Treatment of neuronal ceroid lipofuscinosis 3 (CLN3) with TPP1

Abstract

The present application relates, in general, to methods of using rhTPP1 in the prevention and treatment of symptoms of Neuronal Ceroid Lipofuscinosis (CLN3) disease.

Description

TREATMENT OF NEURONAL CEROID LIPOFUSCINOSIS 3 (CLN3) WITH TPP1 CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority of U.S. Provisional Patent Application No. 63 / 764,273, filed February 27, 2025, herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “70816_SeqListing.xml", which was created on February 26, 2026, and is 3 ,633 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.FIELD

[0003] The present application relates, in general, to treatment of neuronal ceroid lipofuscinosis 3 (CLN3) disease with compositions comprising tripeptidyl peptidase 1 (TPP1).BACKGROUND

[0004] The neuronal ceroid lipofuscinoses (NCLs) are a group of more than a dozen genetically distinct but similar lysosomal storage diseases characterized by an accumulation of autofluorescent storage material within the lysosome. NCLs are neurodegenerative and progressive diseases that are manifested by seizures, loss of vision, and eventual loss of mental capacity. Two common NCLs are late-infantile (LINCL) and juvenile forms (JNCL). LINCL results from mutations in the gene encoding tripeptidyl peptidase 1 (TPP1 , formerly designated CLN2), a soluble lysosomal serine protease. Disease in LINCL typically presents at around 4 years of age, and lifespan is ~8 to 15 years. JNCL is caused by mutations in a gene encoding a lysosomal transmembrane protein, CLN3. JNCL / CLN3 disease typically has a later onset and is more slowly progressing than LINCL, with initial signs of disease (such as problems with vision) at around 4-8 years of age and patients frequently surviving into the second or third decade of life. Despite differences in disease timeline and genetic etiology, LINCL and JNCL have a number of similarities, including the accumulation of lysosomal storage of subunit c of mitochondrial ATP synthase (SCMAS).

[0005] CLN3 disease is a fatal neurologic disease that causes progressive vision loss, epilepsy, dementia, behavioral difficulties, and motor impairment. CLN3 protein is an integral, transmembrane protein and the function CLN3 protein is not well understood. It hasbeen implicated in numerous cellular activities, including lysosomal pH homeostasis, endocytosis, autophagy, apoptosis, lysosomal enzyme transport, and others, but its precise function is yet to be definitively established.

[0006] At least 65 mutations in the CLN3 gene have been associated with CLN3 disease. Several mutations in CLN3 have been suggested to be relevant in autophagic vacuolar myopathies (Wright et al., Ophthalmol Retina. 2020 Apr;4(4):433-445). Acommon CLN3 gene mutation deletes about 1 ,000 base pairs of DNA and is usually referred to as the 1 kb deletion. The 1 kb deletion can occur in both copies of the CLN3 gene. The 1 kb deletion results in a translation of a short, dysfunctional CLN3 protein.SUMMARY

[0007] The present application provides for the treatment of CLN3 disease with recombinant TPP1 (rhTPPI). The disclosure also provides for a composition comprising a recombinant human tripeptidyl peptidase-1 (rhTPPI) for use in treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject, or for use of a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in the manufacture of a medicament for treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject.

[0008] Provided herein is a method of treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject comprising administering to the subject a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in an amount effective to treat the CLN3 disease in the subject.

[0009] Also provided is a method of delaying the onset of Neuronal Ceroid Lipofuscinosis (CLN3) disease stage, or a symptom thereof, in a subject, comprising administering a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI).

[0010] In various embodiments, the composition is administered to the subject via intracerebroventricular, intrathecal, intravitreal, and / or intraocular administration.

[0011] In various embodiments, the composition is administered once every 2 weeks or once every 4 weeks.

[0012] The disclosure further provides for a method of treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject comprising administering to the subject a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in an amount effective to treat the CLN3 disease in the subject, wherein a dose of rhTPPI is administered intracerebroventricularly every 2 weeks and a dose of rhTPPI is administered intravitreal ly every 4 weeks.

[0013] Also provided is a method of delaying the onset of Neuronal Ceroid Lipofuscinosis (CLN3) disease, or a symptom thereof, in a subject, comprising administering a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI), wherein a dose of rhTPPI is administered intracerebroventricularly every 2 weeks and a dose of rhTPPI is administered intravitreally every 4 weeks

[0014] In various embodiments, a dosage of about 300 mg or less is administered to the subject intracerebroventricularly. In various embodiments, a dosage of about 0.20 mg to 5 mg is administered to the subject intravitreally. In various embodiments, a dosage of about 0.20 mg or less is administered to the subject intravitreally.

[0015] In various embodiments, the administration improves one or more symptoms of CNL3 disease. In various embodiments, the one or more symptoms are selected from the group consisting of vision, language function, motor function, language function, cognitive function, physical ability, behavior, number, frequency or severity of seizures, and functional capability.

[0016] In various embodiments, the administration slows or reduces time to progression from one stage of CLN3 to the next as determined by CLN3SS described herein.

[0017] In various embodiments stage 0 CLN3, is based on genetic confirmation of a genetic lesion in CLN3, stage 1 CLN3 is based on genetic confirmation of a genetic lesion in CLN3 gene and clinical presentations of vision loss (e.g., impairment), stage 2 CLN3 is based on genetic confirmation of a genetic lesion in CLN3 gene, vision loss, and one or more seizures, stage 3 CLN3 is based on genetic confirmation of a genetic lesion in CLN3 gene, vision loss, one or more seizures, and inability to walk without assistance.

[0018] In various embodiments, the administration slows or reduces time to progression from one stage of CLN3 to the next as determined by UBDRS described herein.In various embodiments, the stage / disease severity is determined based on UBDRS subscale, capability and physical subscale score of severity and / or age of the subject. In various embodiments, the progression is from stage 0 to stage 1 CLN3, from stage 1 to stage 2 CLN3, or from stage 2 to stage 3 CLN3.

[0019] In various embodiments, the composition is administered intracerebroventricularly and / or intravitreally. In various embodiments, the formulation, composition or dose is administered every other week, monthly or every other month.

[0020] In various embodiments, the composition is administered intravitreally every 4 weeks. In various embodiments, the composition is administered intracerebroventricularly every 2 weeks.

[0021] In various embodiments, the composition is administered at a dose of about 300 mg intracerebroventricularly. In various embodiments, the dose is administered to the subject at a rate less than or equal to 75 mg / hour.

[0022] In various embodiments, the rhTPPI comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof.

[0023] In various embodiments, the subject exhibits a decrease in CLN3 enzyme activity as compared to a healthy subject. In various embodiments, the subject has one or more mutation in a CLN3 gene.

[0024] In various embodiments, the subject receiving treatment has an improved score on UBDRS in at least one domain and / or in at least one UBDRS subscales compared to CLN3 natural history studies.

[0025] In some aspects, the method further comprises administering to the subject an antihistamine with or without an antipyretic before administration of the rhTPPI , optionally, about 30 to about 60 minutes before administration of the rhTPPI .

[0026] In various embodiments, the composition comprises the rhTPPI and at least one pharmaceutically acceptably carrier, diluent or excipient.

[0027] In various embodiments, the composition comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof.

[0028] In various embodiments, the composition for intracerebroventricular administration comprises rhTPPI at a concentration of from about 25 mg / mL to about 35 mg / mL.

[0029] In various embodiments, the composition has a pH of about 6.5.

[0030] In various embodiments, the composition further comprises potassium chloride at a concentration of about 0.01 mg / mL to about 1 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, and calcium chloride dihydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL.

[0031] In various embodiments, the composition further comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, and sodium chloride at a concentration of about 1 mg / mL to about 20 mg / mL.

[0032] In various embodiments, the composition comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration ofabout 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, and calcium chloride dihydrate at a concentration of about 0.21 mg / mL.

[0033] In various embodiments, the methods further comprise administering to the subject a flush solution after administering the composition intracerebroventricularly. In various embodiments, the flush solution comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof. In various embodiments, the flushing solution comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration of about 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, and calcium chloride dihydrate at a concentration of about 0.21 mg / mL.

[0034] In various embodiments, the treatment period is at least 10 weeks, at least 20 weeks, at least 40 weeks, at least 80 weeks, at least 96 weeks, at least 6 months, at least 1 year, at least 2 years, or for the life of the subject.

[0035] Also provided is a method of maintaining, slowing, reducing or improving vision loss in a subject having Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease comprising intraocularly administering to the subject a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in an amount effective to improve vision loss in the subject.

[0036] In various embodiments, assessment of vision loss includes assessment of retinal health. Methods of assessing vision loss include OCT scans, OCT-B scans, Spectral-Domain Optical Coherence Tomography (SD-OCT), Electroretinogram (ERG) and change in average central retinal thickness (ACRT) over time. Changes in vision can be determined based on the Weill Cornell Ophthalmic Severity Scores (WCOSS). In various embodiments, the intravitreal administration reduces or slows time to progression on the WCOSS, e.g., from stage 1 to stage 2, from stage 2 to stage 3, from stage 3 to stage 4, or from stage 4 to stage 5.

[0037] Further provided herein is a method of maintaining brain volume or slowing or reducing a decrease in brain volume in a subject having Neuronal Ceroid Lipofuscinosis (CLN3) disease, comprising administering a therapeutically effective dose of recombinant human tripeptidyl peptidase-1 (rhTPPI) to the subject.

[0038] The disclosure also provides a method of reducing the frequency or severity of seizures in a subject having Neuronal Ceroid Lipofuscinosis (CLN3) disease, comprisingadministering a therapeutically effective dose of recombinant human tripeptidyl peptidase-1 (rhTPPI ) to the subject.

[0039] The disclosure also provides for a composition comprising a recombinant human tripeptidyl peptidase-1 (rhTPPI) for use in treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject, or for use of a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in the manufacture of a medicament for treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject.

[0040] In various embodiments, the composition or use is for intracerebroventricular, intrathecal, intravitreal and / or intraocular administration.

[0041] It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and / or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination.

[0042] Such features or combinations of features apply to any of the aspects of the invention. Where examples of values falling within ranges are disclosed, any of these examples are contemplated as possible endpoints of a range, any and all numeric values between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1. Elevated TPP1 prevents SCMAS accumulation in brain and peripheral tissues at 6 months. Images were acquired from a mixture of male and females. Data were quantified in terms of numbers of inclusions and area of image occluded by inclusions using ImageJ / FIJI. Statistical analyses were conducted by one-way ANOVA using Tukey's multiple comparisons test on log transformed data. Note that while all possible comparisons were tested, comparison bars are only shown for significant differences. p<0.0001 ; ***, p<0.001 ; **, p<0.01 , *, p<0.05. Bars indicate mean and standard deviation.

