Novel gene therapy constructs for CLN2 disease

Abstract

Provided herein are methods for expressing tripeptidyl peptidase 1 (TPP1) in neuronal cells. Also provided herein are modified adeno-associated viruses (AAVs) encoding TPP1 under the control of a promoter that is functional in neuronal cells or ependymal cells but not in dorsal root ganglia. TIFF2026505074000004.tif97128

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Provisional Application No. 63 / 482,495, filed January 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present disclosure relates generally to the fields of medicine, virology, and neurology, and more particularly to targeting peptides that target the delivery of viral vectors to distinct structures in the brain, especially in the treatment of TPP1 deficiency / CLN2 disease. [Background technology]

[0003] 2. Description of Related Technology TPP1 deficiency / CLN2 disease is a pediatric neurodegenerative disorder caused by a deficiency of the soluble lysosomal enzyme TPP1 due to mutations in CLN2. Because patients with CLN2 are unable to produce functional TPP1 themselves, gene replacement is the optimal treatment option. TPP1 is an enzyme decorated with mannose-6-phosphate, which can be used for cross-correction of defective cells by enzyme replacement or gene therapy. To be effective, gene therapy for TPP1 deficiency requires widespread distribution throughout the brain. In dogs with CLN2 disease, transduction of ependymal cells lining the ventricles demonstrated therapeutic efficacy, with improvement in neuropathology, prolonged lifespan, and symptomatic remission (Katz, et al., 2013). However, transduction of the ependymal and deep brain structures in NHP brains is not fully achievable with current AAV serotypes.

[0004] Adeno-associated virus (AAV) has become a strong therapeutic candidate for the treatment of neurodegenerative diseases. AAV is a non-enveloped, single-stranded DNA virus that can infect both dividing and non-dividing cells. After infection, the virus does not exhibit robust integration into the host genome and persists as an episome in the cell nucleus. Expression of AAV cargo is spatially regulated by the transgene promoter at the level of the packaging capsid. The use of AAV for disease treatment may require intervention in diseased tissue, which can be problematic in target tissues containing gene expression profiles different from healthy tissues. Finding the correct promoter sequence to drive therapeutic transgene expression is an important goal.

[0005] Various strategies have been developed to generate AAV vector variants, including rational design and directed evolution. Rational design approaches utilize knowledge of the AAV capsid to add target-specific changes to the capsid to alter transduction efficiency or specificity, for example, adding tyrosine mutations to the capsid surface to increase transduction efficiency. Directed evolution approaches do not require knowledge of the capsid structure and are carried out through random mutagenesis, capsid shuffling, or random peptide insertion. These strategies generally use in vitro systems or mice, which are ideal for cell-based or mouse studies but are not intended for clinical application. In fact, there are no AAV variants that specifically or efficiently target distinct brain structures. Therefore, there is a need for AAV variants that can target distinct brain structures. Summary of the Invention

[0006] overview Thus, according to the present disclosure, there is provided a method for expressing tripeptidyl peptidase 1 (TPP1) in neurons or ependymal cells of a subject, comprising administering to the subject a modified adeno-associated virus (AAV) encoding TPP1 under the control of a promoter functional in neurons or ependymal cells, wherein the AAV comprises a modified capsid protein containing a targeting sequence for targeting ependyma and CNS neurons, and the modified AAV avoids transduction of dorsal root ganglia. The promoter may be a constitutive promoter or a tissue-specific promoter. The promoter may be a CMV early enhancer / chicken β-actin (CAG) promoter.

[0007] The CAG promoter may comprise the sequence of SEQ ID NO: 1. The modified AAV may be AAV1. The targeting sequence may be inserted near or after position 590 of SEQ ID NO: 2. The targeting sequence may comprise or consist of ERDRTRG (SEQ ID NO: 3). TPP1 may comprise or consist of the sequence of SEQ ID NO: 4.

[0008] The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are 2 or 3 amino acids in length. The linker sequence may be SSA at the N-terminus of the targeting sequence and AS at the C-terminus of the targeting sequence. The modified capsid protein may comprise or consist of the sequence of SEQ ID NO:5. The TPP1 sequence may be operably linked to a polyadenylation signal.

[0009] Administration can be by direct delivery into the ventricle, cisterna magna, or intrathecal cavity. The modified AAV can be administered multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. The modified AAV can be administered monthly, annually, every 5 years, or less frequently.

[0010] For example, approximately 1 × 10 per kilogram of patient 6 ~Approx. 1×10 14 vector genomes (vg / kg), or approximately 1 × 10 7 ~1×10 14 , about 1×10 8 ~1×10 14 , about 1×10 9 ~1×10 14 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , or about 1 × 10 12 ~1×10 13 A dose of vg / kg allows a large number of viral particles to be administered.

[0011] The subject may be afflicted with infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease. The subject may be under the age of 4 or between the ages of birth and 25.

[0012] In another embodiment, a modified adeno-associated virus (AAV) encoding TPP1 is provided, the modified AAV containing a modified capsid protein containing a targeting sequence, and the modified AAV does not target the dorsal root ganglion. The promoter may be a constitutive promoter or a tissue-specific promoter. The promoter may be a CMV early enhancer / chicken β-actin (CAG) promoter.

[0013] The CAG promoter may comprise the sequence of SEQ ID NO: 1. The modified AAV may be based on AAV1, comprising a targeting sequence near or after position 590 of SEQ ID NO: 2. The targeting sequence may comprise or consist of ERDRTRG (SEQ ID NO: 3). TPP1 may comprise or consist of the sequence of SEQ ID NO: 4.

[0014] The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are 2 or 3 amino acids in length or more. The linker sequence may be SSA at the N-terminus of the targeting sequence and AS at the C-terminus of the sequence peptide. The modified capsid protein may comprise or consist of the sequence of SEQ ID NO:5. The TPP1 sequence may be operably linked to a polyadenylation signal.

[0015] Pharmaceutical compositions comprising the modified AAV described herein are also provided, as are kits comprising the modified AAV described herein.

[0016] In yet another embodiment, the use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter functional in neuronal cells or ependymal cells for expressing TPP1 in neuronal cells or ependymal cells, wherein said AAV comprises a modified capsid protein comprising a targeting sequence such as ERDRTRG (SEQ ID NO:3), and wherein said modified AAV does not target the dorsal root ganglion; the use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter functional in neuronal cells or ependymal cells for treating infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease in a subject suffering from said disease, wherein said AAV comprises a modified capsid protein comprising a targeting sequence such as ERDRTRG (SEQ ID NO:4). and use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter functional in neurons or ependymal cells in the preparation of a medicament for treating infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease in a subject affected with the disease, wherein the AAV comprises a modified capsid protein comprising the targeting sequence ERDRTRG (SEQ ID NO:3), and wherein the modified AAV does not target the dorsal root ganglion.

[0017] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The word "about" means plus or minus 5% of the stated number.

