell or tissue type, such as neurons. In some embodiments, the affected cells are neuronal cells.

[0165] The level or activity of TPP1 can be measured by determining or estimating protein levels or mRNA levels. Methods for determining or estimating protein levels or mRNA levels are well known in the art. Such methods may include enzyme activity analysis, microscopy, immunofluorescence and nucleic acid hybridization (e.g., using proteins and nucleic acids described in U.S. Serial 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., immunoblot analysis, immunoprecipitation analysis). The mRNA level can be determined by RT-PCR.

[0166] The level of measuring TPP1 can be carried out by analyzing the protein itself (by Western blot, ELISA, RIA and other technologies known to those skilled in the art), by analyzing the mRNA encoding these proteins (such as quantitative PCR, Northern blot, RNA enzyme protection analysis, RNA dot blot and other technologies known to those skilled in the art) or by analyzing the activity of the regulatory element of TPP1 gene. For example, the activity of the regulatory element can be assessed by a reporter construct, which is composed of a DNA segment of a promoter, enhancer and / or intron element coupled with a cDNA encoding a reporter (such as luciferase, beta-galactosidase, green fluorescent protein or other reporter genes that can be easily analyzed). These reporter constructs can be stably or transiently transfected into cells.

[0167] Additional Definitions

[0168] To aid understanding of the detailed description of compositions and methods according to the present disclosure, some clear definitions are provided to facilitate the clear disclosure of various aspects of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0169] The term "agent" is used herein to refer to a compound, a mixture of compounds, a biomacromolecule (such as a nucleic acid, an antibody, a protein or a portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such an agent may make it suitable as a "therapeutic agent," which is one or more biologically, physiologically, or pharmacologically active substances that act locally or systemically in a subject.

[0170] The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a subject. The beneficial effects include achieving 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 counteracting a disease, symptom, disorder, or pathological condition.

[0171] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation to a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0172] "Nucleic acid" or "polynucleotide" refers to a DNA molecule (such as but not limited to cDNA or genomic DNA) or an RNA molecule (such as but not limited to mRNA), and includes DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. DNA or RNA molecules can include non-naturally occurring parts, such as modified bases, modified backbones, deoxyribonucleotides in RNA, etc. Nucleic acid molecules can be single-stranded or double-stranded.

[0173] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, thereby causing 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 in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are adjacent and, if necessary, join two protein coding regions in the same reading frame.

[0174] The term "joint" refers to any member, entity or part for joining two or more entities. The joint can be a covalent joint or a non-covalent joint. The example of a covalent joint includes a covalent bond or a joint part that is covalently connected to one or more proteins or domains to be connected. The joint can also be a non-covalent bond, such as an organometallic bond through a metal center (such as a platinum atom). For a covalent bond, various functional groups can be used, such as amide groups (including carbonic acid derivatives), ethers, esters (including organic esters and inorganic esters), amino groups, carbamates, ureas, etc. In order to provide a connection, the domain can be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide a site for coupling. The method for conjugation is well known to those skilled in the art and is included in the present invention. The joint part includes but is not limited to a chemical joint part or, for example, a peptide joint part (joint sequence).

[0175] "Isolated" nucleic acid molecules or polynucleotides mean nucleic acid molecules, DNA or RNA that have been removed from the natural environment. For example, for the purposes of the present invention, the recombinant polynucleotides encoding therapeutic polypeptides contained in the vector are considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. Isolated polynucleotides include the polynucleotide molecules contained in cells that usually contain polynucleotide molecules, but the polynucleotide molecules are present outside the chromosome or in a chromosomal position different from its natural chromosomal position. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids may further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or may include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0176] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, there is at least about 90% and more preferably at least about 95%, 96%, 97%, 98% or 99% nucleotide sequence identity in nucleotide bases as measured by any of the well-known sequence identity algorithms (such as FASTA, BLAST or GAP) discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an identical or substantially similar amino acid sequence to a polypeptide encoded by the reference nucleic acid molecule.

[0177] When applied to polypeptides, the term "substantially similar" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity, when optimally aligned using default gap weights as by the programs GAP or BESTFIT. Preferably, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted by another amino acid residue having a similar side chain (R group) with chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of proteins. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the similarity percentage or degree can be adjusted upward to correct the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference.

[0178] The term "disease" as used herein is intended to be generally synonymous with and used interchangeably with the terms "disorder" and "condition" (e.g., medical condition), as all of these terms reflect an abnormal condition (e.g., a disease or disorder) of the human or animal body or a part thereof that impairs normal function, typically manifests itself as obvious signs and symptoms, and results in a shortened duration or reduced quality of life in the human or animal.

[0179] As used herein, the term "modulate" means any change in a biological state, ie, increase, decrease, etc.

[0180] As used herein, the term "pharmaceutically 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 nontoxic, i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any components of the composition in which it is contained.

[0181] 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, which are involved in carrying or transporting the compounds of the invention in or to a subject so that the compounds can perform their intended function. Typically, such compounds are carried or transported from one organ or part of the body to another organ or part of the body. 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 that can serve as pharmaceutically 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 waxes; 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 glycerol, 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; solution); ethanol; phosphate buffer solution; diluent; granulating agent; lubricant; binder; disintegrant; wetting agent; emulsifier; colorant; release agent; coating agent; sweetener; flavoring agent; aromatic agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; fixative; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically 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 physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0182] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than in a multicellular organism.