[0044] Figure 2. Elevated TPP1 prevents SCMAS accumulation in brain at 12 months. Images were acquired from a mixture of male and females. Data were quantified in terms of numbers of inclusions and area of image occluded by inclusions using ImageJ / FIJI.Statistical analyses were conducted by one-way ANOVA using Tukey's multiple comparisons test on log transformed data. Note that while all possible comparisons were tested, comparison bars are only shown for significant differences. **“, p<0.0001 , *, p<0.05. Bars indicate mean and standard deviation.

[0045] Figure 3. Increased levels of TPP1 reduces astrocyte activation in JNCL. Astrocyte activation was determined by immunohistochemical detection of GFAP. Animals are 12 months old and images were acquired from a mixture of male and females. Regions with JNCL-specific astrocyte activation were identified as the somatosensory cortex layer 6 and ventral nuclei of the thalamus. Graphs depict area occluded by GFAP staining.Statistical analyses were conducted by one-way ANOVA using Tukey's multiple comparisons test on log transformed data. p<0.0001 ; ***, p<0.001 ; **, p<0.01. Bars indicate mean and standard deviation.

[0046] Figure 4. Increased levels of TPP1 reduces microglial activation in JNCL. Microglial activation was determined by immunohistochemical detection of CD68. Animals are 12 months old and images were acquired from a mixture of male and females. Diffuse CD68 staining was detected throughout the cortex with images derived from the somatomotor / orbital layer 5 region. Intense JNCL-specific glial activation was identified in the ventral nuclei of the thalamus and is shown at two different magnifications. Statistical analyses were conducted by one-way ANOVA using Tukey's multiple comparisons test on log transformed data. Note that while all possible comparisons were tested, comparison bars are only shown for significant differences. p<0.0001 ; ***, p<0.001 ; **, p<0.01. Bars indicate mean and standard deviation.

[0047] Figure 5. Elevated neurofilament light levels in plasma of JNCL mice is restored to wild-type levels by TPP1 overexpression. Statistical analyses were conducted by one-way ANOVA using Tukey's multiple comparisons test on log transformed data. Note that while all possible comparisons were tested, comparison bars are only shown for significant differences. ****, p<0.0001 ; ***, p<0.001 . Bars indicate mean and standard deviation. N=6 animals were analyzed for WT / Tg+ and n=12-14 for other genotypes.

[0048] Figure 6A-6C. Progressive loss of brain mass in JNCL mice is corrected by elevated TPP1 expression. Fig. 6A. Brain weights of animals are plotted against age at euthanasia. Open and closed symbols show male and female animals respectively. Data for male and female animals were analyzed together by simple linear regression and p values for all pairwise comparisons of the slopes were determined and corrected for multiple comparisons using the Bonferroni method. Fig. 6B. Same as (A) but depicting analysis of liver weights. Pairwise comparisons of all combinations of genotypes revealed no significantdifferences. Fig. 6C. Brain weights are compared after assigning animals into three different age groups (per genotype: n=25-32 for 6-9 month group, n=7-15 for 11-16 month group; n=5-8 for 21-26 month group). Statistical analyses were conducted by one-way ANOVA using Tukey's multiple comparisons test on log transformed data. All possible comparisons were tested but unless indicated, comparison bars are only shown for significant differences, ns, not significant; p<0.0001 ; ***, p<0.001 ; **, p<0.01.DETAILED DESCRIPTION

[0049] The present application provides for the treatment of CLN3 disease with recombinant TPP1 (rhTPPI). It is hypothesized that because patients with CLN3 also have disrupted TPP1 activity, administration of rhTPPI may be useful to lessen symptoms of CLN3 disease, delay onset of symptoms of CLN3 disease, or restore certain physical functionalities to CLN3 patients.

[0050] The following definitions may be useful in aiding the skilled practitioner in understanding the disclosure. Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range. For example, the ranges with the term “about”.

[0051] As used in the specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” include plural as well as singular referents unless the context clearly dictates otherwise.

[0052] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1 , 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about" or “approximately” applies to each one of the numerical values in that series

[0053] The term “family history” refers to a subject having a blood relative diagnosed with CLN3 disease, e.g., a sibling, parent, grandparent, great-grandparent, etc.

[0054] The terms “subject” and “patient” are used interchangeably. As used herein, the terms “subject” and “subjects” may refer to a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). In various embodiments, the mammal is a human.

[0055] The term “fragment” refers, in one aspect, to a recombinant protein comprising a portion of the rhTPPI proenzyme amino acid sequence set forth in SEQ ID NO:1. For example, a fragment may contain at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the amino acid sequence set forth in SEQ ID NO:1 . In another aspect, a fragment may comprise the full-length (368 amino acids long; amino acids 177 — 544 of SEQ ID NO:1 ) mature TPP1 enzyme amino acid sequence set forth in SEQ ID NO:2, a portion thereof, and / or at least the catalytic triad formed by the amino acid residues S456, E253, and D341. A fragment retains catalytic activity. For example, a fragment exhibits tripeptidyl exopeptidase activity and / or exhibits catalytic activity that results in the sequential release of tripeptides from the N-terminus of a protein substrate. In certain aspects, a “fragment” of the rhTPPI proenzyme comprises at least 500 consecutive amino acids of SEQ ID NO:1 , at least 450 consecutive amino acids of SEQ ID NO:1 , at least 400 consecutive amino acids of SEQ ID NO:1 , at least 368 amino acids of SEQ ID NO:1 , at least 350 amino acids of SEQ ID NO:1 or at least 300 consecutive amino acids of SEQ ID NO:1. In other aspects, a “fragment” of the rhTPPI proenzyme comprises at least 350 consecutive amino acids of SEQ ID NO:2, at least 325 consecutive amino acids of SEQ ID NO:2, at least 300 consecutive amino acids of SEQ ID NO:2, at least 275 consecutive amino acids of SEQ ID NO:2, at least 250 consecutive amino acids of SEQ ID NO:2 or at least 200 consecutive amino acids of SEQ ID NO:2.

[0056] The term “intracerebroventricular” refers to administration of a composition into the ventricular system of the brain, e.g., via injection, infusion, or implantation (for example, into a ventricle of the brain).

[0057] The term “intraocular” refers to the administration of a composition to the eye region, e.g., via injection, infusion, or implantation (for example, into the eyeball) or topical / ophthalmic administration (for example, using a cream, ointment, gel or liquid drops). “Intravitreal” refers to injection into the vitreous of the eye.

[0058] The term “intrathecal” refers to administration of a composition into the lumbar region, e.g., via injection, infusion, or implantation (for example, into the subarachnoid space of the spinal cord).

[0059] The term “therapeutically effective” refers to any therapeutic benefit that arises as a result of the treatment methods provided by the present disclosure. For example, such an effect can be the beneficial effects that manifest in an appropriate target tissue or organ, where such beneficial physiological effect is compared to that physiological parameter being measured in the absence of the enzyme replacement therapy. Such a therapeutic effect may be any reduction or elimination of one or more clinical or subclinical manifestations of CLN3 disease. For example, a therapeutically effective treatment improves, reverses, delays, prevents, or reduces deterioration of one or more physiological function and / or neurological symptom of CLN3 as described herein.

[0060] The term “stable” or “stabilized” refers to a protein-containing formulation in which the protein component therein essentially retains its physical, functional and / or chemical stability upon storage over time. Stability can be measured at a selected temperature for a selected time period. Preferably, the formulation is stable at room temperature (about 30° C.) or at about 40° C. for at least 1 month and / or stable at about 2° C. to about 8° C. for at least 1 year and preferably for at least 2 years. For example, the extent of protein degradation or aggregation during storage can be used as an indicator of protein stability. Thus, a “stable” formulation may be one wherein less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the protein component is present in a degraded or aggregated form in the formulation following storage. “Stable” formulations retain essentially the same functional or therapeutic characteristics of the newly prepared formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).

[0061] The term “prevents” or “reduces” or grammatical equivalents thereof when used in reference to the prevention or reduction of one or more symptoms or physiological consequences of CLN3 disease in a subject means that the rate of decline of that / those symptom(s) in the treated CLN3 subject is slower than that observed in an untreated CLN3 subject. In this regard, the un-treated CLN3 may be the same subject that is subsequently treated with a composition or method of the present disclosure or may be the average rate of decline of the symptom(s) of interest as observed from the natural history study results disclosed herein.

[0062] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” rhTPPI or a formulation thereof to a human subject refers to medical uses for rhTPPI or a formulation thereof, for example, rhTPPI or a formulation thereof for use in treating CLN3 disease as described herein or use of rhTPPI for the manufacture of a medicament for treating CLN3 disease as described herein. The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, “administering” rhTPPI or a formulation thereof includes both methods practiced on the human body and also the foregoing activities.

[0063] The present disclosure provides formulations and kits comprising rhTPPI , and methods of using the same (i.e., formulations and kits comprising rhTPPI) to treat CLN3 disease. Administration of rhTPPI allows for cellular uptake of the protein by the cation independent mannose 6 phosphate receptor (CI-MPR, IGF2 receptor or CD222) and localization to the lysosomes in cells throughout the central nervous system. The enzyme uptake into the lysosomes and subsequent activation promotes increased catabolism of storage material in affected tissues and organs (e.g., brain, eyes), reduces the progressive accumulation of the lysosomal storage material, and arrests decline of the disease. The formulations and methods of the disclosure provide therapeutic benefits that surpass those of currently approved therapeutic treatments.

[0064] In various embodiments, rhTPPI protein is produced in cell culture as a zymogen (proenzyme), which does not have enzymatic activity. The proenzyme is auto-activated at acidic pH (and by lysosomal proteases) upon uptake to the lysosome. The mature native TPP1 protein is a lysosomal serine protease, and is the only known mammalian member of the sedolisin (serine-carboxyl peptidase) family characterized by a highly conserved Ser-Glu-Asp (SED) catalytic triad. The catalytic triad on rhTPPI is formed by 5456, E253 and D341. The primary activity of the enzyme is as a tripeptidyl exopeptidase with a broad substrate specificity. Activity of the enzyme on its substrate leads to a sequential release of tripeptides from the N-terminus of the protein substrate (Oyama et al., J Biochem. 2005; 138(2): 127-34). A secondary, significantly weaker endoproteolytic activity with a pH optimum of 3 has also been reported (Lin et al., J Biol Chem. 2001 ; 276(3) :2249-55).Formulations

[0065] With regard to the present disclosure, the formulations comprise an amount of rhTPPI and in exemplary aspects, the formulation is suitable for intracerebroventricular, intrathecal, and / or intraocular administration. In one aspect, the rhTPPI comprises SEQ IDN0:1 or a fragment thereof. RhTPPI proteins suitable for use in the formulations and methods described herein, and methods of obtaining the rhTPPI proteins, are described in U.S. Pat. Nos. 6,302,685, 8,277,800, 10,279,015, 10,758,598 and 11,229,687, incorporated herein by reference in their entirety.