[0018] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] [Figure 1] epAAV: Brain parenchyma and ependyma show strong signal throughout. Ependymal / neuron-targeting AAV capsids (epAAV) expressing the fluorophore mTFP1 (cyan) were intracerebroventricularly (ICV) injected at 1.85E13vg into non-human primates. Ependymal cells lining the lateral and fourth ventricles, including the choroid plexus, had strong mTFP1-positive signals as seen by confocal microscopy. In the brain parenchyma, positive cells were present in the subiculum and parasubiculum, as well as in cortical regions throughout the brain (entorhinal cortex, motor cortex, and temporal cortex). Positive mTFP1 signals were also seen in multiple thalamic nuclei (dorsolateral, dorsomedial, and ventral anterior lateral thalamus). Confocal images were taken with a Leica SP8 microscope. Scale bar 50 μm. [Figure 2]epAAV: Dorsal root ganglia (DRG) are transgene-negative. Ependymal-targeting AAV capsids (epAAV) expressing the fluorophore mTFP1 (cyan) were intracerebroventricularly (ICV) injected into non-human primates at 1.85E13vg. DRG toxicity is known to occur in the presence of strong exogenous expression from AAV delivery. Cell nuclei were visualized using DAPI counterstaining (blue; left column). Transcripts of interest were probed by RNAscope fluorescent in situ hybridization (middle column; probes are indicated within the image). A positive control (+ control) was used to detect ubiquitin C (UBC), a transcript known to be expressed in DRG; a scrambled probe (scramble) was used as a negative control (- control); mTFP1 was detected in cervical, thoracic, and lumbar DRGs with a probe against the transgene (mTFP1). Overlay images are shown in the right column. There were no detectable transcripts in either the cervical, thoracic, or lumbar DRG. Therefore, the ependymal / neuron-targeting capsids do not transduce DRG. Confocal images taken with a Leica SP8 microscope. Scale bar 50 μm. [Figure 3] epAAV: The spinal cord is transgene-negative. Ependymal / neuron-targeting AAV capsids (epAAV) expressing the fluorophore mTFP1 (cyan) were intracerebroventricularly (ICV) injected into non-human primates at 1.85E13vg. Cell nuclei were visualized using DAPI counterstaining (blue; left column). Transcripts of interest were probed by RNAscope fluorescent in situ hybridization (center column; probes are indicated in the image). A positive control (+control) was used to detect ubiquitin C (UBC), a transcript known to be expressed in the spinal cord; mTFP1 was detected in the cervical, thoracic, and lumbar regions of the spinal cord with a probe for the transgene (mTFP1; cyan). Overlay images are shown in the right column. No detectable transcripts were present in the cervical, thoracic, or lumbar regions of the spinal cord. Confocal images taken with a Leica SP8 microscope. Scale bar 50 μm. [Figure 4]epAAV: Peripheral organs show low or no density of positive cells. Ependymal / neuron-targeting AAV capsids (epAAV) expressing the fluorophore mTFP1 (cyan) were intravenously injected (ICV) into the CSF of non-human primates at 1.85E13vg. Cell nuclei were visualized using counterstaining with DAPI (blue; left column). Epifluorescence of mTFP1 (cyan) was detected. Cells with high autofluorescence accumulation levels are shown in red (autofluorescence). Overlay images are shown in the right column. The liver (upper panel) had a small number of cells with a positive signal for the transgene mTFP1, whereas the heart (lower panel) showed no transgene detection. All images were taken with a Leica DM6000B microscope. Scale bar 50 μm. [Figure 5] Survival curves showing increased lifespan of Cln2- / - mice treated with novel therapeutic constructs compared with untreated normal or diseased mice. The graph shows survival rates over time for Cln2+ / +, Cln2- / -, and Cln2- / - +5.0E+10vg epAAV mice. Overall, the lowest survival rates were observed in Cln2- / - mice (n=10), with a mean lifespan of 17.1 weeks and a maximum lifespan of 19.7 weeks. Gene therapy treatment with 5.0E+10vg epAAV significantly increased the half-life of Cln2- / - (n=10) mice to a mean of 18.6 weeks and a maximum of 28.6 weeks. 100% survival was observed in Cln2+ / - mice (n=9). *p<0.05 (Mantel-Cox test). [Figure 6]In Cln2- / - mice treated with the novel therapeutic construct, the onset of the resting tremor phenotype was delayed and milder compared to untreated mice. The graph shows the assessment of resting tremor in Cln2+ / + mice, Cln2- / - mice, and Cln2- / -+5.0E+10vg epAAV mice. In contrast to Cln2+ / +, the resting tremor phenotype was detectable in untreated Cln2- / - mice at 13 weeks of age, and the severity significantly increased over time. In Cln2- / - mice treated with 5.0E+10vg epAAV, the onset of resting tremor was significantly delayed until 16 weeks, and the phenotype was significantly attenuated and progressed more slowly over time. [Figure 7] Following treatment with the novel therapeutic constructs, TPP1 activity in the central nervous system is increased relative to endogenous levels. The graph shows TPP1 activity in untreated Cln2+ / - and Cln2- / - mice or Cln2+ / - and Cln2- / - mice treated with unilateral injection of 5.0E+10vg epAAV. Mice injected with epAAV had increased TPP1 activity in various brain regions analyzed when compared with endogenous TPP1 activity in uninjected Cln2- / - mice, and was also significantly elevated compared with normal mice. The heart and spleen of injected Cln2- / - mice, which are not direct targets of therapy, had minimally increased levels of TPP1 activity compared with uninjected Cln2- / - mice. [Figure 8]Figure 8A-D. Quantification of ependymal transduction in five NHPs receiving ICV injection of AAV-EP+. AAV-Ep+ was administered to rhesus macaques (n=5) via intracerebroventricular (ICV) injection at 1E13vg per subject. Transduced ependymal cells and parenchymal neurons were identified by fluorescence and morphology via microscopy of sectioned brains. Total and transduced cell numbers were counted via automated algorithms using Cellpose and QuPath. (Figure 8A) Representative 40x image showing robust transduction and mRuby3 expression in the ependymal layer of cells lining the ventricles. (Figure 8B) Quantification of transduced ependymal and parenchymal cells across 75 images (40x), 15,096 total cells, and 5,852 ependymal cells. (Figure 8C) 10x tile scan image captured showing extensive ependymal transduction in the region of the anterior (slab 4) lateral ventricle ependymal lining. (FIG. 8D) Quantification of 10x tile scan images from the lateral ventricles in slabs 4 and 5 of four NHPs. Eight tile scans and 34,734 total ependymal cells were quantified. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description Provided herein is a method for expressing tripeptidyl peptidase 1 (TPP1) in the ependyma and neuronal cells of a subject, comprising administering to the subject a modified adeno-associated virus (AAV) capsid that encapsidates a transgene encoding TPP1 under the control of a promoter functional in neuronal cells / ependyma, wherein the AAV comprises a modified capsid protein that includes a targeting sequence.

[0022] In some embodiments, the viral vectors each comprise a modified capsid, wherein the modified capsid comprises an amino acid sequence that targets the viral vector to a distinct brain structure. In certain embodiments, the brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, thalamus, or ependymal cells. In certain embodiments, the targeting peptide is ERDRTRG (SEQ ID NO: 3).

[0023] In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, the AAV is derived from AAV1. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO: 1, which does not appreciably transduce the ependyma after delivery to the CSF. In certain aspects, a targeting peptide is inserted at position 590 of the AAV1 capsid. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO: 4, which shows a targeting peptide insertion after position 590 as SSAX7AS, where SSA in front and AS in back are linker sequences, and X7 represents the targeting peptide.

[0024] I. Batten disease / TPPI deficiency In some aspects, the present disclosure may be directed to the use of compounds for the treatment of neurological disorders such as infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease. Batten disease is a fatal neurological disorder that typically begins in childhood, typically between the ages of 5 and 10. It is often autosomal recessive and is the common name for neuronal ceroid lipofuscinosis (NCL). Batten disease is generally considered the juvenile form (or "type 3") of NCL, although some physicians use the term Batten disease to describe all forms of NCL. Historically, NCL was classified based on the age of disease onset as infantile NCL (INCL), juvenile NCL (JNCL), or adult NCL (ANCL). At least 20 genes have been identified associated with Batten disease, but juvenile NCL, the most common form of Batten disease, is associated with mutations in the CLN3 gene.

[0025] Batten disease is a fatal disease. Brineura is the first FDA-approved treatment to delay the loss of walking ability (gait) in symptomatic pediatric patients aged 3 years and older with infantile neuronal ceroid lipofuscinosis type 2 (CLN2), also known as tripeptidyl peptidase-1 (TPP1) deficiency. TPP1, also known as lysosomal pepstatin-insensitive protease, is an enzyme encoded by the TPP1 gene in humans. Mutations in the TPP1 gene lead to infantile neuronal ceroid lipofuscinosis. The human gene TPP1 encodes a member of the sedolisin family of serine proteases. The human gene has 13 exons and is located on chromosome band 11p15.

[0026] The nucleic acid sequence of human TPP1 is shown as SEQ ID NO:4. Human TPP1 contains tripeptidyl peptidase I activity (TPP1 enzyme activity). TPP1 activity includes nonspecific lysosomal peptidase activity, which generates tripeptides from degradation products generated by lysosomal proteinases. Substrate specificity studies have shown that TPP1 primarily cleaves tripeptides from the unsubstituted amino termini of peptides and proteins. Endogenously expressed TPP1 is synthesized as an enzyme without catalytic activity. After targeting to lysosomes, TPP1 is autocatalytically processed into the mature, active enzyme due to the acidic environment. TPP1 activity can be measured and / or quantified in vitro using known methods. See, for example, Junaid et al., 1999.