[0183] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0184] It should be noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0185] Unless stated otherwise, the terms "including," "comprising," "containing," or "having" and variations thereof are intended to encompass the items listed thereafter and equivalents thereof as well as additional subject matter.

[0186] The phrases "in one embodiment," "in various embodiments," "in some embodiments," etc. are used repeatedly. Such phrases are not necessarily referring to the same embodiment, but they may unless the context dictates otherwise.

[0187] The term "and / or" or " / " means any one, any combination of items, or all of the items associated with this term.

[0188] The word "substantially" does not exclude "completely", for example a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0189] As used herein, the term "approximately" or "about", when applied to one or more target values, refers to a value similar to a specified reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​of 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 than or less than) of the specified reference value, unless otherwise specified or obvious from the context (except for such a number will exceed 100% of the possible value). Unless otherwise specified herein, the term "about" is intended to include values ​​close to the range, such as weight percentages, which are equivalent in terms of the functions of individual components, compositions or embodiments.

[0190] It should be understood that whenever values ​​and ranges are provided herein, all values ​​and ranges encompassed by these values ​​and ranges are intended to be included within the scope of the present invention. In addition, this application encompasses all values ​​that are also within these ranges and the upper or lower limit of a series of values.

[0191] As used herein, when used with respect to a collection of items, the term "each" is intended to identify an individual item in the collection, but does not necessarily refer to every single item in the collection. Exceptions may occur if explicit disclosure or context clearly indicates otherwise.

[0192] Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not impose limitations on the scope of the invention. No language in the specification should be construed as indicating any unclaimed element as being essential to practicing the invention. When used in this document, the term "exemplary" means "by way of example" and does not mean that a particular exemplary item is preferred or required.

[0193] Unless otherwise specified herein or otherwise clearly contradictory to the context, all methods described herein are performed in any suitable order. With respect to any method provided, the steps of the method may be performed simultaneously or sequentially. When the steps of the method are performed sequentially, unless otherwise specified, the steps may be performed in any order.

[0194] Where the method comprises a combination of steps, every combination or subcombination of steps is encompassed within the scope of the present disclosure unless otherwise indicated herein.

[0195] Each publication, patent application, patent and other reference cited herein is incorporated by reference in its entirety to the extent that it does not contradict the present disclosure. Publications disclosed herein are provided only for their disclosures before the filing date of the present invention. Anything herein should not be construed as admitting that the present invention cannot have the right to precede such publications due to existing inventions. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0196] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that based on these examples and embodiments, various modifications or changes will occur to those skilled in the art and that these modifications or changes will be included within the spirit and scope of this application and the scope of the appended claims.

[0197] Examples

[0198] Example 1

[0199] This example describes the materials and methods used in the following examples.

[0200] animal

[0201] Mice were housed and used in accordance with a protocol approved by the Rutgers University and Robert Wood Johnson Medical School InstitutionalAnimal Care and Use Committee (“Preclinical evaluation of therapy in an animal model for LINCL”, protocol I09-0274-4). - / - and Cln3 - / -Mice were of C57BL / 6 genetic background and genotyped as described (Sleat DE et al. J Neurosci 24:9117-26 (2004); Greene ND et al. Mol Genet Metab 66:309-13 (1999)). The experimental cohort contained an equal number of male and female animals and was 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 of 0.9% saline. Brains were dissected and frozen on dry ice. For histopathology, mice were anesthetized, perfused with saline, and then perfused and fixed with PBS containing 4% paraformaldehyde. Brains were excised, fixed in 4% paraformaldehyde in PBS for 48 hours, and then transferred to 30% sucrose / PBS at 4°C until the brain sank.

[0202] Quantitative mass spectrometry

[0203] 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 digested sequentially in solution with trypsin (specificity, carboxyl side of K and R) and endoproteinase LysC (specificity, carboxyl side of K), and the resulting peptides were labeled with TMT11-plex isobaric reagent (ThermoFisher Scientific). The labeled samples were combined and analyzed by synchronous precursor selection MS3 on a Lumos Tribrid instrument (ThermoFisher Scientific). Peak lists were generated using Proteome Discoverer 2.2 (Thermo Fisher Scientific), and data were searched using a local implementation of the Global Proteome Machine (GPM) (Craig R, Cortens JP and Beavis RC (2004) Journal of Proteome Research (J Proteome Res) 3:1234-42; Beavis RC (2006) Methods in Molecular Biology 328:217-28). Report 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 representing protein assignments, peptide spectrum matches, and corresponding report ion intensities) were archived in MassIVE (http: / / massive.ucsd.edu) and ProteomeXchange (http: / / www.proteomexchange.org / ) repositories with submission number MSV000087613.