[0066] In one aspect, the rhTPPI comprises the amino acid sequence of SEQ ID NO:1 (amino acids 1-544) or a fragment thereof possessing catalytic activity (i.e., is “functional”). In another aspect, the rhTPPI comprises the amino acid sequence of SEQ ID NO: 2 (amino acids 177-544 of SEQ ID NO: 1) or a fragment thereof possessing catalytic activity. In still another aspect, the rhTPPI has detectable enzyme activity or is processed in vivo to a form of the enzyme that has detectable enzyme activity (i.e., is “functional”) and has at least about 70% sequence identity with SEQ ID NO:1 or SEQ ID NO:2. For example, the functional rhTPPI is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 97% identical, to SEQ ID NO:1 or SEQ ID NO:2.

[0067] In one aspect, the formulation is a liquid formulation that comprises rhTPPI at a concentration of about 1 mg / mL to about 100 mg / mL, for example, about 10 mg / mL to about 50 mg / mL, about 25 mg / mL to about 40 mg / mL, or about 30 mg / mL to about 60 mg / mL. In various aspects, the formulation comprises rhTPPI at a concentration of from about 1 mg / mL to about 100 mg / mL, from about 5 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 20 mg / mL to about 40 mg / mL, from about 25 mg / mL to about 35 mg / mL, more specifically about 1 mg / mL, about 10 mg / ml, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, or about 100 mg / mL. In one aspect, the formulation has a pH of about 5.5 to about 7.5 or about 6.0 to about 7.0, for example, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

[0068] In one aspect, a formulation comprising rhTPPI of the disclosure further comprises one or more excipients that maintains the level of a key electrolyte in the cerebrospinal fluid (CSF) or ocular fluid. For example, in one aspect, in addition to the rhTPPI or fragment thereof, the formulation further comprises potassium chloride at a concentration of about 0.01 mg / mL to about 1 mg / mL, for example, about 0.1 mg / mLto about 0.5 mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.2 mg / mL to about 0.4 mg / mL, about 0.15 mg / mL to about 0.25 mg / mL, or about 0.05 mg / mL to about 0.3 mg / mL. In another aspect, the formulation further comprises magnesium chloride hexahydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, for example, about 0.1 mg / mLto about 0.5 mg / mL, about 0.1 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.15 mg / mL to about 0.25 mg / mL, or about 0.05 mg / mL to about 0.3 mg / mL. In another aspect, theformulation further comprises calcium chloride dihydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, for example, about 0.1 mg / mL to about 0.5 mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.15 mg / mL to about 0.25 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, or about 0.05 mg / mL to about 0.3 mg / mL. In still another aspect, the formulation comprises a combination of all or any of the foregoing.

[0069] In various embodiments, the formulation comprises rhTPPI at a concentration of from about 25 mg / mL to about 35 mg / mL.

[0070] In various embodiments, the formulation has a pH of about 6.5.

[0071] In another aspect, the formulation comprising rhTPPI further comprises one or more buffering agents. For example, in various aspects, the formulation further comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, for example, about 0.1 mg / mL to about 0.5 mg / mL, about 0.05 mg / mL to about 0.4 mg / mL, or about 0.1 mg / mL to about 0.3 mg / mL; and / or sodium phosphate monobasic monohydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, for example, about 0.01 mg / mL to about 0.2 mg / mL, about 0.05 mg / mL to about 0.3 mg / mL, or about 0.08 mg / mL to about 0.4 mg / mL.

[0072] In another aspect, the formulation further comprises an isotonicity agent, such as sodium chloride at a concentration of about 1 mg / mL to about 20 mg / mL, for example, about 1 mg / mL to about 10 mg / mL, about 5 mg / mL to about 15 mg / mL, or about 8 mg / mL to about 20 mg / mL. Other buffering agents and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations of the present disclosure.

[0073] In one aspect, a formulation comprising about 30 mg / mL of rhTPPI further comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration of about 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, calcium chloride dihydrate at a concentration of about 0.21 mg / mL, and a diluent, such as water for injection.

[0074] The rhTPPI formulations of the present disclosure are stable and can be stored for extended periods of time without an unacceptable change in quality, potency, or purity. In one aspect, the formulation is stable at a temperature of about 5° C. (e.g., 2° C. to 8° C.) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about -20° C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24months, at least 36 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about -40° C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about -60° C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.

[0075] In one aspect, a formulation of the disclosure is preservative-free and / or stabilizer-free and thus does not contain any of thimerosal, phenylmercurate salts, chlorhexidene, phenol, benzoic acid, sorbic acid, parabens, alcohols, or other preservatives commonly found in parenteral or ophthalmic formulations.

[0076] In another aspect, the formulation useful in the present methods may comprise one or more preservatives, stabilizers or excipients. In this regard, numerous well known and routinely employed preservatives, stabilizers and excipients useful for protein-containing formulations for intrathecal or ICV delivery are known in the art. More specifically, examples of such additives to enzyme-containing formulations for use in intrathecal or ICV delivery are described in W02013 / 096899 and US Pat. No.10,279,015, which are herein incorporated by reference.Methods

[0077] The disclosure provides methods of treating CLN3 disease comprising administering a therapeutically effective amount of a formulation comprising rhTPPI described herein to a subject in need thereof. The disclosure also provides a composition comprising rhTPPI for use in treating CLN3 disease described herein and use of rhTPPI in the manufacture of a medicament for treating CLN3 disease described herein. In some aspects, a subject exhibits one or more mutations in the CLN3 gene as determined by a genetic test. In another aspect, a subject can exhibit one or more clinical symptoms of CLN3 disease and / or where the subject has a family history of CLN3 disease.

[0078] Natural history studies relating to diagnosis and monitoring of CLN3 are described in Masten et al. (Neurology 94:e2436-e2440, 2020) which established a CLN3 staging system (CLN3SS) useful for cohort stratification and analysis of disease progression.Individuals are assigned to a stage based on the following criteria: stage 0, genetic confirmation; stage 1 , genetic confirmation and vision loss; stage 2, genetic confirmation, vision loss, and one or more seizures; stage 3, genetic confirmation, vision loss, one or more seizures and unable to walk without assistance. Masten {supra) also correlated average age with stage, showing that the average age of an individual in stage 1 is 9.5, stage 2 is 17.75 and stage 3 after 17.75 years. Similarly, Masten et al. correlated UBDRS score with stage ofdisease, summarizing that individuals in stage 1 had an average UBDRS physical subscale score of approximately 5 + / - 5, individuals in stage 2 had an average UBDRS physical subscale score of approximately 17 + / - 10, and stage 3 had an average UBDRS physical subscale score of approximately 50 + / - 15. The capability subscale score in each stage was determined to be: stage 1 , approximately 2.5 + / - 2.5; stage 2, approximately 6 + / - 2.5; stage 3, approximately 10 + / - 2. See Table 1 below.Table 1: CLN3 Disease Specific Staging Scale (CLN3SS)

[0079] In various aspects, the disclosure provides a method of treating CLN3 disease, or one or more clinical symptoms of CLN3 disease, comprising administering a composition comprising a therapeutically effective amount of rhTPPI to a subject in need thereof, use of rhTPPI in the manufacture of a medicament for the treatment of CLN3 disease in a subject, or rhTPPI for use in treating CLN3 disease in a subject.

[0080] The disclosure also provides methods of preventing one or more clinical symptoms of CLN3 disease comprising administering a formulation comprising rhTPPI described herein to a subject in need thereof, optionally wherein the subject has a family history of CLN3 disease. In various aspects, the disclosure provide a method of preventing one or more clinical symptoms of CLN3 disease comprising administering a composition comprising a therapeutically effective amount of rhTPPI to a subject in need thereof, use of rhTPPI in the manufacture of a medicament for the prevention of one or more clinical symptoms of CLN3 disease in a subject, or rhTPPI for use in preventing one or more clinical symptoms ofCLN3 disease in a subject, optionally wherein the subject has a family history of CLN3 disease.

[0081] The disclosure also provides methods of delaying the onset of one or more clinical symptoms of CLN3 disease comprising administering a formulation comprising rhTPPI described herein to a subject in need thereof, optionally wherein the subject has a family history of CLN3 disease. In various aspects, the disclosure provides a method of delaying the onset one or more clinical symptoms of CLN3 disease comprising administering a composition comprising a therapeutically effective amount of rhTPPI to a subject in need thereof, use of rhTPPI in the manufacture of a medicament for the delay of one or more clinical symptoms of CLN3 disease in a subject, or rhTPPI for use in delaying one or more clinical symptoms of CLN3 disease in a subject, optionally wherein the subject has a family history of CLN3 disease. For example, delaying can be maintaining or slowing progression of one or more clinical symptoms of CLN3 disease in a subject.

[0082] The Unified Batten Disease Rating Scale (UBDRS) is a tool used to assess the severity and progression of CLN3 disease, also known as Juvenile Batten Disease (Wibbeler et al., Eur J Paediatr Neurol. 2022 May; 38:62-65). It was developed to assess disease severity in 4 domains: physical, behavior, seizures, and functional capability. See Table 1 below. In the physical subscale there are 28 different items which are each rated on a scale 0 (normal) to 4 (severe impaired). The total range of the physical subscale includes from 0 to 112 points. The higher the points the more severe the status of the disease. The behavior, seizure and functional capability assessments are based on interviews with the parents or primary caregivers. The 9 behavioral items (sad mood, apathy, anxiety, aggression toward others, aggression toward self, stereotyped / repetitive behavior, compulsions, auditory hallucinations, obsessions) are rated by severity (0 = normal, 3 = severe) and frequency (0 = never, 3 = almost always). In the 12 seizure assessment questions relating to form, duration, frequency, complications and medication changes are asked. The patients’ capability items include 5 items: school, chores, play, activities of daily living and overall care levels, asked from the perspective of how the activity would be without the influence of blindness.Table 2: The Unified Batten Disease Rating Scale (UBDRS)

[0083] Elements of the physical assessment that are measured include speech clarity, abnormal speech sounds, tongue protrusion, visual acuity, passive motion in the arms, passive motion in the legs, passive motion in the neck, power in arms, power in legs, hand taps, maximal dystonia, normal spontaneous movements, gait, retropulsion pull test, heel stomping, motor tic stereotypes, myoclonus, rest tremor, tremor with maintained posture or action, dysmetria, appendicular chorea, weight, and motor examination. Seizure assessment includes the following parameters: tonic / clonic seizures-average frequency, tonic / clonic seizures-post ictal period, atonic seizures-average frequency, myoclonic seizures-average frequency, complex partial seizures without generalization and / or absence-average frequency, complex partial seizures without generalization and / or absence-post ictal period, simple partial seizures-average frequency, simple partial seizures-average duration of event, frequency of injury related to seizures, maximal level of care for seizure complications, hospitalization for seizures, anticonvulsant adjustment to control seizures in the past month, and seizure assessor.

[0084] In In various embodiments, the administration slows or reduces time to progression from one stage of CLN3 to the next as determined by CLN3SS described herein. In various embodiments, the stage is determined based on UBDRS severity, capability score severity and / or age of the subject.