[0027] Human TPP1 is 61 kDa in size and consists of 563 amino acids. Alternative splicing generates a 34.5 kDa, 320 amino acid isoform, resulting in the loss of a peptide fragment of 1–243 amino acids. TPP1 contains a subtilisin-like fold, a globular structure containing the catalytic triad Ser475-Glu272-Asp360. It also contains an octahedral Ca2+ complex, a characteristic feature of the S53 sedolisin family of peptidases. 2+ It also contains the binding site. Unlike other S53 peptidases, it has steric constraints in the P4 substrate pocket that may contribute to preferential cleavage of tripeptides from the unsubstituted N-terminus of the protein. Two alternative conformations of the catalytic Asp276 are associated with the activated state of TPP1.

[0028] High expression of TPP1 is found in bone marrow, placenta, lung, pineal gland, and lymphocytes. This protease functions in lysosomes to cleave N-terminal tripeptides from substrates and has relatively weak endopeptidase activity. It is synthesized as a catalytically inactive enzyme that becomes activated and undergoes autoproteolysis when acidified.

[0029] Neuronal ceroid lipofuscinosis (NCL) is a group of inherited neurodegenerative disorders characterized by a pathological phenotype characterized by the presence of autofluorescent lipopigments in neurons and other cell types. Over the past two decades, evidence has accumulated that NCL is caused by mutations in eight different genes, including genes encoding several soluble proteins (cathepsin D, PPT1, and TPP1). Mutations in TPP1, a gene associated with the failure of lysosomal degradation of certain neuropeptides and ATP synthase subunits, result in infantile-type neuronal ceroid lipofuscinosis. Mutations in the TPP1 gene lead to infantile-type neuronal ceroid lipofuscinosis, a fatal childhood neurodegenerative disease. A single intravitreal transplant of autologous bone marrow-derived stem cells transduced with a TPP1 expression construct early in disease progression has been shown to substantially inhibit the development of disease-associated retinal dysfunction and structural changes. These results imply that ex vivo gene therapy using autologous stem cells may be an effective means to achieve sustained delivery of therapeutic compounds to tissues such as the retina where systemic administration is ineffective.

[0030] In some embodiments, TPP1 may be under the control of a promoter such as the CMV early enhancer / chicken β-actin (CAG) promoter. The CAG promoter is a strong synthetic promoter that is frequently used to drive high-level gene expression in mammalian expression vectors. The CAG promoter has the following sequence: (C) cytomegalovirus (CMV) early enhancer element; (A) Promoter, first exon, and first intron of the chicken β-actin gene; (G) Splice acceptor of rabbit β-globin gene Built from.

[0031] The resulting synthetic element was used in the pCAGGS expression vector. The initiation codon located in the proximal region of the second exon was destroyed by digestion with NcoI restriction enzyme and replacing the site with a HindIII linker. The entire construct, commonly referred to as the "CAG promoter," contains part of the transcribed sequence (the first exon and first intron of the chicken β-actin gene) as well as enhancer elements. In addition to the CMV immediate-early enhancer, the chicken β-actin gene intron contains enhancer elements that are highly conserved among vertebrates. The 3' portion of the promoter has a high GC content and is therefore resistant to PCR amplification.

[0032] II. Adeno-associated virus (AAV) vectors Adeno-associated viruses (AAVs) are small, non-pathogenic viruses of the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, with more than 12 isolated from humans or primates. AAVs are distinguished from other members of this family by their dependence on a helper virus for replication.

[0033] The AAV genome can exist extrachromosomally without integrating into the host cell genome; it has a broad host range; it transduces both dividing and non-dividing cells in vitro and in vivo, maintaining high levels of transduced gene expression. AAV viral particles are thermostable; resistant to solvents, detergents, pH changes, and temperature; and can be column-purified and / or concentrated by CsCl gradients or other means. The AAV genome contains single-stranded deoxyribonucleic acid (ssDNA) of either positive or negative strand. The approximately 4.7 kb AAV genome consists of a single segment of single-stranded DNA of either positive or negative polarity. The ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as origins of viral DNA replication.

[0034] The term "AAV genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. AAV particles often contain an AAV genome packaged by AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not include portions of the "plasmid" that do not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone" and is important for cloning and amplification of the plasmid, a process necessary for plasmid growth and production, but is not itself packaged or encapsulated into a viral particle. Thus, the term "AAV vector genome" refers to the nucleic acid that is packaged or encapsulated by the AAV capsid proteins.

[0035] AAV virions (particles) are non-enveloped, icosahedral particles approximately 25 nm in diameter that contain the AAV capsid. AAV particles have icosahedral symmetry, consisting of three related capsid proteins, VP1, VP2, and VP3, which interact together to form the capsid. Most native AAV genomes often contain two open reading frames (ORFs), sometimes referred to as the left and right ORFs. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a 1:1:10 ratio, respectively, but can also occur in different ratios, all derived from the right ORF. The capsid proteins VP1, VP2, and VP3 differ from each other due to alternative splicing and the use of rare start codons. Deletion analysis has shown that removal or modification of VP1, which is translated from alternatively spliced ​​messages, results in reduced yields of infectious particles. Mutations within the VP3 coding region result in single-stranded progeny DNA or failure to produce infectious particles. In certain embodiments, the genome of an AAV particle encodes one, two, or all three VP1, VP2, and VP3 polypeptides.

[0036] The left ORF often encodes nonstructural Rep proteins, Rep 40, Rep 52, Rep 68, and Rep 78, which are involved in the control of replication and transcription as well as the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with preferential integration of the AAV genome into the q arm region of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity, as well as DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals and several potential phosphorylation sites. In certain embodiments, the genome of an AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of an AAV (e.g., rAAV) does not encode a Rep protein. In certain embodiments, one or more of the Rep proteins may be delivered in trans and therefore not included in AAV particles containing nucleic acids encoding polypeptides.

[0037] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that can potentially fold into T-shaped hairpin structures that serve as origins of viral DNA replication. Thus, the AAV genome contains one or more (e.g., a pair of) ITR sequences flanking the single-stranded viral DNA genome. The ITR sequences are often approximately 145 bases long each. Within the ITR region, two elements thought to be central to ITR function have been described: a GAGC repeat motif and a terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for site-specific and strand-specific cleavage at the trs. In addition to their role in replication, these two elements appear to be central to viral integration. The integration locus on chromosome 19 contains a Rep binding site flanked by the trs. These elements have been shown to be functional and necessary for locus-specific integration.

[0038] The term "recombinant," both as a modifier of vectors, such as recombinant viral vectors, e.g., recombinant lentiviral vectors or recombinant parvoviral (e.g., AAV) vectors, and as a modifier of sequences, such as recombinant nucleic acid sequences and recombinant polypeptides, generally means that a composition has been manipulated (i.e., modified) in a manner that does not occur in nature. Specific examples of recombinant vectors, e.g., recombinant AAV vectors, recombinant retroviral vectors, or recombinant lentiviral vectors, are those in which a nucleic acid sequence not normally present in the wild-type viral genome has been inserted into the viral genome. An example of a recombinant nucleic acid sequence is one in which the nucleic acid (e.g., gene) encodes an inhibitory RNA cloned into the vector, with or without the 5', 3', and / or intron regions with which the gene is normally associated in the viral genome. The term "recombinant" is not necessarily used herein with respect to vectors, such as viral vectors, and sequences, such as polynucleotides; however, despite such omission, "recombinant" forms, including nucleic acid sequences, polynucleotides, transgenes, and the like, are expressly included.

[0039] A recombinant viral "vector" is derived from a wild-type viral genome by using molecular methods to remove a portion of the wild-type genome from the virus and replace it with a non-native nucleic acid, e.g., a nucleic acid sequence. Typically, for example, in the case of AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome because a portion of the viral genome has been replaced with a non-native sequence, e.g., a nucleic acid encoding a transactivator, a nucleic acid encoding an inhibitory RNA, or a nucleic acid encoding a therapeutic protein, relative to the viral genome nucleic acid. Therefore, the incorporation of such a non-native nucleic acid sequence defines the viral vector as a "recombinant" vector, and in the case of AAV, a "recombinant" vector is also called a "rAAV vector."

