Novel gene therapy constructs for CLN2 disease

Abstract

Provided herein are methods of expressing tripeptidyl peptidase 1 (TPP1) in neurons. Also provided herein are modified adeno-associated viruses (AAVs) encoding TPP1 under the control of a promoter operable in neuronal or ventricular membrane cells rather than in the dorsal root ganglion.

Description

[0001] Priority Declaration

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 482,495, filed on January 30, 2022, the entire contents of which are hereby incorporated by reference. Background Art 1. Technical Field

[0003] The present disclosure relates generally to the fields of medicine, virology, and neurology. More specifically, the present disclosure relates to targeting peptides that target the delivery of viral vectors to different structures in the brain, particularly in the treatment of TPP1 deficiency / CLN2 disease.

[0004] 2. Description of Related Technology

[0005] TPP1 deficiency / CLN2 disease is a pediatric neurodegenerative disease caused by a deficiency of the soluble lysosomal enzyme TPP1 due to mutations in CLN2. Because CLN2 patients are unable to produce any functional TPP1 themselves, gene replacement is the best treatment option. TPP1 is an enzyme modified by mannose-6-phosphate, which can be used to cross-correct defective cells through enzyme replacement or gene therapy. To be effective, gene therapy for TPP1 deficiency requires widespread distribution throughout the brain. Transduction of ependymal cells lining the ventricles of dogs with CLN2 disease has demonstrated therapeutic efficacy with improved neuropathology, prolonged lifespan, and symptom relief (Katz et al, 2013). However, transduction of the ependyma and deep brain structures in NHP brains is difficult to achieve with current AAV serotypes.

[0006] Adeno-associated virus (AAV) represents a powerful 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 show robust integration in the host genome, but persists in the nucleus as an episome. The expression of AAV cargo is spatially controlled by the level of the packaging capsid and the transgenic promoter. Because the use of AAV to treat diseases may require intervention in diseased tissues, problems may arise in target tissues that contain gene expression profiles that are different from their healthy counterparts. Finding the correct promoter sequence to drive therapeutic transgene expression is an important goal.

[0007] Different strategies have been developed to generate AAV vector variants, including rational design and directed evolution. Rational design methods use knowledge of the AAV capsid to make targeted changes to the capsid to change transduction efficiency or specificity, such as tyrosine mutations on the capsid surface to improve transduction efficiency. Directed evolution methods do not require any knowledge of capsid structure, and the method is accomplished by random mutagenesis, capsid shuffling, or random peptide insertion. These strategies typically use in vitro systems or mice, which are ideal for cell-based or mouse-based studies, but are not meant to be translated into the clinic. In fact, no AAV variant can specifically or effectively target different brain structures. Therefore, there is a need for AAV variants that can target different brain structures. Summary of the Invention

[0008] 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 operable in the neurons or ependymal cells, wherein the AAV comprises a modified capsid protein containing a targeting sequence for targeting ependymal and CNS neurons, and wherein 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 beta actin (CAG) promoter.

[0009] 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.

[0010] The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three amino acids long. The linker sequences may be SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side 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.

[0011] Administration can be by direct intracerebroventricular or intracisternal or intrathecal delivery. The modified AAV can be administered more than once, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The modified AAV administration can occur monthly, annually, every 5 years or less frequently.

[0012] Multiple viral particles can be administered, for example, at a concentration of about 1×10 6 to about 1×10 14 vector genomes / kg (vg / kg), or approximately 1×10 7 to 1×10 14 , about 1×10 8 to 1×10 14 , about 1×10 9 to 1×10 14 , about 1×10 10 to 1×10 14 , about 1×10 10 to 1×10 13 , about 1×10 10 to 1×10 13 , about 1×10 10 to 1×10 11 , about 1×10 11 to 1×10 12 or about 1×10 12 to 1×10 13 vg / kg patient administration.

[0013] The subject may have late infantile neuronal ceroidlipofusincosis type 2 (CLN2) disease / Batten disease. The subject may be younger than 4 years old or between birth and 25 years old.

[0014] In another embodiment, a modified adeno-associated virus (AAV) encoding TPP1 under the control of a promoter operable in neurons or ependymal cells is provided, wherein the AAV comprises a modified capsid protein containing a targeting sequence, and wherein the modified AAV does not target the dorsal root ganglion. The promoter can be a constitutive promoter or a tissue-specific promoter. The promoter can be a CMV early enhancer / chicken beta actin (CAG) promoter.

[0015] The CAG promoter may comprise the sequence of SEQ ID NO: 1. The modified AAV may be based on AAV1 but have 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.

[0016] The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three or more amino acids long. The linker sequences may be SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the sequent. 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.

[0017] Also provided are pharmaceutical compositions comprising a modified AAV as described herein, and kits comprising a modified AAV as described herein.

[0018] In another embodiment, provided is the use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter operable in neurons or ependymal cells, wherein the AAV comprises a modified capsid protein comprising a targeting sequence, such as ERDRTRG (SEQ ID NO: 3), wherein the modified AAV does not target the dorsal root ganglion; and the use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter operable in neurons or ependymal cells for expressing TPP1 in neurons or ependymal cells. The AAV comprises a modified capsid protein comprising a targeting sequence, such as ERDRTRG (SEQ ID NO: 3), wherein the modified AAV does not target the dorsal root ganglion. NO:3), wherein the modified AAV does not target the dorsal root ganglion; and use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter operable in neurons or ependymal cells in the preparation of a medicament for treating late 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 comprising the targeting sequence ERDRTRG (SEQ ID NO:3), wherein the modified AAV does not target the dorsal root ganglion.

[0019] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word without a quantifier can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". The word "about" means plus or minus 5% of the specified number.

[0020] It is contemplated that any method or composition described herein may be implemented for any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate some specific embodiments of the present disclosure, they are provided by way of example only, because, based on this detailed description, various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings constitute part of this specification and are included to further illustrate 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 shown herein.