[0085] In various embodiments, the subject to be treated has a UBDRS score between 0 and 10, or between 5 and 27, or between 25 and 70. In various embodiments, the subject has a capability score between 0 and 5, between 3.5 and 9 or between 7 and 15.

[0086] The disclosure further provides methods of treating CLN3 disease comprising administering rhTPPI to a subject in need thereof at a dose effective to maintain a physiological function or slow or reduce deterioration of a physiological function in the subject, wherein the physiological function is vison, language function, or motor function. The disclosure also provides use of rhTPPI in the manufacture of a medicament for maintaining a physiological function or slowing or reducing deterioration of a physiological function in a subject having CLN3, and rhTPPI for use in maintaining a physiological function or slowing or reducing deterioration of a physiological function in a subject having CLN3 disease; wherein the physiological function is vision, language function, or motor function.

[0087] Provided herein is a method of treating a subject having CLN3 or a family history of CLN3 disease comprising administering a dose of rhTPPI effective to improve vision to thesubject. In one aspect, the improvement in vision is an improvement, for example, lower points in at least one UBDRS domain or at least one UBDRS subscale, compared to a previous rating determined before or during treatment as measured using a UBDRS or compared to natural history studies on CLN3 disease progression. Assessment of vision will be carried out using standard vision tests.

[0088] In various embodiments, assessment of vision or vision loss includes assessment of retinal health. Methods of assessing vision loss include OCT scans, OCT-B scans, Spectral-Domain Optical Coherence Tomography (SD-OCT), Electroretinogram (ERG) and change in average central retinal thickness (ACRT) over time. Changes in vision can be determined based on the Weill Cornell Ophthalmic Severity Scores (WCOSS). In various embodiments, the intravitreal administration reduces or slows time to progression on the WCOSS, e.g., from stage 1 to stage 2, from stage 2 to stage 3, from stage 3 to stage 4, or from stage 4 to stage 5.

[0089] Provided herein is a method of treating a subject having CLN3 or a family history of CLN3 disease comprising administering a dose of rhTPPI effective to motor function in the subject. In one aspect, the improvement in motor function is improvement compared to a previous rating determined before or during treatment as measured using a UBDRS or compared to natural history studies. Exemplary motor functions include walking and other everyday activities. A 50 minute walk and / or cognitive assessment is contemplated to determine change in these parameters before and after administration of a therapeutically effective dose of rhTPPI provided by the disclosure.

[0090] Also provided is a method of treating a subject having CLN3 or a family history of CLN3 disease comprising administering a dose of rhTPPI effective to improve language function and / or cognitive function in the subject. In one aspect, the improvement in cognitive function is an improvement compared to a previous rating determined before or during treatment as measured using a UBDRS or compared to CLN3 natural history studies.

[0091] The disclosure further provides methods of treating CLN3 disease comprising administering rhTPPI to a subject in need thereof at a dose effective to prevent or treat a neurological symptom of the disease, wherein the neurological symptom is a seizure, decrease in brain volume, decrease in gray matter in the brain, or increase of cranial cerebrospinal fluid (CSF). The disclosure also provides use of rhTPPI in the manufacture of a medicament for preventing or treating a neurological symptom in a subject having CLN3 or a family history of CLN3, and rhTPPI for use in preventing or treating a neurological symptom in a subject having CLN3 or a family history of CLN3; wherein the neurologicalsymptom is a seizure, decrease in brain volume, decrease in gray matter in the brain, or increase of cranial CSF.

[0092] Provided is a method of treating a subject with CLN3 disease comprising administering a composition, formulation or dose of rhTPPI effective to maintain or reduce the number of seizures experienced by the subject. In one aspect, the dose is effective to reduce the number of seizures per month that the subject experiences. In another aspect, the dose is effective to increase the seizure rating by at least one point compared to a previous rating determined before or during treatment as measured using a disease rating scale.

[0093] Provided is a method of treating a subject with CLN3 disease comprising administering a composition, formulation or dose of rhTPPI effective to maintain or improve cognitive function or behavior in the subject. In another aspect, the dose is effective to improve cognitive function or behavior by at least one point compared to a previous rating determined before or during treatment as measured using a disease rating scale

[0094] The disclosure provides a method of treating a subject having CLN3 or a family history of CLN3 comprises administering a composition, formulation or dose of rhTPPI effective to maintain brain volume or slow or reduce the decrease in brain volume to a subject. Brain atrophy increases as the disease progresses, resulting in a loss of brain volume and an associated increase in the volume and relative proportion of intracranial CSF. Brain volume can be measured using methods known in the art, including imaging techniques such as magnetic resonance imaging (MRI), computed tomography (CT / CAT), positron emission tomography (PET), single photon emission computerized tomography (SPECT), electroencephalography (EEG), magnetoencephalography (MEG), and near infrared spectroscopy (NIRS). In one aspect, the dose of rhTPPI is effective to slow or reduce the CLN3-associated decrease in brain volume in the subject, which can be demonstrated by maintenance of brain volume for a longer period of time or a smaller decrease in brain volume, compared to what would be expected considering the natural progression of the disease.

[0095] In another aspect, methods of treating a subject having CLN3 or a family history of CLN3 comprise administering a composition, formulation or dose of rhTPPI effective to maintain gray matter in the brain or slow or reduce the decrease of gray matter in the brain to a subject. A loss of gray matter due to brain atrophy occurs as the disease progresses, resulting in a decrease in gray matter as a percentage of brain volume. The amount of gray matter in the brain can be assessed using methods known in the art, for example, imaging techniques such as MRI, CT / CAT, PET, SPECT, EEG, MEG, and NIRS. In one aspect, thedose of rhTPPI is effective to slow or reduce the decrease in gray matter in the subject, which can be demonstrated by maintenance of gray matter volume for a longer period of time or a smaller decrease in gray matter as a percentage of brain volume, compared to what would be expected considering the natural progression of the disease.

[0096] In another aspect, a method of treating a subject having CLN3 or a family history of CLN3 comprises administering a composition, formulation or dose of rhTPPI effective to maintain the volume of cranial CSF or slow the increase in the volume of cranial CSF to a subject. Cranial CSF increases in volume and proportion of total CSF as a result of cerebral atrophy. The amount and proportion of cranial CSF can be assessed using methods known in the art, for example, imaging techniques such as MRI and CT / CAT. In one aspect, the dose of rhTPPI is effective to slow or reduce the increase in cranial CSF in the subject, which can be demonstrated by maintenance of the volume of cranial CSF for a longer period of time or a smaller increase in cranial CSF as a percentage of total CSF, compared to what would be expected considering the natural progression of the disease.

[0097] Additional readouts assessed in a subject include serum, urine and / or cerebrospinal fluid (CSF) biomarkers, such as levels of neurofilament light (NFL), lysosomal storage of subunit c of mitochondrial ATP synthase (SCMAS), glial fibrillary associated protein (GFAP), neurofilament heavy (NFH), neurofilament medium (NFM), cathepsin F (CTSF), acid sphingomyelinase (SMPD1), and Niemann-Pick C1 (NPC1). Also assessed is the amount of neuroinflammation in the brain of the subjects, e.g., using MRI and other brain imaging techniques. Levels of activated neurological cells are also measured, e.g., including microglia and astrocytes, which can be measured by MRI quantitative susceptibility mapping (QSM), diffusion-weighted MRI or.paramagnetic rim lesions (PRLs).

[0098] The foregoing methods, compositions for use, and uses may further comprise any of the following features, alone and in combination.

[0099] In one aspect, a method, composition for use, or use of (i.e., medical use of the rhTPPI or a formulation thereof) the disclosure comprises administering a formulation, composition or dose comprising rhTPPI to a subject continuously or continually over a period of at least about 1 hour, for example, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, or more. In another aspect, a method or use of the disclosure comprises administering a formulation, composition or dose comprising about 20 mg to about 500 mg, about 30 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 200 mg to about 400 mg, about 250 mg to about 350 mg, or about 275 mg to about 325 mg of rhTPPI to a subject in need thereof, for example, about 20 mg, about 30 mg, about 50 mg,about 100 mg, about 200 mg, about 300 mg, about 400 mg, or about 500 mg of rhTPPI . In one aspect, a method or use of the disclosure comprises administering a formulation, composition or dose having a volume of about 20 mL or less, about 15 mL or less, about 10 mL or less, about 7.5 mL or less, or about 5 mL or less, for example, about 20 mL, about 15 mL, about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL about 2 mL, about 1 mL, or about 0.5 mL per dose or administration event.

[0100] In various aspects, a method, composition for use, or use of the disclosure comprises administering a formulation, composition or dose comprising rhTPPI to a subject at a rate of less than or equal to about 2.5 mL of the formulation, composition or dose per hour; less than or equal to about 75 mg of rhTPPI per hour; or less than or equal to about 75 mg of rhTPPI per 2.5 mL of formulation or composition per hour. The formulation, composition or dose is optionally administered continuously or continually over a period of at least about 4 hours.

[0101] In one aspect, a method, composition for use, or use of the disclosure comprises administering a formulation, composition or dose comprising rhTPPI weekly or less frequently, for example, weekly, every other week, or monthly. More specifically, a method, composition for use, or use of the disclosure comprises administering a formulation, composition or dose comprising rhTPPI once every 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days. In one aspect, the formulation, composition or dose is administered intracerebroventricularly. In another aspect, the formulation, composition or dose is administered intrathecal ly. In still another aspect, the formulation, composition or dose is administered intraocularly. In another aspect, the formulation, composition or dose is administered intravitreally. In one aspect, the formulation, composition or dose is administered intracerebroventricularly or intrathecally, as well as intravitreally. In various embodiments, the formulation, composition or dose is administered intracerebroventricularly and intravitreally. Intracerebroventricular delivery allows penetration to the deep gray structures of the brain such as the thalami, striatum and midbrain, due to the physiology of CSF flow in which ventricular delivery allows flow into third and fourth ventricles, but also percolates through the neuropil of the cerebral hemispheres, along a slight pressure gradient from ventricle to subarachnoid space. Intraocular / intravitreal administration provides direct administration of the therapeutic to the eye to improve symptoms of vision loss in subjects. Intrathecal and intracerebroventricular administration of recombinant enzyme to treat lysosomal storage disorders are described in U.S. Patent No. 7,442,372, incorporated herein by reference in its entirety.

[0102] A formulation, composition, or dose of rhTPPI useful in the methods may be administered in a single bolus injection or series of injections (e.g., into the brain, lumbar region, or eye), or as a continuous or continual infusion, e.g., using an infusion pump or other implanted device. In one aspect, a formulation, composition, or dose of rhTPPI is administered using an infusion system comprising tubing, an in-line filter (e.g., about 0.2 pm), a reservoir (e.g., intrathecal or intracerebroventricular), and a catheter. Frequently, when a composition is administered intrathecally or intracerebroventricularly, in order to prevent adverse side effects resulting from artificially increasing intracerebral or intrathecal pressure, a volume of CSF comparable to the volume of composition to be administered is first removed from the subject before the composition is administered.