[0040] In certain embodiments, an AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs adjacent to (i.e., at each of the 5' and 3' ends of) a nucleic acid sequence encoding at least a polypeptide having a function or activity.

[0041] AAV vectors (e.g., rAAV vectors) can be packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo, and are referred to herein as "AAV particles." When a recombinant AAV vector is encapsulated or packaged in an AAV particle, the particle is also referred to as an "rAAV particle." In certain embodiments, the AAV particle is an rAAV particle. An rAAV particle often comprises an rAAV vector or a portion thereof. An rAAV particle can be one or more rAAV particles (e.g., multiple AAV particles). An rAAV particle typically comprises proteins (e.g., capsid proteins) that encapsulate or package the rAAV vector genome. Note that reference to an rAAV vector can also be used to refer to an rAAV particle.

[0042] Any suitable AAV particle (e.g., rAAV particle) can be used for the methods or uses herein. The rAAV particle, and / or the genome contained therein, can be derived from any suitable serotype or strain of AAV. The rAAV particle, and / or the genome contained therein, may be derived from more than one serotype or strain of AAV. Thus, the rAAV can contain proteins and / or nucleic acids or portions thereof of any serotype or strain of AAV, where the AAV particle is suitable for infecting and / or transducing mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8.

[0043] In certain embodiments, the plurality of rAAV particles comprises particles of or derived from the same strain or serotype (or subgroup or variant). In certain embodiments, the plurality of rAAV particles comprises a mixture of two or more different rAAV particles (e.g., of different serotypes and / or strains).

[0044] As used herein, the term "serotype" refers to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such differences in cross-reactivity are generally due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the sequences of VP1, VP2, and / or VP3 of AAV serotypes). AAV variants, including capsid variants, may not be serologically distinct from a reference AAV or other AAV serotypes, but they differ in at least one nucleotide or amino acid residue compared to a reference or other AAV serotype.

[0045] In certain embodiments, an rAAV vector based on a genome of a first serotype corresponds to one or more serotypes of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that make up the AAV vector genome corresponds to the serotype of the capsid that makes up the rAAV particle.

[0046] In certain embodiments, the rAAV vector genome may be based on the genome of an AAV (e.g., AAV2) serotype that is distinct from one or more serotypes of the AAV capsid proteins that package the vector. For example, at least one or more of the three capsid proteins may be derived from a different serotype, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype, or a variant thereof, while the rAAV vector genome may contain nucleic acid (e.g., ITR) derived from AAV1.

[0047] In certain embodiments, an rAAV particle or its vector genome for a reference serotype has a polynucleotide, polypeptide, or subsequence that comprises or consists of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide, or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle. In specific embodiments, the rAAV particle or its vector genome for a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to the capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0048] In certain embodiments, the methods herein include the use, administration, or delivery of particles of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8.

[0049] In certain embodiments, the methods herein include the use, administration, or delivery of rAAV1 particles. In certain embodiments, the rAAV1 particles include AAV2 capsids. In certain embodiments, the rAAV1 particles include one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid proteins of native or wild-type AAV2 particles, for example, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV1 particles comprise capsid proteins VP1, VP2, and VP3 that are at least 75% or more identical to the corresponding capsid proteins of a native or wild-type AAV1 particle, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV1 particles are variants of native or wild-type AAV1 particles. In some aspects, one or more capsid proteins of the AAV1 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of a native or wild-type AAV1 particle.

[0050] In certain embodiments, the rAAV particles contain one or more desired ITR functions (e.g., DNA replication; AAV integration into the host cell genome; and / or AAV-2i8, so long as the ITRs retain the ability to integrate DNA and / or, if desired, form a hairpin to enable packaging.

[0051] In certain embodiments, the rAAV1 particles comprise one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding ITRs of a native or wild-type AAV1 particle, so long as the rAAV1 particles retain one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome; and / or the ability to form a hairpin to enable packaging, if desired).

[0052] rAAV particles can contain ITRs with any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of AAV2 particles contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particles contain ITRs with three "GAGC" repeats. In certain embodiments, the rAAV2 particles contain ITRs with fewer than four "GAGC" repeats. In certain embodiments, the rAAV2 particles contain ITRs with more than four "GAGC" repeats. In certain embodiments, the ITRs of rAAV2 particles contain Rep binding sites in which the fourth nucleotide of the first two "GAGC" repeats is C rather than T.

[0053] An exemplary suitable length of DNA that can be incorporated into an rAAV vector for packaging / encapsidation into an rAAV particle can be about 5 kilobases (kb) or less, hi specific embodiments, the length of the DNA is less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

[0054] rAAV vectors containing nucleic acid sequences directing the expression of RNAi or polypeptides can be produced using suitable recombinant techniques known in the art (see, for example, Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles using an AAV viral packaging system and propagated. Transducible AAV particles can bind to and enter mammalian cells and then deliver nucleic acid cargo (e.g., heterologous genes) to the nucleus of the cells. Thus, intact transducible rAAV particles are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often replication-incompetent and require additional protein machinery for self-replication. Thus, rAAV particles configured to transduce mammalian cells are modified to bind to, enter, and deliver nucleic acid to mammalian cells, where the nucleic acid for delivery is often located between a pair of AAV ITRs within the rAAV genome.

[0055] Suitable host cells for producing transducible AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of heterologous rAAV vectors. Cells derived from the stable human cell line HEK293 (e.g., readily available through the American Type Culture Collection under accession number ATCC CRL1573) can be used. In certain embodiments, modified human embryonic kidney cell lines (e.g., HEK293) that have been transformed with adenovirus type 5 DNA fragments and express the adenovirus E1a and E1b genes are used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles. Methods for producing high-titer AAV particles that can transduce mammalian cells are known in the art.

[0056] In certain embodiments, AAV helper functions are introduced into host cells by transfecting them with an AAV helper construct before or simultaneously with the transfection of the AAV expression vector. Thus, AAV helper constructs may be used to complement missing AAV functions necessary for productive AAV transduction, providing at least transient expression of the AAV rep and / or cap genes. AAV helper constructs often lack AAV ITRs and are unable to replicate or be packaged. These constructs may be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known.

[0057] III. AAV-TTP1 as a Therapeutic Agent In some embodiments, viral gene transfer methods can be used to introduce nucleic acids into mammalian cells. Such methods can be used to administer nucleic acids encoding therapeutic proteins to cultured cells or cells within a host organism. In some embodiments, the therapeutic proteins can be useful for the treatment of infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease. Some embodiments can involve the expression of a polypeptide comprising tripeptidyl peptidase 1 (TPP1) activity.

[0058] A polypeptide comprising TPP1 activity refers to a mammalian TPP1 protein or portion thereof that exhibits at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the peptidase activity of human TPP1 of SEQ ID NO:4, assayed using an appropriate peptide substrate, e.g., by the method of Junaid et al., 1999, or another comparable method. In certain embodiments, a polypeptide comprising TPP1 activity refers to a mammalian TPP1 protein or subsequence or variant thereof that exhibits at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the peptidase activity of human TPP1 of SEQ ID NO:4.

[0059] Polypeptides comprising TPP1 activity can include truncated, mutant, chimeric, or modified forms of TPP1 polypeptides that retain at least partial TPP1 activity. Polypeptides comprising TPP1 activity can include TPP1 proteins or portions thereof obtained from any suitable organism (e.g., mammals, humans, non-human mammals, such as dogs, pigs, cows, etc.). In certain embodiments, polypeptides comprising TPP1 activity have at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the TPP1 encoded by the sequence set forth in SEQ ID NO:4.

[0060] In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid encoding a polypeptide comprising TPP1 activity. In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid that directs the expression and / or secretion of a polypeptide comprising TPP1 activity. In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid that encodes a TPP1 polypeptide or an enzymatically active portion thereof. In certain embodiments, an AAV particle comprises an AAV capsid protein and a nucleic acid that directs the expression and / or secretion of a TPP1 polypeptide or an enzymatically active portion thereof. In certain embodiments, an AAV particle comprises a polypeptide having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the sequence set forth in SEQ ID NO:2 or SEQ ID NO:5. In certain embodiments, the administered nucleic acid encodes TPP1, a TPP1 having substantial identity to wild-type TPP1, and / or a variant, mutant, or fragment of TPP1. In certain embodiments, the nucleic acid encoding TPP1 activity or encoding or directing the expression of a TPP1 polypeptide is a nucleic acid having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the nucleic acid set forth in SEQ ID NO:4.