[0022] Figure 1 .epAAV: The brain parenchyma and ependyma always show strong signals. An AAV capsid (epAAV) targeting the ependyma / neurons expressing the fluorophore mTFP1 (cyan) was injected into non-human primates at 1.85E13 vg intracerebroventricularly (ICV). Ependymal cells lining the lateral ventricles and the fourth ventricle (including the choroid plexus) were observed to have strong mTFP1 positive signals by confocal microscopy. In the brain parenchyma, positive cells were present in the subiculum and parasubiculum and in cortical areas throughout the brain (entorhinal cortex, motor cortex, and temporal cortex). Positive mTFP1 signals were also observed in multiple nuclei of the thalamus (lateral dorsal, medial dorsal, and ventral anterolateral thalamus). Confocal images were captured with a Leica SP8 microscope. Scale bar 50 μm.

[0023] Figure 2.epAAV: Dorsal root ganglia (DRG) are negative for the transgenes of the present invention. An ependyma-targeted AAV capsid (epAAV) expressing the fluorophore mTFP1 (cyan) was injected intracerebroventricularly (ICV) into non-human primates at 1.85E13 vg. Dorsal root ganglion 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). RNAscope fluorescence in situ hybridization was used to detect transcripts of interest (middle 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 DRG; a scramble probe (Scramble) was used as a negative control (- control); and mTFP1 was detected using probes for the transgene of the present invention (mTFP1) in cervical, thoracic, and lumbar DRGs. The merged images are shown in the right column. No transcripts were detectable in the cervical, thoracic, or lumbar regions of the DRG. Therefore, capsids targeting the ependyma / neurons do not transduce the DRG. Confocal images obtained with a Leica SP8 microscope. Scale bar, 50 μm.

[0024] Figure 3 .epAAV: The spinal cord is negative for the transgene of the invention. Ependyma / neuron-targeting AAV capsids (epAAV) expressing the fluorophore mTFP1 (cyan) were injected intracerebroventricularly (ICV) into non-human primates at 1.85E13 vg. Nuclei were visualized using DAPI counterstaining (blue; left column). RNAscope fluorescence in situ hybridization was used to detect the transcript of interest (middle 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 using a probe for the transgene of the invention (mTFP1; cyan). The merged image is shown in the right column. No transcripts were detectable in the cervical, thoracic, or lumbar regions of the spinal cord. Confocal images from a Leica SP8 microscope. Scale bar 50 μm.

[0025] Figure 4.epAAV: Peripheral organs showed few or no positive cells. An ependymal / neuron-targeted AAV capsid (epAAV) expressing the fluorophore mTFP1 (cyan) was injected intracerebroventricularly (ICV) into the CSF of non-human primates at 1.85E13 vg. Cell nuclei were visualized using DAPI counterstaining (blue; left column). Epifluorescence of mTFP1 was detected (cyan). Cells with high levels of autofluorescence accumulation appear red (autofluorescence). The merged images are shown in the right column. The liver (top panel) had a small number of cells with positive signals for the transgene mTFP1 of the present invention, while no transgene of the present invention was detected in the heart (bottom panel). All images were captured on a Leica DM6000B microscope. Scale bar 50 μm.

[0026] Figure 5 . Depicts the Cln2 expression in mice treated with the novel therapeutic constructs of the present invention relative to untreated normal or diseased mice. - / - Survival curves of extended lifespan in mice. + / + 、Cln2 - / - and Cln2 - / - +5.0E+10% survival of vgepAAV mice over time. - / - The lowest survival rate was observed in mice (n=10), with an average of 17.1 weeks and a maximum lifespan of 19.7 weeks. Gene therapy with 5.0E+10 vg epAAV significantly increased Cln2 - / - The half-life of Cln2 was increased to 18.6 weeks on average and 28.6 weeks at most. + / - A 100% survival rate was observed in mice (n=9). *p<0.05 according to Mantel-Cox test.

[0027] Figure 6 . Relative to untreated Cln2 - / - , in Cln2 treated with the novel therapeutic constructs of the present invention - / - The resting tremor phenotype is delayed in onset and less aggressive. + / + 、Cln2 - / - and Cln2 - / - Resting tremor assessment in +5.0E+10vg epAAV mice. + / + , in untreated Cln2 at 13 weeks of age - / - A resting tremor phenotype was detected in mice and increased dramatically in severity over time. - / -The mice significantly delayed the onset of resting tremor up to 16 weeks, and the phenotype was significantly weaker and progressed more slowly over time.

[0028] Figure 7 TPP1 activity in the central nervous system is increased relative to endogenous levels after treatment with the novel therapeutic construct. + / - and Cln2 - / - TPP1 activity in mice. - / - When compared to endogenous TPP1 activity in mice, epAAV-injected mice had elevated TPP1 activity in different analyzed brain regions, which was also significantly higher than that of normal mice. - / - Mice, injected with Cln2 - / - The hearts and spleens of the mice, although not direct targets of treatment, had minimally elevated levels of TPP1 activity.

[0029] Figure 8 A to D. Quantification of ependymal transduction in 5 NHPs receiving ICV-infused AAV-EP+. AAV-Ep+ was administered to (n=5) rhesus monkeys with 1E13 vg per subject by intracerebroventricular (ICV) infusion. Transduced ependymal cells and parenchymal neurons were identified by fluorescence and morphology of microscopy of the sliced ​​brain. Using Cellpose and QuPath, the total number of cells and the number of transduced cells were counted by an automatic algorithm. (Fig. 8A) Representative 40× images show the robust transduction and mRuby3 expression of the ependymal cell layer lining the ventricle. (Fig. 8B) Quantification of transduced ependymal cells and parenchymal cells across 75 images (40×), a total of 15,096 cells and 5,852 ependymal cells. ( FIG8C ) A 10× tile scan image captured shows extensive ependymal transduction across the ependymal lining of the anterior (slab 4) lateral ventricle. ( FIG8D ) Quantification of 10× tile scan images from the lateral ventricle in slices 4 and 5 of four NHPs. Quantification was performed on eight tile scans and a total of 34,734 ependymal cells. DETAILED DESCRIPTION

[0030] Provided herein are methods for expressing tripeptidyl peptidase 1 (TPP1) in the ependyma and neurons of a subject, comprising administering to the subject a modified adeno-associated virus (AAV) capsid encapsidating a transgene encoding TPP1 under the control of a promoter operable in the neurons / ependyma, wherein the AAV comprises a modified capsid protein comprising a targeting sequence.