[0103] In one aspect, a method or use of the disclosure comprises administering about 10 mL of a formulation, composition or dose comprising about 300 mg of rhTPPI intracerebroventricularly over a period of about 4 hours every other week to a subject having CLN3. In various embodiments, a dosage of about 0.20 mg to 5 mg is administered to the subject intravitreally.

[0104] The formulations and compositions of the present disclosure may be directly administered to a subject in need (i.e. , non-isovolumetric) or may be administered subsequent to removal of a defined volume of CSF from the subject prior, wherein that defined volume is approximately the same as the volume of the composition subsequently administered (i.e., isovolumetric).

[0105] In one aspect, a method, composition for use, or use of the disclosure further comprises administering a flushing solution to the subject following administration of the rhTPPI . The flushing solution is administered via the same route as the rhTPPI and using the same delivery system (e.g., an infusion system), to remove any rhTPPI remaining in the delivery system and to ensure the subject received the full intended dose of rhTPPI . In one aspect, the flushing solution is administered (e.g., using the same catheter previously used to administer a composition comprising rhTPPI) to the subject in an amount between about 0.5 mL and about 5 mL, for example, about 0.5 mL, about 1 mL, about 2 mL, about 3 mL, or about 5 mL. In one aspect, the flushing solution comprises the same components as the formulation or composition comprising rhTPPI , but without the rhTPPI . In one aspect, the flushing solution comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration of about 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, calcium chloride dihydrate at a concentration of about 0.21 mg / mL, and a diluent, such as water for injection.

[0106] In various embodiments, the time of maximum concentration (Tmax) of rhTPPI in cerebrospinal fluids is between 4 and 10 hours after end of infusion. In various embodiments, elimination half-life (t-1 / 2) of rhTPPI in cerebrospinal fluid is between 5 and 20 hours.

[0107] The present disclosure is also directed to methods of treating CLN3 disease, or one or more symptoms associated with CLN3 disease, in a subject, and methods of delaying the onset of CLN3 disease, or a symptom thereof, in a subject, wherein the subject is a juvenile subject, e.g., less than about 18 years old, or from about 2 to about 18 years old, or from about 4 to about 18 years old, or from about 2 to about 9 years old, or from about 4 to about 9 years old.

[0108] In exemplary instances, the subject is a sibling of an individual diagnosed with CLN3. In exemplary instances, the subject is not a sibling of an individual diagnosed with CLN3.

[0109] In exemplary aspects, the subject has no prior treatment with a stem cell therapy, gene therapy, or enzyme supplementation therapy. In exemplary instances, the method comprises administering to the subject an antihistamine with or without an antipyretic before administration of the rhTPPI , optionally, about 30 to about 60 minutes before administration of the rhTPPI . In various aspects, the formulation comprises the rhTPPI and at least one pharmaceutically acceptably carrier, diluent or excipient. In various instance, the formulation is any one of the formulations provided herein, including but not limited to being one which comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof. In various instances, the method comprises administering to the subject a flush solution after administering the formulation. In various aspects, the flush solution is any one of those described herein. In various aspects, the flush solution comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof. In various aspects, the treatment period is at least 10 weeks, at least 20 weeks, at least 40 weeks, at least 80 weeks, or at least 96 weeks. In various aspects, the treatment period is longer than 96 weeks. In various aspects, the methods treat the CLN3 disease or delay the onset of the CLN3 disease, or the symptom thereof, without causing serious adverse events (SAEs). In various aspects, the subject has an ICV device. In various aspects, the method comprises implanting an ICV device in the subject.Kits

[0110] The disclosure further provides kits comprising a formulation of rhTPPI described herein, in a dose and form suitable for administration to a CLN3 patient. In one aspect, the kit comprises a formulation comprising about 30 mg / mL of rhTPPI , sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration of about 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, calcium chloride dihydrate at a concentration of about 0.21 mg / mL, and a diluent, such as water for injection. In one aspect, the kit further comprises instructions for the intracerebroventricular, intrathecal, and / or intraocular administration of the therapeutic compositions of the present disclosure, in addition to the therapeutic formulation. In another aspect, the kit further comprises a flushing solution as described herein. In still another aspect, the kit further comprises a system for administering the formulation, comprising any or all of the following components: tubing, an in-line filter, a reservoir for implantation, and a catheter. In one aspect, the kit may comprise catheters, reservoirs, or other devices preloaded with the therapeutic formulations of the present disclosure. For example, catheters preloaded with about 100 mg of rhTPPI , about 200 mg of rhTPPI , about 300 mg of rhTPPI , about 400 mg of rhTPPI , or about 500 mg of rhTPPI , in a pharmaceutically acceptable formulation, are specifically contemplated.Alternatively, the kit may comprise catheters, reservoirs, or other devices that are refillable and appropriate amounts of the rhTPPI enzyme provided by the disclosure for refilling such devices.

[0111] In certain embodiments, kits of the present disclosure may comprise one or more of the following components: an extension line (e.g., product number 536040, Smiths Medical, Dublin OH), an in-line filter (e.g., product number FS116, Smiths Medical), a port needle (e.g., product number 21-2737-24, Smiths Medical), a syringe or two or more syringes (e.g., product number 309604, Becton Dickinson, Franklin Lakes, NJ) or a syringe needle or two or more syringe needles (e.g., product number 305196, Becton Dickinson).

[0112] The present disclosure will be more readily understood by reference to the following Examples, which are provided by way of illustration and are not intended to be limiting.EXAMPLESExample 1-Cinical Study of RhTPPI to Treat CLN3

[0113] Previous preclinical models of CLN3 disease studied the effect of TPP1 transgene in JNCL mice (See e.g., US20240158772). Results showed that animals expressing the TPP1 transgene and making TPP1 protein had less SCMAS accumulation at 6 months in theliver spleen cortex and thalamus of animals. Levels of CD68 were reduced in the cerebellum cortex and thalamus by 45%, 29% and 60%, respectively. GFAP detected in the cortex and thalamus was also reduced by 73% and 79% respectively in JNCL mice expressing TPP! compared to those lacking the transgene.

[0114] In order to determine the effects of administering TPP1 recombinant human protein (rhTPPI) to a subject with CLN3 disease, a Phase 2 study is undertaken in human juvenile aged subjects ranging from 4-9 years age and diagnosed with CLN3 disease. For example, the subject may exhibit one or more mutations in the CLN3 gene as determined by a genetic test.

[0115] Two treatment groups, (1 ) 4-5 yrs of age and (2) 8-9 yrs of age, will be administered rhTPPI via intravitreal and intracerebroventricular administration. Patients will receive 150 mg / 5 mL (30 mg / mL) rhTPPI (BRINEURA®, cerliponase alfa). Intraventricular administration will be as prescribed for BRINEURA.

[0116] The intravitreal injection of cerliponase alfa (0.2mg diluted in 20 ul, e.g., in artificial CSF, commercially available) will be performed in the operating room (OR) under sedation and under sterile technique into the proclaimed study eye(s) in a 4-week interval over 24 months. Unilateral injections will occur for the first 12-months of treatment. Bilateral injections will occur for the remaining 12-months of treatment.

[0117] The intravitreal injection will be performed with the following procedures:

[0118] A sterile1 / 2 inch 30-guage needle will be used for the injection, and this will be on the prepared syringe the pharmacist provides.

[0119] A drop of 5% providone-iodine (diluted 10% Betadine) is applied to the ocular surface. The primary investigator, medical doctor or medical professional will have the option to use chlorhexidine as an alternative for subjects with local irritation or allergy to providone-iodine.

[0120] The eye lids are scrubbed with 5% providone-iodine. A sterile wire eyelid speculum is placed to hold the eyelids open. The fornices (loose arching folds connecting the conjunctival membrane lining the inside of the eyelid with the conjunctival membrane covering the eyeball) are treated with topical 5% povidone-iodine.

[0121] Next, 20 pl total volume of the drug is injected through the inferior temporal sclera (the outer white part of the eye) into the vitreous cavity 3.0 mm posterior to the limbus with a 30-gauge needle. A sterile cotton swab is used to stem reflux of drug while removing the needle. 5% povidone-iodine will be applied to the ocular surface.

[0122] Immediately after injection, the optic nerve will be visualized to confirm retinal perfusion and the eye will be manually checked to confirm it is soft.

[0123] Subjects will be given cyclopentolate 1 % pre-injection and one drop of prednisolone acetate 1% in the operative eye. The subject will continue on the prednisolone acetate 1% drops QID for 1-week post injection. The primary investigator, medical doctor or medical professional will have the option to utilize moxifloxacin and prolong the administration of prednisone to protect against signs of infection.

[0124] Ocular Testing

[0125] Baseline ocular testing will be performed in the OR under anesthesia, as per NCH standard clinical protocols. Baseline ocular testing and assessment of ocular health will include a Spectral-Domain Optical Coherence Tomography (SD-OCT) of the optic nerve and macula to obtain retinal nerve fiber layer thickness, central macular thickness, and central foveal thickness. An OCT Spectralis Flex Module will be used to obtain these measurements. The OCT Spectralis is a non-contact ophthalmic diagnostic imaging device currently FDA approved. The FLEX module allows the Spectralis to be affixed to a movable stand with an adjustable arm, which allows OCT testing to be performed on subjects lying down. The FLEX module is not yet approved by the FDA. It is a non-significant risk device. Due to the characteristics of our study population, subjects will need to be under anesthesia during OCT testing. The FLEX module would allow us to obtain the necessary measurements. Fundus photos will be taken of the posterior pole and periphery. Any component of the baseline eye exam that could not be performed in the office will be completed during baseline ocular testing under anesthesia prior to the intravitreal injection. Ocular testing will then be performed immediately prior to each intravitreal injection. The Weill Cornell Ophthalmic Severity Scores (WCOSS) may serve as an objective marker of ocular disease severity and progression. The WCOSS rates severity on a scale of 1 to 5, with A LINCL ophthalmic score of 1 represents a normal fundus exam by OCT; a score of 2 shows normal retinal layers immediately adjacent at fovea; immediately outside fovea, outer retinal / photoreceptor atrophy; a score of 3 represents outer retinal atrophy / photoreceptor loss extending to 1.0 disc diameter from fovea; retinal layers appear normal outside fovea; a score of 4 shows early buildup of outer retinal hyper-reflective material in macula; more extensive outer retinal abnormalities extending 1.0 to 2.0 disc diameters from fovea; beyond 2.0 disc diameters, outer retina appears normal; a score of 5 shows nearly complete retinal atrophy in both the posterior pole and the peripheral retina with severe retinal thinning with generalized loss of photoreceptors and outer retina, involving the entire macula; extensive clumps of outer retinal hyper-reflective material; thickness map shows central thinning with paracentral thickening, with outer ring of further thinning (with bull's eye appearance); outerretinal changes also found outside the macula. Orlin et al., PLsOne, 2013, Volume 8, Issue 8:e73128.