[0061] Recombinant TTP1 polypeptides may have amino acid deletions and / or substitutions; therefore, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further contain insertions or additions of amino acids, such as fusion proteins or proteins with linkers. A "deletion-modified protein" lacks one or more residues of the native protein, but may retain the specificity and / or activity of the native protein.

[0062] Substitution or exchange variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly effector function and / or bioavailability. Substitutions may or may not be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0063] In addition to deletions or substitutions, modified proteins may also have insertions of residues into the polypeptide, which typically include the addition of at least one residue, which may include the insertion of a targeting peptide or polypeptide, or the insertion of only a single residue.

[0064] The term "biologically functionally equivalent" is well understood in the art and is further defined herein. Thus, it includes sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to those of a reference polypeptide, provided that the biological activity of the protein is maintained. A recombinant protein may, in certain aspects, be biologically functionally equivalent to its native counterpart.

[0065] It will also be understood that amino acid and nucleic acid sequences can be essentially as set forth in one of the sequences disclosed herein, even if they contain additional residues, e.g., additional N- or C-terminal amino acids, or 5' or 3' sequences, so long as the sequence meets the above criteria, including maintenance of biological protein activity, in terms of protein expression. The addition of terminal sequences particularly applies to nucleic acid sequences that may contain, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or various internal sequences known to be present within genes, i.e., introns.

[0066] IV. Method of Administration In some aspects, viral vectors can be administered directly to a patient (in vivo) or used to treat cells in vitro or ex vivo and then administered to a patient. The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., an AAV vector), or other vehicle that can be manipulated by the insertion or incorporation of a nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells so that the nucleic acid sequence therein can be transcribed and, if encoding a protein, then translated by the cell.

[0067] Any suitable cell or mammal can be administered or treated by the methods or uses described herein. Typically, a mammal in need of the methods described herein is suspected of having or expressing an abnormal or aberrant protein associated with a disease state. Alternatively, the recipient mammal may have a condition amenable to gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration to a recipient of exogenous genetic material encoding a therapeutic agent and subsequent in situ expression of the administered genetic material. Thus, the phrase "condition amenable to gene replacement therapy" includes conditions such as genetic diseases (i.e., disease states resulting from one or more gene defects), acquired pathologies (i.e., pathological states not resulting from congenital defects), cancer, and prophylactic processes (i.e., prevention of disease or undesirable medical conditions). Thus, as used herein, the term "therapeutic agent" refers to any agent or material that has a beneficial effect on the recipient mammal. Thus, "therapeutic agent" includes both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0068] Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., a human, pig, goat, sheep, horse, dog, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or in utero mammal). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, e.g., an animal model having or expressing an aberrant protein associated with a disease state, or an animal model having insufficient protein expression that causes a disease state.

[0069] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years of age or older) and children (under 18 years of age). Adults include the elderly. A typical adult is 50 years of age or older. Children range in age from 1 to 2 years, or 2 to 4 years, 4 to 6 years, 6 to 18 years, 8 to 10 years, 10 to 12 years, 12 to 15 years, and 15 to 18 years. Children also include infants. Infants are typically in the 1 to 12 month age range.

[0070] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal described herein, wherein the severity, frequency, progression, or time of onset of one or more symptoms of a disease state, e.g., a neurodegenerative disease, is reduced, decreased, prevented, inhibited, or delayed. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat a deleterious symptom of a disease state, e.g., a neurodegenerative disease. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to stabilize, delay or prevent the deterioration or progression of, or reverse a deleterious symptom of, a disease state, e.g., a neurodegenerative disease.

[0071] In certain embodiments, the method includes administering a multiplicity of viral particles to the central nervous system of a mammal, or a portion thereof as described herein, wherein the severity, frequency, progression, or time to onset of one or more symptoms of a disease state, e.g., a neurodegenerative disease, is reduced, decreased, prevented, inhibited, or delayed by at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about 50 days, or about 50 to about 100 days.

[0072] In certain embodiments, the symptoms or adverse effects include early, mid, or late symptoms; behavioral, personality, or language symptoms; swallowing, movement, seizures, tremors, or fodgetting symptoms; ataxia; and / or cognitive symptoms, e.g., memory, ability to organize.

[0073] In certain embodiments, the method comprises administering or delivering AAV-TPP1 particles to a mammal and administering one or more immunosuppressive agents to the mammal. In certain embodiments, the method comprises administering or delivering AAV-TPP1 particles to a mammal and administering two, three, four, or more immunosuppressive agents to the mammal. In certain embodiments, the method comprises administering or delivering AAV-TPP1 particles to a mammal and administering two immunosuppressive agents to the mammal. In one representative embodiment, the method of treating a mammal comprises administering or delivering AAV-TPP1 particles to the mammal and administering a first and a second immunosuppressive agent to the mammal.

[0074] When two or more immunosuppressive agents are administered, each immunosuppressive agent is separate and / or different (e.g., each agent differs in structure and / or mechanism of action). "Agent" refers to a pharmaceutically active ingredient. In certain embodiments, the immunosuppressive agent is an anti-inflammatory agent. In certain embodiments, the immunosuppressive agent is mycophenolic acid or a derivative thereof. An example of such a mycophenolic acid derivative is mycophenolate mofetil (MMF). In certain embodiments, the immunosuppressive agent is cyclosporine or a derivative thereof. In certain embodiments, a first immunosuppressive agent comprises cyclosporine and a second immunosuppressive agent comprises mycophenolic acid or a derivative thereof (e.g., MMF). In certain embodiments, a first immunosuppressive agent comprises cyclosporine and a second immunosuppressive agent comprises MMF.

[0075] In certain embodiments, the immunosuppressant is administered before, during, and / or after administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered simultaneously with administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered after administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered about 1 to about 60 minutes, about 1 to about 24 hours, about 1 to about 100 days, about 1 to about 12 months, or about 1 to about 5 years after administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered before administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered about 1 to about 60 minutes, about 1 to about 24 hours, about 1 to about 100 days, or about 1 to about 3 months before administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days before administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered at scheduled intervals (e.g., once daily, twice daily, three times daily, every other day, weekly, biweekly, bimonthly, combinations thereof, etc.) before, during, and / or after administration of the AAV-TPP1 particles to the mammal.

[0076] In certain embodiments, a first immunosuppressant is administered to the mammal at least about 1 to about 7 days, or about 1, about 2, about 3, about 4, or about 5 weeks, before administration of the AAV-TPP1 particles to the mammal, and a second immunosuppressant is administered to the mammal about 1 to about 7 days, about 1, about 2, about 3, about 4, or about 5 weeks before, during, and / or within about 10, about 20, about 30, about 40, 19, about 50, about 100, about 200, about 300, about 350, about 400, or about 500 days after administration of the AAV-TPP1 particles. In certain embodiments, cyclosporine is administered to the mammal at least about 1 to about 7 days, or about 1, about 2, about 3, about 4, or about 5 weeks, before administration of the AAV-TPP1 particles to the mammal, and mycophenolic acid or a derivative thereof (e.g., MMF) is administered to the mammal about 1 to about 7 days, about 1, about 2, about 3, about 4, or about 5 weeks before, during, and / or within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 350, about 400, or about 500 days after administration of the AAV-TPP1 particles. In certain embodiments, cyclosporine is administered about 1 to about 7 days, or about 1, about 2, about 3, about 4, or about 5 weeks before administration of the AAV-TPP1 particles, and at regular intervals after treatment, and mycophenolic acid or a derivative thereof (e.g., MMF) is administered to the mammal about 1 to about 7 days, about 1, about 2, about 3, about 4, or about 5 weeks before, during, and / or once within about 10 to about 40 days after administration of the AAV-TPP1 particles.

[0077] The immunosuppressant may be administered at any appropriate dose. In certain embodiments, cyclosporine is administered at a dose of about 1 to about 50 mg / kg, about 1 to about 20 mg / kg, or about 5 to about 10 mg / kg, once daily, twice daily, or three times daily to once every other day. In certain embodiments, cyclosporine is administered at about 10 mg / kg twice daily. In certain embodiments, cyclosporine is administered at about 10 mg / kg twice daily for a period of at least about 1, about 2, about 3, about 4, or about 5 months. In certain embodiments, the dose of cyclosporine is tapered to a dose of less than about 5 mg / kg or less than about 2 mg / kg about 1 to about 2 months after administration of the AAV-TPP1 particles to the mammal.