[0031] 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 different brain structures. 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).

[0032] In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, AAV is derived from AAV1. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO: 1, which does not transduce the ependyma to any appreciable extent after delivery into the CSF. In certain aspects, the 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 that after position 590, the targeting peptide is inserted as SSAX7AS, where the leading SSA and trailing AS are linker sequences and X7 represents the targeting peptide.

[0033] I. Batten Disease / TPP1 Deficiency

[0034] In some aspects, the present disclosure may relate to uses for treating neurological disorders such as late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease. Batten disease is a fatal neurological disease that typically begins in childhood, usually between the ages of 5 and 10. It is typically autosomal recessive and is the common name for neuronal ceroid lipofuscinosis (NCL). Although Batten disease is often considered juvenile NCL (or "type 3"), some physicians use the term Batten disease to describe all forms of NCL. Historically, NCLs have been classified based on age of onset as infantile NCL (INCL), late infantile NCL (LINCL), juvenile NCL (JNCL), or adult NCL (ANCL). At least 20 genes have been identified as associated with Batten disease, but the most common form of Batten disease, juvenile NCL, is associated with mutations in the CLN3 gene.

[0035] Batten disease is a terminal illness. Brineura is the first FDA-approved treatment to slow the loss of ambulation (walking) in symptomatic pediatric patients 3 years of age and older with late 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 that in humans is encoded by the TPP1 gene. Mutations in the TPP1 gene cause late 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.

[0036] The nucleic acid sequence of human TPP1 is shown in SEQ ID NO:4. Human TPP1 comprises tripeptidyl peptidase I activity (TPP1 enzyme activity). TPP1 activity comprises nonspecific lysosomal peptidase activity, which produces tripeptides from the degradation products produced by lysosomal proteases. Substrate specificity studies have shown that TPP1 mainly cuts tripeptides from the unreplaced amino termini in peptides and proteins. Endogenously expressed TPP1 is synthesized as an enzyme without catalytic activity. After targeting the lysosome, due to the acidic environment, TPP1 is automatically catalytically processed into a mature active enzyme. The activity of TPP1 can be measured and / or quantified in vitro using known methods. For example, see Junaid et al, 1999.

[0037] Human TPP1 is 61 kDa and consists of 563 amino acids. A 34.5 kDa, 320 amino acid isoform is generated by alternative splicing, and a peptide fragment lacking amino acids 1 to 243 is missing. TPP1 contains a globular structure with a Ser 475-Glu 272-Asp 360 catalytic triad with a subtilisin-like fold. It also contains an octahedral coordinated Ca 2+ The binding site is characteristic of the S53 sedolisin peptidase family. Unlike other S53 peptidases, it exhibits steric constraints on the P4 substrate pocket, which may contribute to its preferential cleavage of tripeptides from the unsubstituted N-terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation state of TPP1.

[0038] High expression of TPP1 is found in the bone marrow, placenta, lung, pineal gland, and lymphocytes. This protease functions in lysosomes to cleave N-terminal tripeptides from substrates and has weak endopeptidase activity. It is synthesized as a catalytically inactive enzyme that becomes activated upon acidification and undergoes autoproteolysis.

[0039] Neuronal ceroid lipofuscinosis (NCL) is a group of inherited neurodegenerative disorders characterized by the presence of autofluorescent lipopigments in neurons and other cell types. Over the past two decades, increasing evidence has demonstrated that NCL is caused by mutations in eight different genes, including those encoding several soluble proteins (cathepsin D, PPT1, and TPP1). Mutations in the gene TPP1 cause late infantile neuronal ceroid lipofuscinosis, which is associated with an inability to degrade specific neuropeptides and ATP synthase subunits in the lysosome. Mutations in the TPP1 gene cause late infantile neuronal ceroid lipofuscinosis, a fatal childhood neurodegenerative disease. A single intravitreal injection of autologous bone marrow-derived stem cells transduced with a TPP1 expression construct has been shown to significantly suppress the development of disease-associated retinal functional defects and structural changes at an early stage of disease progression. This result suggests 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, for which systemic administration would be ineffective.

[0040] In some embodiments, TPP1 can be under the control of a promoter, such as the CMV early enhancer / chicken beta actin (CAG) promoter. The CAG promoter is a strong synthetic promoter commonly used to drive high levels of gene expression in mammalian expression vectors. The CAG promoter is constructed from the following sequence:

[0041] (C) Cytomegalovirus (CMV) early enhancer element,

[0042] (A) The promoter, first exon and first intron of the chicken β-actin gene,

[0043] (G) Splice acceptor of the rabbit β-globin gene.

[0044] The resulting synthetic element is used in the pCAGGS expression vector. The start codon located in the proximal region of the second exon is destroyed by digestion with the NcoI restriction enzyme and replacing this site with a HindIII linker. Although the entire construct is commonly referred to as the "CAG promoter", it includes a portion of the transcription sequence (the first exon and first intron of the chicken beta actin gene) and an enhancer element. In addition to the CMV early enhancer, the introns of the chicken beta actin gene contain enhancer elements that are highly conserved in vertebrates. The 3' portion of the promoter has a high GC content and is therefore difficult to PCR amplify.

[0045] II. Adeno-associated virus (AAV) vectors

[0046] Adeno-associated virus (AAV) is a small, non-pathogenic virus of the Parvoviridae family. To date, many serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV differs from other members of the family in that it relies on a helper virus for replication.