[0126] Electroretinogram (ERG) assessment is also contemplated. An ERG measures the electrical activity of the retina in response to a light stimulus and can aid in assessing retinal cell health and overall retinal function.

[0127] OCT B-scans are also used to assess eye deterioration. OCT-B uses cross-sectional images of the retina created using OCT by laterally combining a series of axial depth scans (A-scan) to provide a 3D image of the retina. OCT-B is performed in patients in a non-eye reflexive state.

[0128] Change in average central retinal thickness (ACRT) over time is also used to determine progression of ophthalmic symptoms.

[0129] Ophthalmological Assessments

[0130] Subjects will have a baseline eye exam performed prior to the first intravitreal injection. The baseline eye exam will include an evaluation of vision by a preferential looking test (PLT). The PLT will be done using Teller Acuity Cards at a distance of 55cm. The PLT will first be performed binocularly. If subject is cooperative and can tolerate an eye patch, the test will then be performed monocularly, beginning with the right eye. A form of linear Visual Acuity (VA) testing will be attempted based on the subject’s level of cooperation. The linear VA testing options include E-ETDRS, HOTV, counting fingers, hand motions, light perception, fix and follow, and central steady maintained. The baseline eye exam will also include confrontation visual field testing, ocular motility evaluation, pupil exam, intra ocular pressure measurement, anterior segment exam, and dilated fundus exam and a refraction.

[0131] Subjects will have an eye exam 1 -day post intravitreal injection. Vision will be evaluated by a PLT. A form of linear visual acuity (VA) testing will be done based on the subject’s level of cooperation. The eye exam will also include confrontation visual field testing, ocular motility evaluation, pupil exam, intra ocular pressure measurement, anterior segment exam, and dilated fundus exam. The fundus will be examined for signs of inflammation, infection, or any other treatment related event. If exam findings indicate inflammation, the subject will continue treatment with topical eye prednisolone acetonide 1% drops If findings indicate infection, subject will be treated with antibiotics. Any other treatment related events will be treated at the investigator’s discretion. Adverse events and concomitant medications will be recorded.

[0132] Subjects will have an eye exam 7 days (+ / - 1 day) post intravitreal injection. The subject will have a clinic dilated exam in Pediatric Ophthalmology Associates. Vision will beevaluated by a PLT. A form of linear visual acuity (VA) testing will be done based on the subject’s level of cooperation. The eye exam will also include confrontation visual field testing, ocular motility evaluation, pupil exam, intra ocular pressure measurement, anterior segment exam, and dilated fundus exam. The fundus will be examined for signs of inflammation, infection, or any other treatment related event. If exam findings indicate inflammation, subject will continue prednisolone acetonide 1%, otherwise, these will be stopped. If findings indicate infection, subject will be treated with antibiotics. Any other treatment related events will be treated at the investigator’s discretion. Postoperative drops will be discontinued at this visit if clinically indicated. Adverse events and concomitant medications will be recorded.

[0133] Patients may also be assessed for gait and language change, brain volume, cerebral atrophy, and total disease assessment as outlined in the UBDRS for physical ability, behavior, seizures, and functional capability. Biomarkers, such as NFL, in the CSF and other biological samples from the subject are also measured.

[0134] Results herein are compared to natural history data to determine efficacy of treatment.Example 2: Analysis of TPP1 in Mouse model of CLN3

[0135] There is evidence to suggest a role for TPP1 in JNCL disease. Using mouse models of LINCL deficient in TPP1 (Tppl'' (Sleat et al., J Neurosci. 2004;24(41 ):9117-26), and JNCL deficient in CLN3 protein (Gins'') (Mitchison et al., The Batten Mouse Model Consortium [corrected]. Neurobiol Dis. 1999;6(5):321 -34) to investigate the effect of TPP1 genotype on disease progression in the JNCL mouse (Sleat et al., J Inherit Metab Dis. 2023. Epub 20230420), it was found that heterozygosity for a Tpp1 knockout allele has no effect on survival of an otherwise wild-type mouse but it significantly shortens the lifespan of a ClnS7' JNCL mouse. One possible explanation is that loss of CLN3 could alter the lysosomal microenvironment in such a way that partially impairs TPP1 function. This would result in a slow accumulation of undegraded SCMAS in the lysosome reflecting or contributing to neuronal death. Consistent with this, reduction of TPP1 activity by heterozygosity for a null allele would accelerate storage and exacerbate disease. Support for this hypothesis comes from the observation that TPP1 activity is elevated in JNCL (Mitchison, supra; Sleat 2023, supra; Junaid et al., Neurosci Lett. 1999;264(1 -3):157-60), which may be a compensatory response to the loss of CLN3 impairing the ability of TPP1 to degrade SCMAS. If this hypothesis is correct, JNCL might be regarded, in some respects, to be a later onset and more slowly progressing form of LINCL. Consistent with this, there are LINCL patients withhypomorphic TPP1 mutations that resulted in slowly progressing disease which was originally diagnosed as JNCL (Sleat et al., Am. J. Human Genetics. 1999;64(6):1511-23).

[0136] If TPP1 function is impaired in JNCL, contributing to disease, it was hypothesized that elevating TPP1 levels could correct such deficiencies. This is tested using a genetic approach in which a transgenic mouse that constitutively over-expresses TPP1 (Nemtsova et al., PLoS One.2018;13(2):e0192286) was crossed with a C / n3' / ' JNCL mouse to determine the effect of elevated TPP1 levels on SCMAS accumulation and disease progression. The experimental strategy was to determine the effect of transgenic overexpression of TPP1 on JNCL disease progression in a mouse model. Four experimental cohorts of littermate mice were established: wild-type (WT) Cln3* / +animals with or without the TPP1 transgene (Tg) and C / n ' (JNCL) animals with or without the TPP1 transgene. These groups are designated as: WT / Tg+, WT / Tg-, JNCL / Tg+ and JNCL / Tg-, respectively. The TPP1 transgenic overexpresses murine TPP1 from a CAG-promoter driven transgene integrated at the ROSA26 locus. Based on enzyme activity measurements, this mouse constitutively expresses TPP1 at levels ~10-fold and 50-fold higher than normal wild-type brain and liver, respectively. In brain, expression is ubiquitous, including cortex and hippocampus (Nemtsova, supra).

[0137] MATERIALS AND METHODS

[0138] Experimental design

[0139] All studies (brain weight measurements, neuropathology, behavior and Nfl measurements) were conducted a blinded manner where the respective researchers had access only to animal tag numbers which convey no information regarding genotype.Genotypes were revealed only after collection of data. Cohort sizes of n=6 mice were used for all studies except analysis of brain weight loss and plasma Nfl (see respective legends for details). Experimental cohorts were divided approximately equally among male and female animals.

[0140] Animals

[0141] Mice were maintained and used following protocols approved by the Rutgers University and Robert Wood Johnson Medical School Institutional Animal Care and Use Committee (“Preclinical evaluation of therapy in an animal model for LINCL,” protocol I09-0274-4). Both the JNCL model and the TPP1 transgenic were in a C57BL / 6 genetic background, and genotyping was conducted as described (Nemtsova, supra). Animals were deeply anesthetized with sodium pentobarbital / phenytoin (a 1 :4 dilution of Euthasol;Delmarva Laboratories, Midlothian, VA) and euthanized by transcardial perfusion with 0.9% saline for biochemical analyses. For histopathology, mice were anesthetized, perfused withsaline then perfusion-fixed with 4% paraformaldehyde in PBS. Brains were excised, fixed for 48 hours in 4% paraformaldehyde in PBS, and then transferred to 30% sucrose / PBS at 4°C until they sunk.

[0142] Neuropathology

[0143] Immunohistochemistry was conducted by NeuroScience Associates, Inc.(Knoxville, TN). In brief, mouse brains were embedded within a single block and 35 pm sagittal sections were cut. SCMAS was visualized using an affinity-purified rabbit anti-polyclonal antibody raised against SCMAS amino-terminal peptide which was custom-prepared by Pacific Immunology (Ramona, GA) (Xu et al., Mol Ther. 2011 ;19(10):1842-8). IHC analysis of GFAP and CD68 were conducted using rabbit anti-GFAP (DAKO), and rat anti-mouse CD68 (Bio-Rad). Sections were prepared from tissue blocks, immunostained with the primary antibodies overnight and visualized using a biotinylated secondary antibody (goat anti-rabbit IgG, Vector Labs) then avidin-biotin-HRP complex (Vector Labs) and diaminobenzidine tetrahydrochloride to create a visible reaction product as described (Sleat et al., Neurobiol Aging. 2022;118:106-7).

[0144] Measurement of Nfl

[0145] Plasma NfL concentration was measured with the Simple Plex Murine NF-L cartridge on the Ella Automated Immunoassay System according to manufacturer's instructions (Bio-techne GmbH, Minneapolis, USA). Plasma NfL results were extrapolated using the in-cartridge factory standard curve, with a quantification range of 2.7 - 10290 pg / ml. Quality control (QC) samples at three concentration levels and a pooled mouse plasma sample were included in each run to monitor assay performance. Six QC samples were prepared by spiking sample diluent at 16.08, 643.13 and 6431.3 pg / ml, and spiking pooled plasma (BiolVT, NY, USA) at 16.08, 643.13 and 6431.3 pg / ml with Human / Mouse NfL control (ProteinSimple, CA, USA). Two sets of each QC were processed along with unknown samples, and the plate results were accepted if the Analytical Recovery of the QCs fell within the range of 80-120% at each QC concentration. Analysis was conducted blinded to mouse genotype.

[0146] Statistics

[0147] Data were analyzed using GraphPad Prism 10. Experimental endpoints measured were compared using ANOVA with Tukey’s test for multiple comparisons using log transformed data comparing the mean of each genotype with the mean of all other genotypes. Data are plotted on a linear scale using the adjusted p values obtained using log transformed data. No statistical methods were used to predetermine cohort sizes.

[0148] RESULTS

[0149] SOMAS accumulation

[0150] SOMAS storage in peripheral tissues liver and spleen were initially investigated at 6 months of age by immunohistochemistry (Fig. 1). In wild-type controls (WT / Tg-) or wildtype animals expressing the TPP1 transgene (WT / Tg+), no staining was observed for SOMAS in liver or spleen.

[0151] In contrast, JNCL mice lacking the TPP1 transgene (JNCL / Tg-) showed characteristic dark punctate inclusions of accumulated SOMAS storage material. However, JNCL mice expressing the TPP1 transgene (JNCL / Tg+) showed little or no detectable accumulation of SOMAS. Similar results were obtained in brain (Fig. 1). In JNCL / Tg-animals, storage was widespread throughout the brain with some regions of particularly intense staining, e.g., pyramidal cells of the CA3 region of the hippocampus and Purkinje cells of the cerebellum while no SOMAS storage was detectable in control WT / Tg- and WT / Tg+ animals. Again, JNCL mice expressing the TPP1 transgene (JNCL / Tg+) showed no detectable SOMAS storage in the brain. These data indicate that in brain and peripheral organs, accumulation of SOMAS can be prevented by increased levels of TPP1.