[0204] Normalization and statistical analysis of mass spectrometry data

[0205] TMT-16 reporter ion intensity was normalized and analyzed as previously described (Sleat DE et al. (2017) Journal of Proteome Research 16: 3787-3804). In short, the reporter ion intensity data was extracted from the peak list file using a customized internal script (https: / / github.com / cgermain / IDEAA), and the mass spectrum was then normalized relative to the total reporter ion intensity of each channel to correct for differences in labeling efficiency and / or labeled protein quantity. First, peptides were filtered for complete trypsin cleavage, no missed cleavage sites, and complete iTRAQ tags for lysine and amino termini. Peptides were then filtered to remove peptides containing post-translational modifications (i.e., asparagine or glutamine deamidation, methionine dioxidation, tryptophan monooxidation and dioxidation, and isobaric labeling of tyrosine at positions other than the amino terminus) that may increase data variability. For selected comparisons, expression ratios and q values ​​corrected for multiple comparisons using the Benjamini-Hochberg procedure were generated using a nested procedure that takes into account variability at the peptide and protein levels (YB and YH (1995) Journal of the Royal Statistical Society: Series B (Statistical Methodology) 57:289-300).

[0206] Immunostaining

[0207] For immunohistochemistry, one of six serial coronal brain sections from each mouse was stained for astrocyte marker glial fibrillary associated protein (GFAP, rabbit anti-GFAP, 1:1000, DAKO) and microglial marker CD68 (rat anti-mouse CD68, 1:400, Bio-Rad) using a modified immunofluorescence protocol (Nelvagal HR et al. Neuropathol Appl Neurobiol 47:251-267). Briefly, 40 μm coronal sections were mounted on Superfrost Plus slides (Fisher Scientific) and air dried for 30 minutes. The slides were then blocked for 1 hour in 2% TBS-T solution (1× Tris buffered saline, pH 7.6, with 2% Triton-X100, Fisher Scientific) containing 15% serum. The slides were then incubated for 2 hours in primary antibodies in 2% TBS-T containing 10% serum, washed three times in 1× TBS, and incubated for 2 hours in 2% TBS-T containing 10% serum with fluorescent Alexa-Fluor labeled secondary antibodies (Alexa-Fluor goat anti-rabbit 488, goat anti-rat 546, Invitrogen). The slides were washed three times in 1× TBS and incubated for 2 minutes in 1× solution of TrueBlack lipofuscin autofluorescence quencher (Biotium, Fremont, CA) in 70% ethanol, followed by 1× TBS rinse. The slides were coverslipped in Fluoromount-G mounting medium with DAPI (Southern Biotech, Birmingham, AL).

[0208] Threshold image analysis

[0209] In order to analyze the degree of activation of glial cells in gray matter (GFAP positive astrocytes + CD68 positive microglia), the semi-automated threshold image analysis method of Image-Pro Premier software (Media Cybernetics) was used. In brief, the stained sections were scanned using Zeiss AxioScan Z1 slide scanner at the Washington University Center for Cellular Imaging (WUCCI), and each of the six series of sections of each animal was magnified 10 times, followed by division of all relevant areas. The images were then analyzed using Image-Pro Premier (Media Cybernetics) with an appropriate threshold for selecting foreground immunoreactivity above the background. This threshold was then applied as a constant to all subsequent images analyzed by animal batch and reagent used to determine the specific immunoreactive area of ​​each antigen. The measurements of histological processing were performed without knowledge of the genotype, and statistical analysis was performed using GraphPad Prism 8.0.0 for MacOS. Data were analyzed using two-way ANOVA and post hoc Bonferroni correction, where p values ​​≤ 0.05 were considered significant.

[0210] Survival statistics

[0211] Survival curves were compared using the log-rank test in GraphPad Prism 9.1 (GraphPad Software, San Diego, California USA, www.graphpad.com) with Bonferroni's method corrected for multiple comparisons.

[0212] Example 2

[0213] Breeding strategy

[0214] Initially, in order to produce all the desired genotypes as littermates, double crosses were performed using animals that were doubly heterozygous for both the Tpp1 and Cln3 mutations. Transmission ratio analysis showed that offspring were not produced at the predicted ratio (data not shown), with a significant reduction in the number of double mutant and double wild-type animals. This reflects the genetic linkage to Tpp1 and Cln3 located on mouse chromosome 7 (Tpp1 position: Chr 7E3; 7 55.97 cM, Cln3 position: Chr 7F3; 7 69.16 cM). Subsequently, through Tpp1 - / - ;Cln3 - / - ×Tpp1 - / - ;Cln3- / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - Double knockout animals were generated by mating.

[0215] Survival

[0216] Survival curves and statistical comparisons are shown in Figure 1 and 2 and Table 1. Given the significant differences in survival, Figure 1 Display Tpp1 - / - animals in the background, and Figure 2 shows that Tpp1 + / - or Tpp1 + / + Animals on background.