[0047] The AAV genome can exist in an extrachromosomal state without integrating into the genome of the host cell; it has a wide host range; it transduces both dividing and non-dividing cells in vitro and in vivo, and maintains high levels of expression of the transduced genes. AAV virus particles are heat-stable; they are resistant to solvents, detergents, pH changes, and temperature; and can be column purified and / or concentrated on a CsCl gradient or by other means. The AAV genome contains positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7kb genome of AAV consists of a segment of single-stranded DNA at the positive or negative pole. At the end of the genome are short inverted terminal repeats (ITRs), which can fold into a hairpin structure and serve as the starting point for viral DNA replication.

[0048] AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form AAV particles. AAV particles typically contain an AAV genome packaged with AAV capsid proteins. In the case of using a recombinant plasmid to construct or prepare a recombinant vector, the AAV vector genome does not contain a portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called a "plasmid backbone," which is important for cloning and amplification of the plasmid (processes required for plasmid proliferation and production), but is not itself packaged or encapsulated in viral particles. Therefore, the AAV vector "genome" refers to the nucleic acid packaged or encapsulated by the AAV capsid protein.

[0049] AAV virions (particles) are non-enveloped icosahedral particles with a diameter of about 25 nm that include the AAV capsid. AAV particles contain icosahedral symmetry and are composed of three related capsid proteins (VP1, VP2, and VP3), which interact to form capsids. The genomes of most natural AAVs typically contain two open reading frames (ORFs), sometimes referred to as left and right ORFs. The right ORF typically encodes capsid proteins VP1, VP2, and VP3. These proteins typically exist in a ratio of 1:1:10, but can exist in different ratios and all derive from the right ORF. VP1, VP2, and VP3 capsid proteins differ from each other by using alternative splicing and unique start codons. Deletion analysis shows that the removal or alteration of VP1 translated from the information of alternative splicing results in a reduced yield of infectious particles. Mutations in the VP3 coding region result in the inability to produce any single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of the AAV particles encodes one, two, or all three VP1, VP2, and VP3 polypeptides.

[0050] The left ORF usually encodes nonstructural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which are involved in the regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins are related to the preferential integration of the AAV genome into the region of the q arm of human chromosome 19. It has been shown that Rep68 / 78 has NTP binding activity and DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals and several potential phosphorylation sites. In certain embodiments, the genome of AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of AAV (e.g., rAAV) does not encode Rep proteins. In certain embodiments, one or more of the Rep proteins can be delivered in trans, and therefore are not included in the AAV particles containing the nucleic acid encoding the polypeptide.

[0051] The end of the AAV genome contains short inverted terminal repeats (ITRs), which have the potential to fold into a T-type hairpin structure, which is used as the starting point for viral DNA replication. Therefore, the genome of AAV contains one or more ITR sequences (e.g., ITR sequence pairs), which are located on the flanks of the single-stranded viral DNA genome. The length of the ITR sequence is generally about 145 bases per sequence. Within the ITR region, two elements considered to be the core of ITR function, the GAGC repeat motif and the terminal resolution site (trs), have been described. When the ITR is in a linear or hairpin conformation, the repeat motif shows binding to Rep. This binding is considered to be positioning Rep68 / 78 at trs for cutting, which occurs in a site-specific and chain-specific manner. In addition to their role in replication, these two elements also show that they are the core of viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with adjacent trs. These elements have been shown to be functional and are essential for locus-specific integration.

[0052] The term "recombinant" as a modifier of vector (e.g., recombinant virus, such as lentiviral or parvoviral (e.g., AAV) vector) and sequence (e.g., recombinant nucleic acid sequence and polypeptide) means that the composition has been manipulated (i.e., modified) in a manner that does not normally occur in nature. A specific example of a recombinant vector (e.g., AAV, retroviral, or lentiviral vector) is one in which a nucleic acid sequence not normally found in the wild-type viral genome is inserted into the viral genome. An example of a recombinant nucleic acid sequence is a nucleic acid (e.g., a gene) encoding an inhibitory RNA cloned into a vector with or without the 5', 3', and / or intronic regions normally associated with the gene in the viral genome. Although the term "recombinant" is not always used herein to refer to vectors (e.g., viral vectors) and sequences (e.g., polynucleotides), "recombinant" forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included, notwithstanding any such omission.

[0053] Recombinant viral "vectors" are derived from the wild-type genome of a virus by using molecular methods to remove a portion of the wild-type genome from the virus and replacing it with non-natural nucleic acids (e.g., nucleic acid sequences). Generally speaking, for example, for AAV, one or two inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is different from a viral (e.g., AAV) genome because a portion of the viral genome has been replaced by a non-natural sequence for a viral genomic nucleic acid (e.g., a nucleic acid encoding a transactivator or a nucleic acid encoding an inhibitory RNA or a nucleic acid encoding a therapeutic protein). Therefore, the incorporation of such non-natural nucleic acid sequences defines a viral vector as a "recombinant" vector, which in the case of AAV may be referred to as a "rAAV vector."

[0054] In certain embodiments, AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, AAV (e.g., rAAV) comprises an ITR pair. In certain embodiments, AAV (e.g., rAAV) comprises an ITR pair flanking (i.e., at each of the 5' and 3' ends) at least a nucleic acid sequence encoding a polypeptide having a function or activity.

[0055] AAV vectors (e.g., rAAV vectors) can be packaged and referred to herein as "AAV particles" for subsequent infection (transduction) of cells in vitro, in vitro, or in vivo. In the case where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle may also be referred to as a "rAAV particle." In certain embodiments, the AAV particle is a rAAV particle. The rAAV particle typically comprises an rAAV vector or a portion thereof. The rAAV particle can be one or more rAAV particles (e.g., a plurality of AAV particles). The rAAV particle typically comprises a protein (e.g., a capsid protein) that encapsulates or packages the rAAV vector genome. It should be noted that reference to rAAV vectors can also be used to refer to rAAV particles.