[0078] In certain embodiments, mycophenolic acid or a derivative thereof (e.g., MMF) is administered at a dose of about 1 to about 100 mg / kg, about 1 to about 50 mg / kg, about 1 to about 25 mg / kg, or about 5 to about 20 mg / kg, once daily, twice daily, or three times daily to once every other day. In certain embodiments, mycophenolic acid or a derivative thereof (e.g., MMF) is administered at about 10 to about 20 mg / kg once daily. In certain embodiments, the dosage of mycophenolic acid or a derivative thereof (e.g., MMF) is reduced to a dose of less than about 5 mg / kg or less than about 2 mg / kg about one to two months after administration of the AAV-TPP1 particles to the mammal. The immunosuppressant may be formulated into any appropriate formulation suitable for the specific route of administration. Various pharmaceutically acceptable formulations of immunosuppressants are commercially available and readily available to healthcare professionals.

[0079] The immunosuppressant may be administered by any suitable route. In certain embodiments, the immunosuppressant is administered orally. In certain embodiments, mycophenolic acid or a derivative thereof, such as mycophenolate mofetil (MMF), is administered orally. In certain embodiments, cyclosporine is administered orally. The immunosuppressant may be administered parenterally (e.g., intramuscularly, intravenously, subcutaneously) or by injection into the brain, spinal cord, or a portion thereof (e.g., injected into the CSF).

[0080] In certain embodiments, the method comprises administering one or more (e.g., multiple) AAV-TPP1 particles to the central nervous system of a mammal (e.g., a mammal with LSD). In certain embodiments, the central nervous system comprises the brain, spinal cord, and cerebrospinal fluid (CSF). In certain embodiments, the method comprises administering one or more AAV-TPP1 particles to the brain or spinal cord or CSF of a mammal. In certain embodiments, the AAV-TPP1 particles are administered to a portion of the brain or spinal cord. In certain embodiments, a composition comprising AAV-TPP1 particles and an immunosuppressant is administered to the cisterna magna of a mammal, and / or the ventricles, subarachnoid space, and / or the spinal cavity and / or the ependyma of a mammal. For example, the AAV-TPP1 particles can be delivered directly to the cisterna magna, ventricular cavity, ventricles, subarachnoid space, the spinal cavity, or the ependyma. In certain embodiments, the method comprises administering one or more AAV-TPP1 particles to the ependyma of a mammal.

[0081] In certain embodiments, AAV-TPP1 particles are administered to one or more cells that are in contact with mammalian CSF, for example, by contacting cells with AAV-TPP1 particles.Non-limiting examples of cells that are in contact with CSF include ependymal cells, pial membrane cells, endothelial cells, and / or meningeal cells.In certain embodiments, AAV-TPP1 particles are administered to ependymal cells.In certain embodiments, AAV-TPP1 particles are delivered to ependymal cells, for example, by contacting ependymal cells with AAV-TPP1 particles.

[0082] In certain embodiments, AAV-TPP1 particles are delivered locally. "Local delivery" refers to the delivery of an active agent directly to a target site within a mammal (e.g., directly into a tissue or body fluid). For example, an agent can be delivered locally by direct injection into an organ, tissue, or specific anatomical location. In certain embodiments, one or more AAV-TPP1 particles are delivered or administered by direct injection into the brain, spinal cord, or their tissues or body fluids (e.g., CSF, e.g., ependymal cells, leptomeningeal cells, endothelial cells, and / or meningeal cells). For example, AAV-TPP1 particles can be delivered directly to the CSF, cisterna magna, intraventricular space, ventricles, subarachnoid space, and / or thecal cavity, and / or ependyma by direct injection. In certain embodiments, AAV-TPP1 particles are contacted with brain or spinal cord tissues, body fluids, or cells by direct injection into brain or spinal cord tissues or body fluids. In certain embodiments, the AAV-TPP1 particles are not delivered systemically, for example, by intravenous, subcutaneous, or intramuscular injection, or intravenous infusion. In certain embodiments, the AAV-TPP1 particles are delivered to brain or spinal cord tissues or fluids by stereotactic injection.

[0083] In certain embodiments, one or more AAV-TPP1 particles are delivered or administered by direct injection of the AAV-TPP1 particles into the brain, spinal cord, or tissues or fluids thereof (e.g., CSF, such as the ependyma). In specific aspects, the AAV-TPP particles transduce ependymal cells, pial membrane cells, endothelial cells, and / or meningeal cells.

[0084] As will be clear to those skilled in the art, taking into consideration the teachings herein, such as the dosage ranges provided herein, the effective amount of AAV-TPP1 particles can be empirically determined.Administration can be carried out in one dose continuously or intermittently throughout the course of treatment.The effective dose of administration can be determined by those skilled in the art, and can vary according to the AAV serotype, virus titer, and the body weight, condition and species of the mammal being treated.A single administration or multiple administrations can be carried out, with the dose level, target and timing selected by the treating physician.

[0085] In certain embodiments, a large number of AAV-TPP1 particles are administered. As used herein, a large number of AAV particles is about 1 x 10 5 ~Approx. 1×10 8 Refers to individual particles.

[0086] In certain embodiments, the AAV-TPP1 particles are about 1 x 10 in about 1 to about 5 ml. 5 ~Approx. 1×10 16 at a dose of 1 x 10 vg / ml; approximately 1 to 3 ml of 1 x 10 7 ~Approx. 1×10 14 at a dose of 1 x 10 vg / ml; or about 1 to about 2 ml of 1 x 10 8 ~Approx. 1×10 13 In certain embodiments, the AAV-TPP1 particles are administered at a dose of about 1 x 10 vg / ml relative to the body weight of the mammal being treated. 8 ~Approx. 1×10 15 For example, AAV-TPP1 particles are administered at a dose of about 1×10 vg / kg relative to the body weight of the mammal being treated. 8 vg / kg, approx. 5×10 8 vg / kg, approximately 1×10 9 vg / kg, approx. 5×10 9 vg / kg, approximately 1×10 10 vg / kg, approx. 5×10 10 vg / kg, approximately 1×10 11 vg / kg, approx. 5×10 11 vg / kg, approximately 1×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 1×1013 vg / kg, approx. 5×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 5×10 14 vg / kg, or approximately 1 × 10 15 It may be administered at a dose of 1000 mg / kg.

[0087] Administration of AAV-TPP1 particles can be in one dose or multiple doses, e.g., multiple doses can be administered as needed to maintain adequate enzyme activity.

[0088] V. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable composition, formulation, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical formulations and compositions can be used, for example, in administering viral particles to a subject.

[0089] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension vehicles, coatings, isotonic agents, and absorption enhancers or delayers that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) may also be incorporated into the formulations and compositions.

[0090] Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, such as mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0091] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0092] Pharmaceutical forms suitable for injection or infusion of AAV-TPP1 particles can include sterile aqueous solutions or dispersions suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final form should be sterile, liquid, and stable under the conditions of manufacture, use, and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, the maintenance of the required particle size in the case of dispersions, or the use of surfactants. Isotonic agents, such as sugars, buffers, or salts (e.g., sodium chloride), can be included. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, such as aluminum monostearate and gelatin, in the composition.

[0093] The solution or suspension of AAV-TPP1 particles may optionally contain the following components: a sterile diluent, e.g., water for injection, saline solution, e.g., phosphate-buffered saline (PBS), artificial CSF, fixed oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents; antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, etc.; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetic acid, citric acid, or phosphoric acid, and agents for adjusting osmotic pressure, e.g., sodium chloride or dextrose.

[0094] Pharmaceutical formulations, compositions, and delivery systems suitable for use with the compositions, methods, and methods of the present disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20 th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., 2004).

[0095] AAV-TPP1 particles and compositions can be formulated into dosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unit suitable as a unit dose for each individual to be treated; each unit contains a predetermined amount of active compound, calculated to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier.Dosage unit form depends on the amount of AAV-TPP1 particles that is considered necessary to produce the desired effect.The required amount can be formulated into a single dose or multiple dosage units.The dosage can be adjusted to the appropriate AAV-TPP1 particle concentration, and can be optionally combined with an anti-inflammatory agent and packaged for use.