[0217] Tpp1 generated by mating of double mutants - / - ;Cln3 - / - 、Tpp1 - / - ;Cln3 +- and Tpp1 - / - ;Cln3 + / + The median survival of littermates ranged from 120 to 124 days (Table 1) and was not significantly different from each other (after correction for multiple comparisons). - / - ;Cln3 + / + Compared with mice, Tpp1 - / - ;Cln3 - / - The survival curve of mice was slightly rectangular. This may be due to the effect of CLN3 mutation on Tpp1 - / - The phenotypic effects may reflect subtle differences in the genetic background of the animals. The Tpp1 mutant allele used in these studies was backcrossed to C57BL / 6 (Sleat DE et al. (2004) Journal of Neuroscience 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 differed in that Tpp1 - / - Mouse is Nnt + / + , while Cln3 - / - Mouse is Nnt - / - (Sleat DE et al. Mol Cell Proteomics 18:2244-2261). The Nnt mutation is different in different C57BL / 6 sublines. - / - Genotypes were generated at the Jackson Laboratory https: / / pubmed.ncbi.nlm.nih.gov / 19448337 / and were designated C57BL / 6J, while Nnt+ / + The strain was designated C57BL / 6N. Therefore, double mutant animals are a mixed C57BL / 6N × C57BL / 6J substrain. Littermates generated by mating double Tpp1 / Cln3 mutants Tpp1 - / - ;Cln3 + / + Mice were mated with littermates generated by single Tpp1 mutants Tpp1 - / - ;Cln3 + / + However, the main conclusion is that Tpp1 - / - Cln3 - / - Double mutant animals and Tpp1 - / - Cln3 + / + There were no significant differences in the survival phenotypes of single mutant animals.

[0218] Figure 2 shows that Tpp1 + / - or Tpp1 + / + Survival of animals with mutations in the background. Data from C57BL / 6J animals from another study (the “Yuan dataset”) (Yuan R et al. (2012) PNAS 109:8224-9) were also included for analysis. The survival of Tpp1 heterozygous animals (median survival 843 days) was not significantly different from that of Yuan dataset wild-type animals (median survival 901 days) (Figure 2A). Given that heterozygosity for Tpp1 does not affect survival, it is predicted that Cln3 heterozygotes will have no significant difference in survival compared with wild-type. Tpp1 + / - ;Cln3 + / - The survival of double heterozygous mice (median 748 days) was similar to that of single Cln3 - / - Tpp1 heterozygosity results in CLN3 - / - The animals had a shortened survival (median 584 days), which was significantly shorter than that of Tpp1 + / + ;Cln3 - / - Collectively, these data suggest that Tpp1 heterozygosity leads to the survival of Cln3 + / - and Cln3 - / - The animal's life span is shortened.

[0219] Our Tpp1 + / + ;Cln3 - / - The survival of the mice (median 719 days) was not significantly different from historical data from a different but similar JNCL knockout mouse model (742 days) (Figure 2B). However, both Cln3 mutants had significantly reduced survival compared with wild-type mice.

[0220] Pathology

[0221] Animals were euthanized at approximately 120 days to investigate the effects of Tpp1 and Cln3 genotype on brain pathology. Sections were analyzed by immunofluorescence to simultaneously detect markers of glial activation in two brain regions (CD68 in microglia and GFAP in astrocytes) where significant glial activation was consistently observed in multiple NCL mouse models: the somatosensory barrel field (S1BF) cortex and the medial and lateral ventroposterior nuclei (VPM / VPL) regions of the thalamus. As expected, in mice with Tpp1, glial activation was significantly increased, regardless of Cln3 genotype. + / - or Tpp1 + / + In animals with the genotype with either GFAP or CD68, immunostaining was present at very low levels. In these animals, only very faintly stained GFAP-positive astrocytes were present ( Figure 3 ) or CD68-positive microglia ( Figure 4 ). - / - Cln3 + / + Astrocytes were detected in S1BF and VPM / VPL of animals ( Figure 3 ) and microglia ( Figure 4 ) were significantly activated, with these markers significantly upregulated and corresponding changes in cell morphology. - / - Cln3 - / - Similar levels of significant glial activation were detected in the S1BF and VPM / VPL of animals, with the extent of cellular hypertrophy of astrocytes and microglia being similar to that of Tpp1 - / - Cln3 + / + Threshold image analysis confirmed these qualitative observations, with Tpp1 - / - Cln3 + / + and Tpp1 - / - Cln3 - / - GFAP and CD68 immunoreactivity was significantly increased in both brain regions of the animals ( Figure 5 ). Although Tpp1 - / - Cln3 + / + Animals and Tpp1 - / - Cln3 - / - Individual levels of GFAP and CD68 immunoreactivity varied somewhat between animals, but there were no significant differences for either antigen in either brain region between animals of these genotypes. - / - Cln3 + / + Animals and Tpp1 - / - Cln3 - / - The broad similarities between animals in the extent of glial activation are consistent with the similarities observed in the survival phenotypes of these mutants.

[0222] Proteomic analysis

[0223] Proteomic analysis of brain samples from Tpp1 and Cln3 mutant mice was performed with the goal of identifying changes that might correlate with disease survival and possibly reveal corresponding cellular functions of these proteins. - / - Any informative brain expression changes in mice, hypothesized to be associated with the Cln3 mutation, may be exacerbated and therefore more easily detected in the Tpp1 mutant background. Proteins were identified and quantified in brain extracts from animals at approximately 105 days of age using isobaric labeling mass spectrometry. Figure 6 In , various related genotypes were compared using a volcano plot (a scatter plot that allows comparison of effect sizes versus the probability of significant differences). Q values ​​(i.e., p-values ​​adjusted for multiple comparisons found using the Benjamin-Hochberg false discovery rate method) were calculated by comparing the reporter ion intensities of all individual mass spectra of a given protein across all biological replicates assigned to each genotype.