[0056] Any suitable AAV particles (e.g., rAAV particles) can be used for the methods or uses herein. rAAV particles and / or the genomes contained therein can be derived from any suitable serotype or strain of AAV. rAAV particles and / or the genomes contained therein can be derived from two or more serotypes or strains of AAV. Therefore, rAAV can comprise proteins and / or nucleic acids or parts thereof of any serotype or strain of AAV, wherein the AAV particles are suitable for infection and / or transduction of mammalian cells. Some 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.

[0057] In certain embodiments, the plurality of rAAV particles comprise particles of, or are derived from, the same strain or serotype (or subgroup or variant). In certain embodiments, the plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., different serotypes and / or strains).

[0058] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctness is determined based on the lack of cross-reactivity between antibodies to one AAV and antibodies to another AAV. Such cross-reactivity differences are typically due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of the AAV serotypes). Although it is possible that an AAV variant (including a capsid variant) may be serologically indistinguishable from a reference AAV or other AAV serotype, they may differ from the reference AAV or other AAV serotype by at least one nucleotide or amino acid residue.

[0059] In certain embodiments, the rAAV vector based on the first serotype genome corresponds to the serotype of the one or more capsid proteins that package the vector. For example, the serotype of the one or more AAV nucleic acids (e.g., ITRs) comprising the AAV vector genome corresponds to the serotype of the capsid that comprises the rAAV particle.

[0060] In certain embodiments, the rAAV vector genome can be based on a serotype genome of AAV (e.g., AAV2) that is different from the serotype of one or more AAV capsid proteins that package the vector. For example, the rAAV vector genome can comprise nucleic acids (e.g., ITRs) derived from AAV1, while at least one or more of the three capsid proteins are derived from a different serotype, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotypes, or variants thereof.

[0061] In certain embodiments, a rAAV particle or its vector genome associated with a reference serotype has a polynucleotide, polypeptide, or subsequence thereof comprising or consisting 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 some embodiments, the rAAV particles or their vector genomes related to a reference serotype have a capsid or ITR sequence comprising or consisting 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 capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0062] In certain embodiments, the methods herein comprise the use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0063] In certain embodiments, the methods herein include using, administering or delivering rAAV1 particles. In certain embodiments, rAAV1 particles comprise AAV1 capsids. In certain embodiments, rAAV1 particles comprise one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or higher identical to the corresponding capsid proteins of natural or wild-type AAV1 particles, such as 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 particle comprises VP1, VP2, and VP3 capsid proteins that are at least 75% identical to or more than 100% identical to the corresponding capsid proteins of a native or wild-type AAV1 particle, such as 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% identity. In certain embodiments, the rAAV2 particle is a variant of a native or wild-type AAV1 particle. In some aspects, one or more capsid proteins of an AAV2 variant have 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20, or more amino acid substitutions compared to the capsid proteins of a native or wild-type AAV1 particle.

[0064] In certain embodiments, the rAAV particle comprises one or two ITRs (e.g., an ITR pair) that are at least 75% identical to the corresponding ITRs of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8. %, 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% identity, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0065] In certain embodiments, the rAAV1 particle comprises one or two ITRs (e.g., an ITR pair) that are at least 75% or more identical to the corresponding ITRs of a native or wild-type AAV1 particle, 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% identity, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0066] rAAV particles may comprise ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of the AAV2 particles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs comprising three "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs having fewer than four "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs having more than four "GAGC" repeats. In certain embodiments, the ITRs of the rAAV2 particles comprise a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is a C instead of a T.

[0067] An exemplary suitable length of DNA that can be incorporated into an rAAV vector for packaging / encapsulation into rAAV particles can be about 5 kilobases (kb) or less. In some 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.

[0068] The rAAV vector comprising the nucleic acid sequence for guiding the expression of RNAi or polypeptide can be produced using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are usually packaged into AAV particles with transduction ability and propagated using the AAV viral packaging system. AAV particles with transduction ability can bind to and enter mammalian cells, and then deliver nucleic acid cargo (e.g., heterologous genes) to the nucleus of the cell. Therefore, complete rAAV particles with transduction ability are configured to transduce mammalian cells. The rAAV particles configured to transduce mammalian cells generally do not have replication ability and require additional protein mechanisms to replicate from themselves. Therefore, the rAAV particles configured to transduce mammalian cells are modified to bind to and enter mammalian cells, and deliver nucleic acid to cells, wherein the nucleic acid for delivery is generally located between the AAV ITR pairs in the rAAV genome.

[0069] Suitable host cells for producing AAV particles with transduction capacity include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of heterologous rAAV vectors. Cells from a stable human cell line HEK293 (easily obtained, for example, by the American Type Culture Collection with accession number ATCC CRL1573) can be used. In certain embodiments, a modified human embryonic kidney cell line (e.g., HEK293) transformed with type 5 adenovirus DNA fragments and expressing adenovirus E1a and E1b genes is used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles therein. Methods for producing high-titer AAV particles capable of transducing mammalian cells are known in the art.

[0070] In certain embodiments, before or simultaneously with transfection of the AAV expression vector, the AAV helper functions are introduced into the host cells by transfecting the host cells with an AAV helper construct. Therefore, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the AAV functions of the deletions necessary for productive AAV transduction. AAV helper constructs typically lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of plasmids, phages, transposons, clays, viruses, or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 encoding both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known.

[0071] III. AAV-TTP1 as a Therapeutic Agent

[0072] 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 in a host organism. In some embodiments, therapeutic proteins can be used to treat late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease. Some embodiments may involve the expression of a polypeptide comprising tripeptidyl peptidase 1 (TPP1) activity.

[0073] A polypeptide comprising TPP1 activity refers to a mammalian TPP1 protein, or a 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, as determined using a suitable peptide substrate, for example, as determined 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 a 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.