[0096] In one embodiment, the pharmaceutical composition contains sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate the symptoms of the disease state, or an amount sufficient to confer the desired benefit. Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included, such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0097] As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated; each unit contains a predetermined amount calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle, or filler). Unit dosage forms can be contained, for example, in ampoules and vials, which can contain liquid compositions or freeze-dried or lyophilized compositions, and, for example, a sterile liquid carrier can be added before in vivo administration or delivery. Individual unit dosage forms can also be contained in multi-dose kits or containers. Thus, for example, virus particles and pharmaceutical compositions thereof can be packaged in single-unit dosage forms or multiple-unit dosage forms for ease of administration and uniformity of dosage.

[0098] A formulation containing AAV-TPPl particles contains an effective amount of rAAV particles in a medium, which effective amount can be easily determined by one skilled in the art. The AAV-TPPl particles typically range from about 1% to about 95% (w / w) of the composition, or more if appropriate. The amount to be administered depends on factors such as the age, weight, and physical condition of the mammalian or human subject being treated. Effective dosages can be established by one skilled in the art through routine trials to establish dose-response curves.

[0099] VI.Definition "Promoter" refers to a nucleotide sequence, generally upstream (5') of a coding sequence, that directs and / or regulates expression of the coding sequence by providing recognition for RNA polymerase and other factors necessary for proper transcription. In some embodiments, the promoter comprises a sequence having at least 50% identity, at least 60% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the nucleic acid set forth in SEQ ID NO:1.

[0100] An "enhancer" is a DNA sequence capable of stimulating transcriptional activity and may be an endogenous element of a promoter or a heterologous element that enhances the level or tissue specificity of expression. It can function in either orientation (5'->3' or 3'->5') and when located either upstream or downstream of the promoter.

[0101] A promoter and / or enhancer may be derived entirely from a native gene, or may be composed of different elements derived from different elements found in nature, or even composed of synthetic DNA segments. A promoter or enhancer may contain DNA sequences that are involved in the binding of protein factors that modulate / regulate the effectiveness of transcription initiation in response to stimuli, physiological, or developmental conditions.

[0102] "Transgene" is used herein for convenience to refer to a nucleic acid sequence / polynucleotide intended or introduced into a cell or organism. A transgene includes any nucleic acid, e.g., a gene encoding an inhibitory RNA polypeptide or protein (e.g., TTP1), and is generally heterologous to the naturally occurring genomic sequence.

[0103] The term "transduce" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Thus, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. When the introduced transgene is integrated into the nucleic acid (genomic DNA) of a recipient cell or organism, it can be stably maintained in that cell or organism and further inherited or inherited by the progeny cells or progeny organisms of the recipient cell or organism. Finally, the introduced transgene may exist extrachromosomally or only transiently in the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a transgene has been introduced or its progeny. The transduced cell can be propagated, the transgene can be transcribed, and the encoded protein can be expressed. In the case of gene therapy uses and methods, the transduced cell can be present in a mammal.

[0104] As used herein, the term " modify " or " variant " and its grammatical variations refer to nucleic acid, polypeptide, or their subsequence deviating from reference sequence.Therefore, modified sequence and variant sequence can have substantially the same, more or less expression, activity or function as reference sequence, but at least retain the partial activity or function of reference sequence.A specific type of variant is mutant protein, and mutant protein refers to the protein that is coded by gene that has mutation, for example, missense mutation or nonsense mutation.

[0105] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that is genetically altered compared to the wild-type. A sequence can be genetically modified without changing the encoded protein sequence. Alternatively, a sequence can be genetically modified to encode a variant protein, e.g., a variant TPP1 protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence that has been codon-modified to encode a protein that still retains at least partial sequence identity to a reference sequence, e.g., a wild-type protein sequence, and that has been codon-modified to encode a variant protein. For example, some codons in such a nucleic acid variant are altered without changing the amino acids of the TPP1 protein encoded thereby, and some codons in the nucleic acid variant are altered to change the amino acids of the protein encoded thereby.

[0106] The terms "protein" and "polypeptide" are used interchangeably herein. A "polypeptide" encoded by a "nucleic acid" or "polynucleotide" or "transgene" disclosed herein includes partial or full-length native sequences, naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide (e.g., TPP1) retains some function or activity. Thus, in the methods and uses of the present disclosure, such polypeptides encoded by nucleic acid sequences need not be identical to defective endogenous proteins or endogenous proteins whose activity, function, or expression is insufficient, missing, or absent in the mammal being treated.

[0107] Non-limiting examples of modifications include substitution of one or more nucleotides or amino acids (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000, or more nucleotides or residues).

[0108] An example of amino acid modification is conservative amino acid substitution or deletion. In a specific embodiment, the modified or variant sequence retains at least part of the function or activity of the unmodified sequence (e.g., wild-type sequence). Another example of amino acid modification is the introduction of targeting peptides into the capsid protein of viral particles. Peptides that target recombinant viral vectors to the central nervous system, for example, distinct brain regions, have been identified.

[0109] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a native molecule. In the case of a nucleotide sequence, variants include sequences that encode the same amino acid sequence of a native protein due to the degeneracy of the genetic code. Naturally occurring allelic variants, for example, can be identified by using molecular biology techniques, for example, polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences include synthetically obtained nucleotide sequences, such as nucleotide sequences generated by site-directed mutagenesis, that encode native proteins, and also include those that encode polypeptides with amino acid substitutions. Generally, nucleotide sequence variants of the present disclosure have at least 40%, 50%, 60% to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% to 79%, typically at least 80%, e.g., 81% to 84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% to 98% sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0110] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide, compared to a reference sequence using one of the alignment programs described, contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame positioning, etc. Substantial amino acid sequence identity for these purposes typically means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

[0111] The term "substantial identity" in reference to a polypeptide refers to a polypeptide comprising a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence in a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with an antibody raised against the second polypeptide. Thus, for example, if two peptides differ only by conservative substitutions, the polypeptide is identical to the second polypeptide.

[0112] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures aimed at preventing, inhibiting, slowing, or reducing the onset, progression, or worsening of an undesirable physiological change or disorder, e.g., a disorder. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, which may or may not be detectable, reduction in the extent of disease, stabilization of symptoms or adverse effects of disease (i.e., no worsening or progression), delay or slowing of disease progression, remission or alleviation of the disease state, and recovery (partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those with a predisposition thereto (e.g., as determined by genetic assays).

[0113] VII. Kit The present disclosure provides kits that contain packaging materials and one or more components therein. The kits typically contain labels or packaging inserts that include descriptions of the components or instructions for the in vitro, in vivo, or ex vivo use of the components. The kits can contain a collection of such components, for example, nucleic acids, recombinant vectors, and / or viral particles.

[0114] A kit refers to the physical structure that houses one or more components of the kit. The packaging material is capable of maintaining the components sterility and may be made from materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0115] The label or insert may include identification information of one or more components therein, dosage amounts, and clinical pharmacology of the active ingredients, e.g., mechanism of action, pharmacokinetics, and pharmacodynamics. The label or insert may include information identifying the manufacturer, lot number, location and date of manufacture, and expiration date. The label or insert may include information identifying the manufacturer information, lot number, location and date of manufacture. The label or insert may include information regarding diseases for which the components of the kit can be used. The label or insert may include instructions for a physician or subject regarding the use of one or more of the components of the kit in a method, use, or treatment protocol or therapeutic regimen. The instructions may include the amount, frequency, or duration of administration, and instructions for carrying out any of the methods, uses, treatment protocols, or preventative or therapeutic regimens described herein.

[0116] Label or insert can include information about any benefit that the component can provide, for example, preventive or therapeutic benefit.Label or insert can include information about possible harmful side effects, complications or reactions, for example, warnings to subjects or doctors about the situation in which it is not appropriate to use a particular composition.When a subject receives, plans to receive, or is currently receiving one or more other drug treatments that may be incompatible with the composition, or when a subject receives, plans to receive, or is currently receiving another treatment protocol or treatment plan that is incompatible with the composition, harmful side effects or complications may also occur, and therefore the instructions can include information about such incompatibility.