[0224] When Tpp1 - / - When animals were compared to wild-type, a number of significant changes in protein expression were detected, including increases in GPNMB, LYZ2, SERPINA3N, and various lysosomal proteins including several cathepsins (Table 2). - / - ;Cln3 - / - When animals were compared to wild-type, very similar changes in protein expression were identified, such as Tpp1 - / - ;Cln3 + / + and Tpp1 - / - ;Cln3 - / - This is shown by the high correlation between the fold change in expression compared to wild-type animals (Figure 7A). - / + or Tpp1 + / + Background, in Cln3 - / - Relatively few changes were detected in mutant animals, but NPC1 and CTSF were consistently increased, while NEU4 and SMPD1 were decreased ( Figure 7B ).

[0225] TPP1 activity in mutant mice.

[0226] like Figure 8 As shown, TPP1 activity in Cln3 mutants was found to be approximately 2-fold higher than that measured in wild-type animals, and irrespective of Cln3 genotype, Tpp1 - / - No activity was detected in animals. As expected, Tpp + / -The TPP1 activity in animals is about 50% of that in wild type animals. + / - Cln3 - / - The animals were approximately 100% of wild type. This again indicates that loss of CLN3 increases TPP1 activity by approximately 2-fold.

[0227] The overall goal of this study is to determine the phenotype of the double Tpp1 and Cln3 mutant mouse model, which can provide useful information about the cellular role of CLN3 and the potential functional interaction between TPP1 and CLN3. To a large extent, the phenotype of these double knockout mice is similar or identical to that of single Tpp1 mutants: the difference in the median survival of the two models is within five days, and it shows a similar degree of characteristic NCL pathology in the form of astrogliosis and microglial activation. In addition, the proteomic changes in the brain of the double knockout are very similar to those in the single Tpp1 knockout (Figure 7A). Consistent with these observations, one possibility is that the effect of the disease in JNCL may be caused at least in part by the secondary effect of CLN3 on TPP1. Although the loss of CLN3 does not seem to directly affect the TPP1 activity measured in vitro, the loss of CLN3 may affect the acquisition of TPP1 to its physiological substrates (possibly including SCMAS). Two observations provide support for this possibility. First, accumulation of SCMAS is present in both LINCL and JNCL (although it also occurs in other lysosomal storage diseases). Second, in this study, an approximately 2-fold increase in TPP1 activity was observed in the absence of CLN3 ( Figure 8 ), indicating the presence of a compensatory response.

[0228] The lifespan of the JNCL mouse model was shortened (median 719 days) compared with wild-type (median 901 days), and this is consistent with previous analyses of different Cln3 knockout mouse models (Cln3 - / - However, heterozygosity for Tpp1 and Cln3 - / - The double heterozygous Tpp1 + / - ;Cln3 + / - It is unclear why heterozygosity for Tpp1 exacerbates Cln3 - / - phenotype of the animals, or a survival phenotype in Cln3 heterozygotes, but this is consistent with the possibility that CLN3 deficiency may have downstream effects on TPP1 function. Another possibility is that the compensatory increase in TPP1 activity detected in the absence of CLN3 may actually provide neuroprotection. Thus, heterozygosity for Tpp1 would attenuate such protection, thereby allowing Cln3 to - / -Mice have a shorter lifespan.

[0229] Glial activation is a consistent pathological feature in NCL diseases, including LINCL and JNCL. Often, localized glial activation precedes the onset of neuronal loss and is used to predict the location of subsequent neurodegeneration. Indeed, it has been proposed that glial dysfunction may contribute to neuronal loss in a variety of NCLs. - / - Cln3 + / + and Tpp1 - / - Cln3 - / - Quantitative analysis of the mice revealed that the extent of astrogliosis and microglial activation was very similar between these genotypes.

[0230] Regardless of the underlying basis for lifespan shortening, Tpp1 + / - Cln3 - / - The shortened survival of mutants may be suitable for testing therapeutic strategies for JNCL. Survival provides a clear and objective endpoint when developing any therapeutic strategy: for example, in evaluating several different strategies for LINCL, survival studies in mouse models have highlighted promising approaches (e.g., gene therapy, intrathecal 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. Animal models of LINCL have significantly shortened lifespans; therefore, proof of principle for treatment in terms of survival is readily achievable. In contrast, the survival of Cln3 mutant mice approaches that of wild-type mice, complicating survival as an endpoint. Therefore, behavioral models have been widely used to analyze disease progression and the effects of potential treatments in Cln3 mutant mice. Although many studies have characterized the motor deficits of Cln3 mouse mutants, the behavioral phenotypes are subtle and depend on the mouse strain and sex.

[0231] In Cln3 - / - ;Tpp1 + / -Evaluation of potential therapeutic agents for JNCL in mouse mutants will provide a relatively robust survival phenotype to measure efficacy. In addition, behavioral phenotypes may be exacerbated at a younger age. It is expected that effective therapies to address CLN3 deficiency (e.g., extensive gene therapy of the entire brain) will extend the survival of this mouse mutant to be similar to the survival of Tpp1 heterozygotes that are indistinguishable from wild type. A similar approach was performed using mutant Cln3 mice expressing human amyloid precursor protein (APP) with familial Alzheimer's disease mutations (Centa JL et al. (2020) Nature Medicine (Nat Med) 26: 1444-1451). However, even without CLN3 deficiency, mutant APP confers a severe survival phenotype. Therefore, active treatment of CLN3 deficiency will essentially improve the severe phenotype rather than restore wild-type survival.