[0074] The polypeptide comprising TPP1 activity may comprise a truncated, mutated, chimeric or modified form of a TPP1 polypeptide that retains at least a portion of the TPP1 activity. The polypeptide comprising TPP1 activity may comprise a TPP1 protein obtained from any suitable organism (e.g., obtained from a mammal, a human, a non-human mammal, e.g., obtained from a dog, a pig, a cow, etc.), or a portion thereof. In certain embodiments, the polypeptide comprising TPP1 activity has 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 shown in SEQ ID NO: 4.

[0075] In certain embodiments, the AAV particle comprises an AAV capsid protein and a nucleic acid encoding a polypeptide containing TPP1 activity. In certain embodiments, the AAV particle comprises an AAV capsid protein and a nucleic acid that directs the expression and / or secretion of a polypeptide containing TPP1 activity. In certain embodiments, the 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, the 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, the 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 with the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5. In certain embodiments, the nucleic acid administered 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.

[0076] Recombinant TTP1 polypeptides may have amino acid deletions and / or substitutions; thus, proteins with deletions, proteins with substitutions, and proteins with both deletions and substitutions are modified proteins. In some embodiments, these proteins may also contain insertions or added amino acids, such as, for example, proteins with fusion proteins or proteins with linkers. A "modified deletion protein" lacks one or more residues of a native protein but may possess the specificity and / or activity of the native protein.

[0077] Substitution or substitution variants typically comprise an exchange of one amino acid with another at one or more sites within a protein and can be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. A substitution may or may not be conservative, i.e., an amino acid is replaced by one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: 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.

[0078] In addition to deletions or substitutions, the modified protein may have an insertion of residues, which generally involves adding at least one residue to the polypeptide. This may include the insertion of a targeting peptide or polypeptide or just a single residue.

[0079] The term "biologically functional equivalent" is well known in the art and is further defined in detail herein. Thus, sequences are encompassed 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 the amino acids of the reference polypeptide, provided that the biological activity of the protein is maintained. In certain aspects, a recombinant protein can be biologically functionally equivalent to its native counterpart.

[0080] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, and still be substantially as shown in a sequence disclosed herein, so long as the sequence meets the criteria set forth above, including, in the context of protein expression, maintaining biological protein activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, contain various non-coding sequences flanking the 5' or 3' portion of the coding region, or may contain various internal sequences known to exist within genes, i.e., introns.

[0081] IV. Method of Administration

[0082] In some aspects, viral vectors can be directly administered to a patient (in vivo), or they can be 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, plasmid, virus (e.g., AAV vector), or other carrier that can be manipulated by inserting or incorporating nucleic acid. Vectors (e.g., viral vectors) can be used to introduce / transfer a nucleic acid sequence into a cell so that the nucleic acid sequence therein is transcribed and, if encoding a protein, is subsequently translated by the cell.

[0083] Any suitable cell or mammal can be applied or treated by the methods or uses described herein. Generally speaking, the mammals that need the methods described herein are suspected of having or expressing abnormal or abnormal proteins associated with the disease state. Alternatively, the mammal recipient may suffer from a disease suitable for gene replacement therapy. "Gene replacement therapy" used herein refers to the exogenous genetic material encoding the therapeutic agent administered to the recipient and then the genetic material administered in situ is expressed. Therefore, the phrase "disease suitable for gene replacement therapy" covers such diseases, such as hereditary diseases (that is, disease conditions due to one or more gene defects), acquired conditions (that is, pathological conditions not due to congenital defects), cancer and preventive processes (that is, prevention of disease or undesirable medical conditions). Therefore, the term "therapeutic agent" used herein refers to any agent or substance that has a beneficial effect on the mammal recipient. Therefore, "therapeutic agent" covers both therapeutic molecules and preventive molecules with nucleic acid or protein components.

[0084] Some 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), farm animals (e.g., horses, cows, goats, sheep, pigs), and experimental 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, adolescent, child, infant, or mammal in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, for example, an animal model having or expressing an abnormal or abnormal protein associated with a disease state, or an animal model in which a protein is not fully expressed, resulting in a disease state.

[0085] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years of age or older) and children (less than 18 years of age). Adults include the elderly. Representative adults are 50 years of age or older. Children are 1 to 2 years of age, or 2 to 4, 4 to 6, 6 to 18, 8 to 10, 10 to 12, 12 to 15, and 15 to 18 years of age. Children also include infants. Infants are typically 1 to 12 months of age.

[0086] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, wherein the severity, frequency, progression, or onset of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, lowered, blocked, inhibited, or delayed. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat adverse symptoms 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 adverse symptoms of a disease state (e.g., a neurodegenerative disease), or to reverse adverse symptoms of a disease state (e.g., a neurodegenerative disease).

[0087] In certain embodiments, the method comprises administering a plurality of viral particles to the central nervous system, or a portion thereof, of a mammal as described herein, and the severity, frequency, progression, or onset of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, decreased, arrested, inhibited, or delayed for 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.

[0088] In certain embodiments, symptoms or adverse effects comprise early, intermediate, or late symptoms; behavioral, personality, or language symptoms; swallowing, movement, seizure, tremor, or restlessness symptoms; ataxia; and / or cognitive symptoms such as memory and organizational skills.

[0089] In certain embodiments, the method comprises administering or delivering AAV-TPP1 particles to a mammal and administering one or more immunosuppressants 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 immunosuppressants to the mammal. In certain embodiments, the method comprises administering or delivering AAV-TPP1 particles to a mammal and administering two immunosuppressants to the mammal. In one representative embodiment, the method of treating a mammal comprises administering or delivering AAV-TPP1 particles to a mammal and administering a first and a second immunosuppressant to the mammal.