[0117] A label or insert includes "printed matter," e.g., paper or cardboard, that is separate from or attached to a component, kit, or packaging (e.g., box), or that is attached to an ampoule, tube, or vial containing a component of the kit. A label or insert can also include computer-readable media, e.g., a printed label with a barcode, a disk, an optical disk, e.g., a CD or DVD-ROM / RAM, a DVD, an MP3, or an electrical storage medium, e.g., RAM and ROM, or a hybrid thereof, e.g., magnetic / optical storage medium, flash memory, hybrid, and memory-type card. [Example]

[0118] VIII. Working Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be understood by those of skill in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the present disclosure and, therefore, are to be considered to constitute preferred modes for its practice. However, in light of the present disclosure, those of skill in the art should understand that many changes can be made to the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the present disclosure.

[0119] Example 1 – Improved AAV capsids for gene therapy of CLN2 disease A novel AAV capsid was developed that preferentially transduces not only neurons but also ependymal cells in NHPs (described in PCT / US2020 / 061464). Subsequently, a gene therapy for CLN2 disease was designed using the AAV capsid and a ubiquitous neuronal promoter sequence (CAG) to drive the expression of tripeptidyl peptidase 1 (TPP1) in neurons throughout the brain (previously described in a prior patent, but packaged in AAV2). As shown in Appendix 1, in TPP1-deficient mice, transduced hTPP1 secreted from the ependyma was stably expressed widely in the brain and produced well-tolerated, dose-dependent therapeutic benefits. Peptide-modified AAV resulted in robust improvements in hTPP1 biodistribution in mice and improved the phenotype at a significantly lower dose (5E10vg) than the conventional serotype (1E11vg).

[0120] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and steps or the order of steps described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure, as defined by the appended claims.

[0121] IX. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2026505074000002.tif177146TIFF2026505074000003.tif71146

Claims

1. A method for expressing tripeptidyl peptidase 1 (TPP1) in neurons and / or ependymal cells of a subject, comprising administering to the subject a modified adeno-associated virus (AAV) encoding TPP1 under the control of a promoter functional in neurons, e.g., central nervous system neurons, or ependymal cells, wherein the AAV comprises a modified capsid protein including the targeting sequence ERDRTRG (SEQ ID NO:3), and the modified AAV does not target dorsal root ganglia.

2. The method of claim 1, wherein the promoter can be a constitutive promoter.

3. The method of claim 1, wherein the promoter can be a tissue-specific promoter.

4. 2. The method of claim 1, wherein the promoter is a CMV early enhancer / chicken beta actin (CAG) promoter.

5. The method of claim 4, wherein the CAG promoter comprises the sequence of SEQ ID NO:

1.

6. The method of any one of claims 1 to 5, wherein the modified AAV is derived from AAV1.

7. 7. The method of any one of claims 1 to 6, wherein the targeting sequence is inserted near or after position 590 of SEQ ID NO:

2.

8. 8. The method of any one of claims 1 to 7, wherein the targeting sequence consists essentially of ERDRTRG (SEQ ID NO:3).

9. 8. The method of any one of claims 1 to 7, wherein the targeting sequence consists of ERDRTRG (SEQ ID NO:3).

10. The method of any one of claims 1 to 9, wherein TPP1 comprises the sequence of SEQ ID NO:

4.

11. The method of any one of claims 1 to 9, wherein TPP1 consists of the sequence of SEQ ID NO:

4.

12. 12. The method of any one of claims 1 to 11, wherein the targeting sequence is flanked by linker sequences, the linker sequences on each side of the targeting sequence being 2 or 3 amino acids in length.

13. 13. The method of claim 12, wherein the linker sequence is SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence.

14. 14. The method of any one of claims 1 to 13, wherein the modified capsid protein comprises the sequence of SEQ ID NO:

5.

15. 14. The method of any one of claims 1 to 13, wherein the modified capsid protein consists of the sequence of SEQ ID NO:

5.

16. 16. The method of any one of claims 1 to 15, wherein the administering step is direct intraventricular or intracisternal or intrathecal delivery.

17. The method of any one of claims 1 to 16, wherein the step of administering the modified AAV is performed multiple times.

18. The method of claim 17, wherein the step of administering the modified AAV comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations.

19. 18. The method of claim 17, wherein the step of administering the modified AAV is performed monthly, every other month, every two months, every three months, every four months, every six months, every year, every other year, every three years, every four years, or every five years.

20. 20. The method of any one of claims 1 to 19, wherein a large number of viral particles are administered.

21. The step of administering is about 1×10 per kilogram 6 ~Approx. 1×10 14 21. The method of claim 20, comprising administering at least one vector genome (vg / kg).

22. The step of administering is about 1 x 10 per kilogram of patient. 7 ~1×10 14 , about 1×10 8 ~1×10 14 , about 1×10 9 ~1×10 14 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , or approximately 1 × 10 12 ~1×10 13 22. The method of claim 21, comprising administering 10 vector genomes (vg / kg).

23. 23. The method of any one of claims 1 to 22, wherein the TPP1 sequence is operably linked to a polyadenylation signal.

24. 24. The method of any one of claims 1-23, wherein the subject is afflicted with infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease.

25. 25. The method of any one of claims 1 to 24, wherein the subject is under 4 years old or between 4 and 25 years old.

26. A modified adeno-associated virus (AAV) encoding TPP1 under the control of a promoter functional in neurons or ependymal cells, the modified AAV comprising a modified capsid protein containing the targeting sequence ERDRTRG (SEQ ID NO:3), and the modified AAV not targeting the dorsal root ganglion.

27. The modified AAV of claim 26, wherein the promoter may be a constitutive promoter.

28. The modified AAV of claim 26, wherein the promoter can be a tissue-specific promoter.

29. The modified AAV of claim 26, wherein the promoter is a CMV early enhancer / chicken beta actin (CAG) promoter.

30. The modified AAV of claim 29, wherein the CAG promoter comprises the sequence of SEQ ID NO:

1.

31. The modified AAV of any one of claims 26 to 29, which is AAV1.

32. The modified AAV of any one of claims 26 to 31, wherein the targeting sequence is inserted near or after position 590 of SEQ ID NO:

1.

33. The modified AAV of any one of claims 26 to 32, wherein the targeting sequence consists essentially of ERDRTRG (SEQ ID NO:3).

34. The modified AAV of any one of claims 26 to 32, wherein the targeting sequence consists of ERDRTRG (SEQ ID NO:3).

35. The modified AAV of any one of claims 26 to 34, wherein TPP1 comprises the sequence of SEQ ID NO:

4.

36. The modified AAV of any one of claims 26 to 34, wherein TPP1 consists of the sequence of SEQ ID NO:

4.

37. 37. The modified AAV of any one of claims 26 to 36, wherein the targeting sequence is flanked by linker sequences, and the linker sequences on each side of the targeting sequence are 2 or 3 amino acids in length.

38. The modified AAV of claim 37, wherein the linker sequence is SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence.

39. The modified AAV of any one of claims 26 to 38, wherein the modified capsid protein comprises the sequence of SEQ ID NO:

5.

40. The modified AAV of any one of claims 26 to 38, wherein the modified capsid protein consists of the sequence of SEQ ID NO:

5.

41. A pharmaceutical composition comprising the modified AAV of any one of claims 26 to 40 and a pharmaceutically acceptable carrier.

42. A kit comprising the modified AAV of any one of claims 26 to 40.

43. 1. Use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter functional in neuronal cells for expressing TPP1 in neuronal cells or ependymal cells, wherein the AAV comprises a modified capsid protein containing the targeting sequence ERDRTRG (SEQ ID NO:3), and the modified AAV does not target the dorsal root ganglion.

44. Use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter functional in neurons or ependymal cells for treating infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease in a subject suffering from the disease, wherein the AAV comprises a modified capsid protein including the targeting sequence ERDRTRG (SEQ ID NO:3), and the modified AAV does not target the dorsal root ganglion.

45. Use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter functional in neurons or ependymal cells in the preparation of a medicament for treating infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease in a subject suffering from the disease, wherein the AAV comprises a modified capsid protein including the targeting sequence ERDRTRG (SEQ ID NO:3), and the modified AAV does not target the dorsal root ganglion.

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