[0232] Finally, one goal of this study is to determine whether double Tpp1 and Cln3 knockouts can highlight proteomic changes in the brain, which may potentially provide a platform for clinically useful biomarkers in either or both of LINCL and JNCL. Based on studies of plasma from TPP1 patients and dog models, neurofilament light chain (NEFL in mice) has been proposed as a treatment responsiveness biomarker, and its levels and those of other neurofilament proteins (NEFH and NEFM) have been found to increase in mouse CSF. However, in TPP1 and CLN3 mouse models, brain levels of NEFL, NEFH and NEFM were unchanged or decreased. This is consistent with the unchanged findings of brain NEFL in mouse models of other neurodegenerative diseases that exhibit elevated NEFL plasma and CSF levels. It was also found that several lysosomal proteins (CTSF, SMPD1 and NPC1) were increased in Cln3. - / - The expression of SMPD1 and CTSF in JNCL mice was significantly altered in 142 mice and, although the expression changes were less dramatic, they may be more pronounced in older mice (animals were analyzed at approximately 120 days in this study). These changes may be a compensatory response to CLN3 loss and may potentially provide useful information about its biological function. In addition, the soluble lysosomal proteins SMPD1 and CTSF may also be worthy of further study as potential biomarkers in JNCL.

[0233] Table 1. Tpp1 - / - Animal survival statistics. Unless otherwise stated, survival data were obtained from littermates of crosses between double Tpp1 and Cln3 mutant crosses (* indicates single mutant crosses). The significance of the log-rank tests performed below was assessed after correcting the P values ​​for multiple comparisons using the Bonferroni method.

[0234]

[0235] Table 2. Comparison of proteomic changes in Tpp1-mutant mouse models compared to wild-type. The indicated proteins were significantly altered (FDR 1%) in at least one model compared to wild-type, with a change magnitude ≥ 1.5-fold, and with consistent direction of change in both models. Significant changes are indicated in bold. Note that NNTs are censored (see Examples).

[0236]

[0237]

[0238] Example 3

[0239] One of the goals of this study was to determine whether overexpression of TPP1 could reduce the accumulation of subunit C mitochondrial ATP synthase (SCMAS) in JNCL mice. A transgenic mouse model (R26Tg TPP1+ / 0 )(Nemtsova, Y. et al., PLoS One, 2018.13(2):p.e0192286). This allowed us to test this hypothesis using genetic methods.

[0240] Transgenic mice were derived from a CAG promoter-driven transgene integrated at the ROSA26 locus (Tg TPP1+ ) overexpressed murine TPP1 (Fig. 9A). This mouse expressed TPP1 in the brain at a level at least 10 times higher than normal (Fig. 9B). TPP1 overexpression was constitutive and ubiquitous throughout the brain, including the cortex and hippocampus (Fig. 9B). TPP1 overexpression had no obvious harmful effects on the animals (Fig. 9C). Transgenic TPP1 was expressed in both neurons and microglia, colocalizing with the corresponding markers NeuN (Fig. 9D) and Iba1 (Fig. 9E).

[0241] We used our transgenic mice overexpressing TPP1 to cross-react with Cln3 - / - The JNCL mouse model was crossed to determine whether elevated TPP1 levels had any positive effects on disease progression. Four littermate cohorts were established: 1) Tg-Cln3 + / + ; 2) Tg-Cln3 - / - ; 3) Tg+Cln3 + / + and; 4) Tg+Cln3 - / - . SCMAS storage was examined in the liver and spleen of 6-month-old JNCL mice. SCMAS accumulation in the brain was also examined at this time point.

[0242] method

[0243] Animals. Animals were anesthetized with a euthanasia needle (sodium pentobarbital and sodium phenytoin) and then euthanized by exsanguination / transcardial perfusion with PBS and dissected. One 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.

[0244] Histological embedding, sectioning and staining

[0245] Immunohistochemistry was performed by NeuroScience Associates (Knoxville, TN). Tissue samples (brain, liver, spleen) were treated with 20% glycerol and 2% dimethyl sulfoxide overnight. The samples were embedded in gelatin matrix by technology. After solidification by immersion in 2-methylbutane cooled with crushed dry ice, the blocks were quickly frozen and mounted on the freezing stage of an AO 860 sliding microtome, and the blocks were sliced. All slices were cut along the entire length of the sample section and collected in sequence into a series of 24 containers containing an antigen preservation solution (50% PBS pH 7.0, 50% ethylene glycol, 1% polyvinyl pyrrolidone).