[0090] In the case of administering two or more immunosuppressants, each immunosuppressant is distinct and / or different (e.g., each agent differs in structure and / or mechanism of action). "Agent" refers to an active pharmaceutical ingredient. In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is mycophenolate mofetil or a derivative thereof. An example of such a mycophenolate mofetil derivative is mycophenolate mofetil (MMF). In certain embodiments, the immunosuppressant is cyclosporine or a derivative thereof. In certain embodiments, the first immunosuppressant comprises cyclosporine, and the second immunosuppressant comprises mycophenolate mofetil or a derivative thereof (e.g., MMF). In certain embodiments, the first immunosuppressant comprises cyclosporine, and the second immunosuppressant comprises mycophenolate mofetil or a derivative thereof (e.g., MMF). In certain embodiments, the first immunosuppressant comprises cyclosporine, and the second immunosuppressant comprises MMF.

[0091] In certain embodiments, an immunosuppressant is administered before, during, and / or after the AAV-TPP1 particles are administered to a mammal. In certain embodiments, an immunosuppressant is administered while the AAV-TPP1 particles are administered to a mammal. In certain embodiments, an immunosuppressant is administered after the AAV-TPP1 particles are administered to a mammal. In certain embodiments, an immunosuppressant is administered from about 1 to about 60 minutes, from about 1 to about 24 hours, from about 1 to about 100 days, from about 1 to about 12 months, or from about 1 to about 5 years after the AAV-TPP1 particles are administered to a mammal. In certain embodiments, an immunosuppressant is administered before the AAV-TPP1 particles are administered to a mammal. In certain embodiments, an immunosuppressant is administered from about 1 to about 60 minutes, from about 1 to about 24 hours, from about 1 to about 100 days, or from about 1 to about 3 months before the AAV-TPP1 particles are administered to a mammal. In certain embodiments, the immunosuppressant is administered about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days prior to administration of the AAV-TPP1 particles to the mammal. In certain embodiments, the immunosuppressant is administered at predetermined intervals (e.g., 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.

[0092] In certain embodiments, a first immunosuppressant is administered to the mammal at least about 1 to about 7 days prior to, or about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks prior to, administration of the AAV-TPP1 particles to the mammal; and a second immunosuppressant is administered about 1 to about 7 days prior to, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks prior to, during, and / or within about 10 days, about 20 days, about 30 days, about 40 days, about 50 days, about 100 days, about 200 days, about 300 days, about 350 days, about 400 days, or about 500 days after, administration of the AAV-TPP1 particles to the mammal. In certain embodiments, cyclosporine is administered to the mammal at least about 1 to about 7 days before, or about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks before, administration of the AAV-TPP1 particles to the mammal; and mycophenolate mofetil or a derivative thereof (e.g., MMF) is administered about 1 to about 7 days before, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks before, during, and / or within about 10 days, about 20 days, about 30 days, about 40 days, about 50 days, about 100 days, about 200 days, about 300 days, about 350 days, about 400 days, or about 500 days after administration of the AAV-TPP1 particles to the mammal. In certain embodiments, cyclosporine is administered about 1 to about 7 days before, or about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks before, and at fixed intervals thereafter, administration of the AAV-TPP1 particles; and mycophenolate mofetil or a derivative thereof (e.g., MMF) is administered once about 1 to about 7 days before, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks before, during, and / or within about 10 to about 40 days after administration of the AAV-TPP1 particles to the mammal.

[0093] The immunosuppressant may be administered at any suitable dose. In certain embodiments, cyclosporin is administered at a frequency of once, twice, or three times a day to once every other day 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. In certain embodiments, cyclosporin is administered at about 10 mg / kg twice a day. In certain embodiments, cyclosporin is administered at about 10 mg / kg twice a day for a period of at least about 1 month, about 2 months, about 3 months, about 4 months, or about 5 months. In certain embodiments, the dose of cyclosporin is gradually reduced to a dose of less than about 5 mg / kg or less than about 2 mg / kg about 1 to about 2 months after the AAV-TPP1 particles are administered to a mammal.

[0094] In certain embodiments, mycophenolate mofetil or a derivative thereof (e.g., MMF) is administered at a frequency of once, twice, or three times a day to once every other day 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. In certain embodiments, mycophenolate mofetil or a derivative thereof (e.g., MMF) is administered once a day at about 10 to about 20 mg / kg. In certain embodiments, the dose of mycophenolate mofetil 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 1 to about 2 months after the AAV-TPP1 particles are administered to a mammal. The immunosuppressant can be formulated into any suitable formulation suitable for a particular route of administration. A variety of pharmaceutically acceptable formulations of immunosuppressants are commercially available and readily available to medical practitioners.

[0095] The immunosuppressant may be administered by any suitable route. In certain embodiments, the immunosuppressant is administered orally. In certain embodiments, mycophenolate mofetil or a derivative thereof (e.g., mycophenolate mofetil (MMF)) is administered orally. In certain embodiments, cyclosporine is administered orally. The immunosuppressant may also be administered parenterally (e.g., intramuscularly, intravenously, subcutaneously), or by injection into the brain, spinal cord, or a portion thereof (e.g., by injection into CSF).

[0096] In certain embodiments, the method includes administering one or more (e.g., multiple) AAV-TPP1 particles to the central nervous system of a mammal (e.g., a mammal suffering from LSD). In certain embodiments, the central nervous system includes the brain, spinal cord, and cerebrospinal fluid (cerebral spinal fluid, CSF). In certain embodiments, the method includes administering one or more AAV-TPP1 particles to the brain or spinal cord or CSF of a mammal. In certain embodiments, AAV-TPP1 particles are administered to a part of the brain or spinal cord. In certain embodiments, a composition comprising AAV-TPP1 particles and an immunosuppressant is administered to the cerebellomedullary cistern and / or the ventricles, subarachnoid space, and / or intrathecal space and / or ependyma of a mammal. For example, AAV-TPP1 particles can be delivered directly to the cerebellomedullary cistern, intraventricular space, ventricles, subarachnoid space, intrathecal space, or ependyma. In certain embodiments, the method includes administering one or more AAV-TPP1 particles to the ependyma of a mammal.