[0152] To determine if elimination of SOMAS is persistent, the analysis of SOMAS storage in brain at 12 months was repeated (Fig. 2) in JNCL mice with or without the TPP1 transgene and WT controls (given the absence of pathology in WT mice expressing the TPP1 transgene at 6 months, this control was omitted in subsequent studies). Robust SOMAS storage was detected throughout the brain of the JNCL mice but not in wild-type controls. Consistent with findings at 6 months, expression of the TPP1 transgene essentially prevented SOMAS accumulation in the JNCL mouse.

[0153] A hallmark of NCL diseases is the autofluorescence associated with the storage material. Despite clearly preventing SOMAS accumulation, elevated TPP1 levels did not prevent autofluorescence, which presumably therefore arises from other components of storage material (e.g., lipids).

[0154] Neuroinflammation

[0155] Given the uncertainty regarding the role of SOMAS accumulation in disease progression, it was necessary to determine whether prevention of SOMAS storage by elevated TPP1 was associated with functional correction of disease. This goal was complicated by the extremely attenuated phenotype of the CLN3 knockout mouse (Mitchison, supra). For example, survival of the JNCL mouse (Cotman et al., Hum. Mol. Genet..2002;11 (22):2709-21 ; Katz et al., Neurobiol Dis. 2008;29(2):242-53) is onlymarginally truncated compared to wild-type controls, and while the JNCL mouse has been reported to have locomotor-behavioral defects, most reports indicate that they are nuanced and highly variable, with dramatically different data reported by different groups (Kovacs et al., Dis Model Meeh. 2015;8(4):351-61). Six and 12-month JNCL mice were analyzed using open field activity, RotaRod and pole test but animals failed to display any robust behavioral or locomotor phenotype associated with disease. This was not unexpected and has been reported previously by others (Kovacs, supra).

[0156] As an alternative approach to measure the effect of TPP1 overexpression on disease progression in JNCL, neuroinflammation was considered. A number of recent studies (reviewed in Takahashi et al., Front Neurol. 2022; 13:886567) have suggested that glial dysfunction may be a key driver of pathogenesis and thus therapeutically targeting both neurons and glia may provide the most successful approaches to NCL diseases. The effect of TPP1 transgene expression on glial activation (Cooper et al., Biochim Biophys Acta. 2015:1852(10 Pt B):2256-61, Pontikis et al., Brain Res. 2004 ;1023(2) :231 -42) in the JNCL mouse was investigated.

[0157] Activation of astrocytes was determined by immunohistochemical staining for GFAP (Fig. 3). GFAP was detected throughout the brains of both JNCL and control mice but there were several regions with JNCL-specific staining including somatosensory cortex layers 6a / b and ventral nucleus regions of the thalamus, as reported previously (Sleat 2023, supra, Takahashi, supra). While not completely restoring GFAP staining to wild-type levels, expression of the TPP1 transgene resulted in a major and significant reduction in GFAP immunoreactivity in these two brain regions. Activated microglia were analyzed by immunostaining for CD68 (Fig.4). Sporadic CD68 staining was detected throughout the brain of wild-type animals and this was increased in the JNCL animals as reported previously (Sleat 2023, supra, Takahashi, supra). Elevated TPP1 resulted in a minor but significant decrease in sporadic CD68 staining observed throughout the cerebral cortex. The most extensive microglial activation in the JNCL mice was found in the ventral nuclei of the thalamus and this was decreased significantly by elevated TPP1 levels. Taken together, these data demonstrate that TPP1 overexpression results in a significant reduction in neuroinflammation in the JNCL mouse model.

[0158] Neurofilament light (Nfl)

[0159] Nfl is an established biomarker of neurodegeneration in a wide range of neurological diseases (reviewed in Yuan et al., Front Neurosci. 2021 ;15:689938) including JNCL (Dang Do AN et al., Genet Med. 2021 ;23(4):751-7; Dang Do AN et al., J Proteome Res. 2023;22(7):2493-508). Nfl is a major structural protein of neurons and is released intocerebrospinal fluid and plasma in increasing amounts during neurodegeneration, inflammation and other disease-related processes. In Fig. 5, Nfl was measured by immunoassay in plasma from 12-month JNCL and WT mice, with or without transgenic TPP1 overexpression. As expected, it was found that Nfl levels in plasma from the JNCL mice were significantly elevated (~2.7-fold) compared to WT animals. Expression of the TPP1 transgene had no effect on Nfl expression in WT mice but in JNCL animals, TPP1 overexpression restored plasma Nfl to WT levels. These data suggest that elevated TPP1 reduces or prevents neurodegeneration and / or neuroinflammation in the JNCL mice.

[0160] Brain mass

[0161] Studies (Katz, supra, Palmieri et al., Nat Commun. 2017;8: 14338) have shown that the brains of the JNCL mice are ~8% smaller in mass than age-matched wild-type animals at 12 months of age, presumably reflecting cortical atrophy and neuronal loss that is observed in both JNCL patients (Autti et al., Neuroradiology. 1996;38(5):476-82; Autti et al., J Neurol.2008 ;255(8): 1226-30) and mice (Greene et al., Eur J Paediatr Neurol. 2001 ;5 Suppl A:103-7). Brain weights in WT and JNCL mice, with or without transgenic TPP1 overexpression, were analyzed (Fig. 6, Panels A and C). Consistent with previous studies, at ~7, 12 and 24 months, the brain mass of the JNCL mice was found to be significantly lower than wild-type animals with or without the TPP1 transgene. However, elevated TPP1 levels corrected the decrease in brain mass associated with JNCL and restored brain mass to that measured in WT mice. In contrast, liver weights in the four genotypes of animals were not significantly different (Fig. 6, Panel B).

[0162] DISCUSSION

[0163] It was demonstrated herein that elevated levels of TPP1 essentially prevent accumulation of SCMAS, the major protein component of storage material, in cells within the brain and other tissues of the Cln3-knockout mouse model of JNCL. This study indicates that elimination of SCMAS in the JNCL mouse by elevated TPP1 is correlated with a partial but major reduction in neuroinflammation, which has been identified as a key driver of neuronal death (Takahashi, supra). In addition, transgenic TPP1 expression corrected other phenotypes of disease including elevated levels of the neurodegeneration biomarker Nfl and a progressive loss of brain mass.

[0164] It is possible that elevated TPP1 provides therapeutic benefits in JNCL independent of its role in the degradation of SCMAS, thus lysosomal SCMAS storage may simply represent an informative biomarker for disease. An alternate explanation is that the accumulation of insoluble SCMAS deposits within neurons is neurotoxic and contributes to neuronal death. For example, many cellular abnormalities have been linked with JNCL(Kollmann et al., Biochim Biophys Acta. 2013;1832(11 ):1866-81 ; Shematorova et al., Int J Mol Sci. 2020;21(21)) including defects in membrane trafficking and autophagy (Fossale et al., BMC Neurosci. 2004;5:57; Wavre-Shapton et al., Hum Mol Genet. 2015;24(24):7060-74; Zhong et al., Biochim Biophys Acta Mol Basis Dis. 2020; 1866(10): 165883; Cao et al. J Biol Chem.2006;281(29):20483-93; Petcherski et al., Cells. 2019;8(12)), and it is plausible that SCMAS accumulation could compromise lysosomal function, which in turn could contribute to such phenotypes. It is not suggested that SCMAS accumulation is the sole driver of disease as transgenic overexpression of TPP1 almost completely prevented SCMAS accumulation at 12 months but, while significantly reduced, some level of disease-associated neuroinflammation persisted. The pathogenic contribution of this residual neuroinflammation is difficult to evaluate, but it is worth noting that other phenotypes (Nfl and brain mass) were completely corrected.

[0165] Finally, it is worth emphasizing that discussion of whether or not SCMAS itself is a driver of neuronal death may be irrelevant from a potential translational perspective. As noted above, the data shows that constitutively elevated TPP1 prevents SCMAS storage but also corrects multiple other phenotypes that are directly linked to disease progression.

[0166] Effect of small molecule drugs on TPP1 function in JNCL

[0167] Several studies in Cln3 knockout mice of small molecule drugs that stimulate TFEB-mediated expression of lysosomal proteins, promoting lysosome biogenesis and clearance of accumulated storage material (Palmieri, supra; Jana et al., J Neurosci. 2023. Epub 20230125; Soldati et al., EMBO Mol Med. 2021 ;13(10):e13742), have shown promise in ameliorating disease phenotype. One of these molecules, gemfibrozil, is also thought to reduce neuroinflammation36. Given our findings, the therapeutic effects of inducers of TFEB-mediated transcription of lysosomal genes in JNCL mouse models may simply reflect an increase in TPP1 activity and consistent with this, gemfibrozil treatment of JNCL mice has been shown to result in elevated TPP1 (Jana, supra).

[0168] Mechanisms underlying the effects of CLN3 loss on TPP1 function

[0169] It remains to be established why the loss of CLN3 impairs TPP1 function, resulting in the lysosomal accumulation of SCMAS. Loss of CLN3 does not simply result in a downregulation of TPP1 expression or loss of catalytic function as TPP1 activity is actually elevated in JNCL patients and mice. One possibility is that disturbance of the lysosomal microenvironment in the absence of CLN3 might interfere with TPP1 catalytic function (in a manner not detectable after cell lysis and processing for in vitro enzyme assay) or access to SCMAS. For example, a recent study (Laqtom et al. Nature. 2022 ;609(7929): 1005-11) indicated that there is a lysosomal accumulation of glycerophosphodiesters in JNCL, whichmay act as inhibitors of enzymes involved in glycerophospholipid catabolism, resulting in an additional accumulation of lysophospholipids (Nyame aet al., Mol Cell. 2024;84(7): 1354-64). It is possible that locally elevated levels of these various lipids might inhibit TPP1 , although they have no apparent effect on TPP1 activity in vitro. Alternatively, these lipids could induce conformational changes in SCMAS that lower the rate of its degradation by TPP1.