[0246] Immunohistochemistry

[0247] Lysosomal storage of SCMAS was visualized using an affinity-purified rabbit anti-polyclonal antibody (PAC3601 / 3602; Pacific Immunology, Ramona, CA) raised against the amino-terminal peptide of SCMAS (Xu, S. et al., Mol Ther, 2011. 19(10): p. 1842-8). Free-floating sections were stained with the desired stain. All incubation solutions starting with the primary antibody used Tris-buffered saline (TBS) containing Triton X-100 as the vehicle; all rinses were performed with TBS. After hydrogen peroxide treatment, free-floating sections were immunostained with anti-SCMAS antibody at a dilution of 1:500 overnight at room temperature. The vehicle solution contained Triton X-100 for permeabilization. After rinsing, a biotinylated secondary antibody (anti-rabbit IgG, prepared in goat by Vector Labs) was applied at a dilution of 1:1000. After further washing, Vector Lab's ABC solution (avidin-biotin-HRP complex; see Vector Lab's Elite ABC instructions). The sections were rinsed again and then treated with diaminobenzidine tetrahydrochloride (DAB) with nickel and hydrogen peroxide to produce a visible reaction product. After further rinsing, the sections were mounted on gelatin-coated slides and air-dried. The slides were dehydrated in alcohol, cleared in xylene and covered with a cover slip.

[0248] Imaging

[0249] Slides were scanned using the TissueScope LE system from Huron Digital Pathology. The entire slide was scanned at 20X resolution (0.4 μm / pixel). SCMAS accumulation was quantified using ImageJ (Schneider, CA et al., Nat Methods, 2012. 9(7): 671-5).

[0250] result

[0251] Animals were euthanized at 6 months of age, and SCMAS accumulation in spleen, liver, and brain was assessed.

[0252] In the liver from control animals (Tg-Cln3 + / + and Tg+Cln3 + / + ) in the liver sections. Fig.10 ). In the absence of the TPP1 transgene, Cln3 - / - Mouse (Tg-Cln3 - / - ), a large amount of SCMAS was detected in the liver. - / - Mouse (Tg+Cln3 - / - ), no SCMAS accumulation was detected.

[0253] In spleens from control animals (Tg-Cln3 + / + and Tg+Cln3 + / + ) in spleen sections, no SCMAS staining was detected ( Fig.11 ). In the absence of the TPP1 transgene, Cln3 - / - Mouse (Tg-Cln3 - / - ), a large amount of SCMAS was detected in the spleen. - / - Mouse (Tg+Cln3 - / - ), no SCMAS accumulation was detected.

[0254] Brain. We analyzed SCMAS storage in four different brain regions: the CA3 region of the hippocampus, cerebellum, thalamus, and cortex. + / + and Tg+Cln3+ / + No SCMAS staining was detected in any brain region sections from Fig.12 ). In the absence of the TPP1 transgene, Cln3 - / - Mouse (Tg-Cln3 - / - ), large amounts of SCMAS were specifically detected in the CA3 region of the hippocampus and in the Purkinje cells of the cerebellum. Extensive accumulation was observed in the thalamus and cortex. In JNCL mice expressing the TPP1 transgene (Tg+Cln3 - / - ), no SCMAS accumulation was detected in any of these regions.

[0255] The accumulation of SCMAS in the liver, cortex, and thalamus was quantified based on the area of ​​the image obscured by inclusions ( Fig.13 ). The effect of transgene expression on SCMAS storage is obvious.

[0256] JNCL mice expressing TPP1 transgene (Tg+Cln3 - / - ) showed no detectable accumulation of SCMAS in the liver, spleen, or four regions of the brain at 6 months of age. These results suggest that elevated TPP1 activity can prevent the appearance of storage material in JNCL and provide support for TPP1 enhancement as a potential treatment for JNCL and other diseases that may accumulate SCMAS.

[0257] The scope of the present disclosure is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will also become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method for treating a subject suffering from a disease or condition characterized by accumulation of SCMAS in the lysosomes of affected cells, comprising 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 condition.

2. The method of claim 1, wherein the disease or condition is selected from juvenile neuronal ceroid lipofuscinosis (CLN3) disease, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, variant 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 Kufor-Rakeb syndrome, Sanfilippo D syndrome (mucopolysaccharidosis type IIID) and autosomal recessive osteopetrosis type 4 (OPTB4).

3. The method according to any one of the preceding claims, wherein the disease or disorder is characterized by a functional defect of the CLN3 protein.

4. The method of any one of the preceding claims, wherein the agent reduces the accumulation level of the SCMAS in the lysosomes of the affected cells.

5. The method according to any one of the preceding claims, wherein the affected cells are neuronal cells.

6. The method according to any one of the preceding claims, wherein the agent comprises recombinant human TPP1 protein or a nucleic acid molecule comprising a nucleotide sequence encoding TPP1 or a variant thereof. The method according to claim 6 , wherein the TPP1 protein is an inactive zymogen.

8. The method according to any one of claims 6 to 7, wherein the TPP1 protein is mannose-6-phosphorylated.

9. The method of any one of claims 6 to 8, wherein the therapeutically effective amount of the TPP1 protein is such that the affected cells receive about 1.0 to about 100 nM of recombinant human TPP1 protein.

10. The method according to any one of claims 6 to 9, wherein the agent is administered by injection.

11. The method of any one of claims 10, wherein the injection is an intracranial injection.

12. The method of any one of the preceding claims, wherein the agent is administered in a controlled release system.

13. The method of any one of the preceding claims, wherein the agent is delivered to the lysosomes of the affected cells.