[0170] Interestingly, a mouse model of mucopolysaccharidosis III B also accumulates SCMAS even though the primary defect lies in degradation of heparin sulfate (Ryazantsev et al., Mol. Genet. Metab. 2007;90(4):393-401) and the authors of this study speculated that “it is likely that some unfavorable condition in lysosomes slows down the degradation of SCMAS, allowing this very hydrophobic protein to pack into closely stacked arrays that may be even harder to digest”. Finally, it may be possible that rather than directly impairing the ability of TPP1 to degrade SCMAS, loss of CLN3 could result in increased delivery of SCMAS to the lysosome, overwhelming the capacity for TPP1 to degrade it efficiently. Of possible relevance, loss of CLN3 has been reported to interfere with mitochondrial function (Fossale, supra) and cellular autophagic pathways (Fossale, supra; Wavre-Shapton, supra; Cao, supra Chandrachud et al., J Biol Chem. 2015;290(23):14361-80; Chang et al., J Neurochem. 2011 ;116(4) :659-68; Vidal-Donet et al., PLoS One. 2013;8(2):e55526) which could potentially result in increased mitochondrial turnover. Regardless of whether CLN3 loss impairs the ability of TPP1 to degrade SCMAS or increases lysosomal influx of SCMAS, increasing TPP1 levels would be predicted to reduce SCMAS storage, as demonstrated herein.

[0171] Translational implications - TPP1 augmentation as a potential therapeutic route for JNCL

[0172] Universal correction of the primary defect (i.e. replacing the mutant CLN3 with a functional copy) would be an effective route to therapy for JNCL. However, this remains a substantial challenge given that CLN3 is a transmembrane protein, thus enzyme replacement therapy is not feasible and gene therapy to correct such cell-autonomous defects continues to be challenging. The results suggest that supplementing TPP1 levels in JNCL patients may be a feasible alternative therapeutic strategy. While this does not address the primary defect, this approach focuses on a secondary pathogenic event that may, at least in part, drive disease progression. A major strength of this approach as a therapeutic strategy is that there are established methods to achieve TPP1 augmentation. One approach would be to conduct intracerebroventricular delivery of recombinant TPP1 in much the same way that LINCL patients are currently treated (Markham A. Drugs.2017;77(11):1247-9; Schulz et al., N Engl J Med. 2018;378(20): 1898-907).

[0173] TPP1 augmentation as general approach in lysosomal diseases that store SCMAS

[0174] In addition to LINCL and JNCL, there are other NCLs that accumulate SCMAS including CLN5, CLN6, CLN7 and CLN8 (Pamlmer D., Biochim Biophys Acta. 2015;1852(10 Pt B):2287-91) as well as a number of unrelated lysosomal storage diseases, including mucopolysaccharidosis (MPS) I, II and IIIA, multiple sulfatase deficiency and, to variable but lesser degree, in some lysosomal lipidoses (Eleder et al., Acta Neuropathol. 1997;93(4):379-90). If impaired TPP1 function reflected by SCMAS accumulation is a common pathogenic trigger in these diseases, then TPP1 augmentation could have application in a range of currently intractable lysosomal disorders. This is especially true for those arising from defects in transmembrane proteins, where gene and enzyme replacement therapy may be difficult or impossible and additional proof-of-principle studies using mouse or other animal models may be warranted for such disorders. In conclusion, this study demonstrates that the ability of TPP1 to degrade its substrate SCMAS is either impaired or overwhelmed in JNCL, providing a mechanistic explanation for the accumulation of this material. Importantly, it was also demonstrated that elevated levels of TPP1 overcomes this impairment, essentially preventing SCMAS storage but also significantly correcting other phenotypes of disease in the JNCL mouse model.

[0175] It was found that elevated TPP1 essentially prevents SCMAS storage in the JNCL mouse in multiple tissues, and this is associated with a significant decrease in neuroinflammatory markers and correction of other phenotypes of disease These results indicate that TPP1 dysfunction plays a role in the pathogenesis in JNCL and provide a rationale to investigate TPP1 augmentation as a potential therapeutic strategy for patients with mutations in CLN3. If this is a generally applicable model for pathogenesis in lysosomal disorders that accumulate SCMAS, TPP1 augmentation may also be useful in other neuronal ceroid lipofuscinoses, especially those due to deficiencies in membrane proteins such as CLN6, CLN7 and CLN8 where correction of the primary defect remains a significant obstacle.

[0176] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing inventions provided by the disclosure have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is claimed is:

1. A method of treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject comprising administering to the subject a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in an amount effective to treat the CLN3 disease in the subject.

2. A method of delaying the onset of Neuronal Ceroid Lipofuscinosis (CLN3) disease, or a symptom thereof, in a subject, comprising administering a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI).

3. The method of claim 1 or 2, wherein the composition is administered to the subject via intracerebroventricular, intrathecal, intravitreal, and / or intraocular administration.

4. The method of any one of the preceding claims, wherein the composition is administered once every 2 weeks or once every 4 weeks.

5. A method of treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject comprising administering to the subject a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in an amount effective to treat the CLN3 disease in the subject, wherein rhTPPI is administered intracerebroventricularly every 2 weeks and rhTPPI is administered intravitreally every 4 weeks.

6. A method of delaying the onset of Neuronal Ceroid Lipofuscinosis (CLN3) disease, or a symptom thereof, in a subject, comprising administering a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI), wherein rhTPPI is administered intracerebroventricularly every 2 weeks and rhTPPI is administered intravitreally every 4 weeks7. The method of any one of the preceding claims, wherein a dosage of about 300 mg is administered to the subject intracerebroventricularly.

8. The method of any one of the preceding claims, wherein a dosage of about 0.2 mg to 5 mg is administered to the subject intravitreally.

9. The method of any one of the preceding claims wherein the administration improves one or more symptoms of CNL3 disease.

10. The method of claim 9, wherein the one or more symptoms are selected from the group consisting of vision, language function, motor function, language function,cognitive function, physical ability, behavior, number, frequency or severity of seizures, and functional capability.

11. The method of any one of claims 1 -10, wherein the administration slows or reduces time to progression from one stage of CLN3 to the next as determined by CLN3 Staging System (CLN3SS).

12. The method of claim 11 , wherein the stage is based on UBDRS severity, Capability score severity and / or age of the subject.

13. The method of any one of the preceding claims, wherein the composition is administered intracerebroventricularly, intraocularly, intravitreally, and / or intrathecally.

14. The method of any one of the preceding claims, wherein the composition is administered intracerebroventricularly and / or intravitreally.

15. The method of any one of the preceding claims, wherein the composition is administered intravitreally every 2 weeks.

16. The method of any one of the preceding claims, wherein the composition is administered at a dose of about 300 mg intracerebroventricularly.

17. The method of claim 16, wherein the dose is administered to the subject at a rate less than or equal to 75 mg / hour.

18. The method of any one of the preceding claims, wherein the rhTPPI comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof.

19. The method of any one of the preceding claims, wherein the formulation, composition or dose is administered every other week, monthly or every other month.

20. The method of any one of the preceding claims, wherein the subject exhibits a decrease in CLN3 enzyme activity as compared to a healthy subject.

21. The method of any one of the preceding claims, wherein the subject has at least one mutation in a CLN3 gene.

22. The method of any one of the preceding claims, wherein the subject has an improved score on the UBDRS motor and language subscales compared to CLN3 natural history studies.

23. The method of any one of the preceding claims, comprising administering to the subject an antihistamine with or without an antipyretic before administration of the rhTPPI , optionally, about 30 to about 60 minutes before administration of the rhTPPI .

24. The method of any one of the preceding claims, wherein the composition comprises the rhTPPI and at least one pharmaceutically acceptably carrier, diluent or excipient.

25. The method of claim 24, wherein the composition comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof.

26. The method of any one of the preceding claims, wherein the composition for intracerebroventricular administration comprises rhTPPI at a concentration of from about 25 mg / mL to about 35 mg / mL.

27. The method of any one of the preceding claims, wherein the composition has a pH of about 6.5.

28. The method of any one of the preceding claims, wherein the composition further comprises potassium chloride at a concentration of about 0.01 mg / mL to about 1 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, and calcium chloride dihydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL.

29. The method of any one of the preceding claims, wherein the composition further comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.01 mg / mL to about 1 mg / mL, and sodium chloride at a concentration of about 1 mg / mL to about 20 mg / mL.

30. The method of any one of the preceding claims, wherein the composition comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration of about 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, and calcium chloride dihydrate at a concentration of about 0.21 mg / mL.

31. The method of any one of the preceding claims, comprising administering to the subject a flush solution after administering the composition intracerebroventricularly.

32. The method of claim 31 , wherein the flush solution comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof.

33. The method of claim 32, wherein the flushing solution comprises sodium phosphate dibasic heptahydrate at a concentration of about 0.11 mg / mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08 mg / mL, sodium chloride at a concentration of about 8.77 mg / mL, potassium chloride at a concentration of about 0.22 mg / mL, magnesium chloride hexahydrate at a concentration of about 0.16 mg / mL, and calcium chloride dihydrate at a concentration of about 0.21 mg / mL.

34. The method of any one of the preceding claims, wherein the treatment period is at least 10 weeks, at least 20 weeks, at least 40 weeks, at least 80 weeks, at least 96 weeks, at least 6 months, at least 1 year, at least 2 years, or for the life of the subject.

35. A method of maintaining, slowing, reducing or improving vision loss in a subject having Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease comprising intravitreally administering to the subject a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in an amount effective to improve vision loss in the subject.

36. A method of maintaining brain volume or slowing or reducing a decrease in brain volume in a subject having Neuronal Ceroid Lipofuscinosis (CLN3) disease, comprising administering a therapeutically effective dose of recombinant human tripeptidyl peptidase-1 (rhTPPI ) to the subject.

37. A method of reducing the frequency or severity of seizures in a subject having Neuronal Ceroid Lipofuscinosis (CLN3) disease, comprising administering a therapeutically effective dose of recombinant human tripeptidyl peptidase-1 (rhTPPI) to the subject.

38. The method of any one of the preceding claims comprising measuring levels of at least one biomarker associated with CLN3 in the subject.

39. The method of claim 38, wherein the at least one biomarker is selected from the group consisting of neurofilament light (NFL), lysosomal storage of subunit c of mitochondrial ATP synthase (SCMAS), glial fibrillary associated protein (GFAP),neurofilament heavy (NFH), neurofilament medium (NFM), cathepsin F (CTSF), acid sphingomyelinase (SMPD1), Niemann-Pick C1 (NPC1) protein, and neuroinflammation.

40. The method of claim 38 or 39, wherein the biomarker is measured in a biological sample.

41. The method of claim 40, wherein the biological sample is serum, urine and / or cerebrospinal fluid (CSF) or a tissue sample.

42. The method of any one of claims 38-41 wherein the TPP1 administration improves levels of at least one of the one or more biomarker in the subject.

43. The method of any one of the preceding claims wherein the TPP1 administration reduces or slows brain mass loss and / or levels of neuroinflammation in the subject.

44. A composition comprising a recombinant human tripeptidyl peptidase-1 (rhTPPI) for use in treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject.

45. Use of a composition comprising recombinant human tripeptidyl peptidase-1 (rhTPPI) in the manufacture of a medicament for treating Neuronal Ceroid Lipofuscinosis 3 (CLN3) disease in a subject.

46. The composition or use of claim 44 or 45, wherein the composition is for intracerebroventricular, intrathecal, intravitreal, and / or intraocular administration.

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