14. The method according to any one of the preceding claims, wherein the subject is a human.

15. An animal model for studying a disease or condition, comprising: (i) a tripeptidyl peptidase 1 (Tpp1) gene heterozygous knockout (Tpp1 + / - ); and (ii) homozygous knockout of Cln3 gene (Cln3 - / - ), where the mouse model had a shortened lifespan compared to wild-type animals.

16. The animal model of claim 15, wherein the animal is a mouse.

17. The animal model of any one of claims 15 to 16, wherein the disease or condition is selected from late infantile neuronal ceroid lipofuscinosis (CLN2) disease, juvenile neuronal ceroid lipofuscinosis (CLN3) disease, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, variant 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 Kull-Ray syndrome, San Philip D syndrome (mucopolysaccharidosis type IIID) and autosomal recessive osteopetrosis type 4 (OPTB4).

18. The animal model of any one of claims 15 to 17, wherein the lifespan of the animal is shortened by at least 25% compared to the wild-type animal.

19. The animal model according to any one of claims 15 to 18, wherein the Tpp1 gene or the Cln3 gene comprises at least one mutation selected from the group consisting of a deletion, an insertion, a frameshift mutation, a rearrangement or a substitution.

20. The animal model of claim 19, wherein the mutation is constitutive.

21. The animal model of claim 19, wherein the mutation is conditional.

22. The animal model of any one of the preceding claims, wherein the Tpp1 gene comprises a deletion of at least a portion of an exon within the Tpp1 gene.

23. The animal model of any one of claims 15 to 22, wherein the Tpp1 gene comprises a neo insertion in intron 11 and an Arg446His missense mutation in exon 11 immediately upstream of the neo insertion.

24. The animal model according to any one of claims 15 to 23, wherein the Tpp1 gene is located at Chr 7E3; 755.97 cM.

25. The animal model of any one of claims 15 to 24, wherein the Cln3 gene comprises a deletion of at least a portion of an exon within the Cln3 gene.

26. The animal model according to any one of claims 15 to 25, wherein the Cln3 gene comprises a deletion of all or part of exons 1-6 in the Cln3 gene.

27. The animal model according to any one of claims 15 to 26, wherein the Cln3 gene is located at Chr 7F3; 769.16cM.

28. The animal model of any one of claims 15 to 27, wherein the animal model has increased levels of lysosomal accumulation of mitochondrial ATP synthase subunit c (SCMAS).

29. The animal model of claim 28, wherein the lysosomal accumulation level of SCMAS in the animal model is increased by at least 50%.

30. The animal model according to any one of claims 15 to 29, wherein the animal model has increased expression levels of Niemann-Pick disease type C1 (NPC1) and / or cathepsin F (CTSF) or decreased expression of SMPD1.

31. The animal model according to claim 30, wherein at least the expression level of NPC1 and / or CTSF in the animal model is increased by 40% or the expression level of acid sphingomyelinase (SMPD1) is decreased by 40%.

32. The animal model of any one of claims 15 to 31, wherein the animal model is characterized by defects in locomotor activity.

33. An offspring of the animal model according to any one of claims 15 to 32.

34. A cell, tissue or cell line derived from the animal model according to any one of claims 15 to 32 or the progeny according to claim 33.

35. A method for obtaining an animal model according to any one of claims 15 to 32, comprising: (a) crossing an animal in which Tpp1 is knocked out with a second animal in which Cln3 is knocked out to obtain an animal having double heterozygosity (Tpp1 + / - ;Cln3 + / - ); and (b) by mating Tpp1 - / - ;Cln3 - / - ×Tpp1 - / - ;Cln3 - / - or Tpp1 - / - ;Cln3 - / + ×Tpp1 + / - ;Cln3 - / - , and the double heterozygous animals are crossed.

36. A method of identifying an agent for treating a disease or condition in a subject, comprising: (i) administering a candidate agent to the animal model according to any one of claims 15 to 32 or the offspring according to claim 33, and evaluating the effect of the candidate agent on the phenotype of the animal model; or (ii) contacting the cell, tissue or cell line of claim 34 with a candidate agent and evaluating the effect of the candidate agent on the cell, tissue or cell line.

37. The method of claim 36, wherein the disease or disorder is characterized by accumulation of SCMAS in lysosomes of affected cells of the animal model.

38. according to the method described in any one of claims 36 to 37, wherein the disease or illness is selected from late infantile neuronal ceroid lipofuscinosis (CLN2) disease, juvenile neuronal ceroid lipofuscinosis (CLN3) disease, variant late infantile neuronal ceroid lipofuscinosis type 5 (CLN5) disease, variant 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 Ku-Ray syndrome, San Philip D syndrome (mucopolysaccharidosis type IIID) and autosomal recessive osteopetrosis type 4 (OPTB4).

39. The method of claims 36 to 38, wherein the phenotype is the lifespan of the animal model.

40. The method of any one of claims 36 to 39, wherein the effect is characterized by an increase in the lifespan of the animal model.

41. The method of any one of claims 36 to 40, wherein the effect is characterized by a reduction in the level of lysosomal accumulation of SCMAS.

42. The method of any one of claims 36 to 41, wherein the effect is characterized by a decrease in the expression level of NPC1 and / or CTSF.

43. The method of any one of claims 36 to 42, wherein the candidate agent comprises a protein, a peptide, a peptide mimetic, a nucleic acid, or a small molecule.

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