[0097] In certain embodiments, AAV-TPP1 particles are administered to one or more cells contacting CSF in a mammal, for example, by contacting the cells with AAV-TPP1 particles. Some non-limiting examples of cells contacting CSF include ependymal cells, pial 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.

[0098] In certain embodiments, AAV-TPP1 particles are delivered locally." local delivery " refers to that active agent is delivered directly to the target site (for example, directly delivered to tissue or fluid) in mammalian body. For example, medicament can be locally delivered to organ, tissue or specific anatomical position by direct injection. In certain embodiments, one or more AAV-TPP1 particles are delivered to or applied to brain, spinal cord or its tissue or fluid (for example, CSF, such as ependymal cells, pia mater cells, endothelial cells and / or meningeal cells) by direct injection. For example, AAV-TPP1 particles can be directly delivered to CSF, cerebellomedullary cistern, intraventricular space, ventricle, subarachnoid space and / or intrathecal space and / or ependyma by the mode of direct injection. In certain embodiments, by being directly injected into the tissue or fluid of brain or spinal cord, AAV-TPP1 particles are contacted with the tissue, fluid or cell of brain or spinal cord. In certain embodiments, AAV-TPP1 particles are not delivered systemically by, for example, intravenous, subcutaneous or intramuscular injection or by intravenous infusion. In certain embodiments, AAV-TPP1 particles are delivered to tissue or fluid of the brain or spinal cord by stereotactic injection.

[0099] 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 a specific aspect, the AAV-TPP particles transduce ependymal cells, pia mater cells, endothelial cells, and / or meningeal cells.

[0100] In view of the teachings herein, such as the dosage ranges provided herein, it will be apparent to one skilled in the art that an effective amount of AAV-TPP1 particles can be determined empirically. Administration can be achieved continuously or intermittently in a single dose throughout the course of treatment. The effective dose administered can be determined by one skilled in the art and can vary depending on the AAV serotype, viral titer, and weight, condition, and species of mammal being treated. Single and multiple administrations can be performed at the treating physician's choice of dosage level, target, and timing.

[0101] In certain embodiments, a plurality of AAV-TPP1 particles is administered. As used herein, a plurality of AAV particles refers to about 1×10 5 to about 1×10 8 particles.

[0102] In certain embodiments, AAV-TPP1 particles are present at a concentration of about 1×10 5 to about 1×10 16 vg / ml dosage; about 1 to about 3 ml 1×10 7 to about 1×10 14 vg / ml; or in a dose of about 1 to about 2 ml 1×10 8 to about 1×10 13 In certain embodiments, AAV-TPP1 particles are administered at a dose of about 1×10 8 to about 1×10 15 vg / kg of the body weight of the mammal being treated. For example, AAV-TPP1 particles can be administered at a dose of about 1×10 8 vg / kg, about 5×10 8 vg / kg, about 1×10 9 vg / kg, about 5×10 9 vg / kg, about 1×10 10 vg / kg, about 5×10 10 vg / kg, about 1×10 11 vg / kg, about 5×10 11 vg / kg, about 1×10 12 vg / kg, about 5×10 12 vg / kg, about 1×10 13 vg / kg, about 5×10 13 vg / kg, about 1×10 14 vg / kg, about 5×10 14 vg / kg or about 1×10 15 vg / kg body weight of the treated mammal.

[0103] Administration of AAV-TPP1 particles can be performed in one or more doses. For example, multiple doses can be administered as needed to maintain adequate enzyme activity.

[0104] V. Pharmaceutical Compositions

[0105] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery routes, or exposure routes. A "pharmaceutically acceptable" or "physiologically acceptable" composition is not a biologically or otherwise undesirable substance, e.g., a substance that 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, to administer viral particles to a subject.

[0106] 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 media, coatings, isotonic agents, and absorption enhancers or absorption delayers that are compatible with drug administration or in vivo contact or in vivo delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into the formulations and compositions.

[0107] Pharmaceutical compositions typically include a pharmaceutically acceptable excipient. Such excipients include any agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive 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, for example: inorganic 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. In addition, auxiliary substances such as surfactants, wetting agents or emulsifiers, pH buffering substances, and the like may be present in such carriers.

[0108] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery as indicated herein or known to those skilled in the art. Thus, the pharmaceutical composition comprises a carrier, diluent or excipient suitable for administration or delivery by a variety of routes.

[0109] The pharmaceutical form suitable for injection or infusion of AAV-TPP1 particles may include a sterile aqueous solution or dispersion, which is suitable for the temporary preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. In all cases, the final form should be a sterile fluid and stable under preparation, use and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using a surfactant. Isotonic agents may be included, such as sugars, buffers, or salts (e.g., sodium chloride). The extended absorption of the injectable composition can be achieved by using delayed absorption agents such as aluminum monostearate and gelatin in the composition.

[0110] The solution or suspension of AAV-TPP1 particles may optionally include the following components: a sterile diluent such as 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 such as parabens, chlorobutanol, phenol, ascorbic acid, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose.

[0111] Pharmaceutical formulations, compositions and delivery systems suitable for use with the compositions, methods and uses of the present disclosure are known in the art (see, e.g.,

[0112] Remington:The Science and Practice of Pharmacy(2003)20 th ed., MackPublishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed.,MackPublishing Co.,Easton,PA;The Merck Index(1996)12 thed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), TechnonicPublishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., 2004).

[0113] For ease of administration and uniformity of dosage, AAV-TPP1 particles and compositions can be formulated in dosage unit form. As used herein, dosage unit form refers to a physically discrete unit suitable as a single dose for an individual to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit form depends on the amount of AAV-TPP1 particles necessary to produce the desired effect. The necessary amount can be formulated in a single dose, or it can be formulated in multiple dosage units. The dose can be adjusted to an appropriate AAV-TPP1 particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0114] In one embodiment, the pharmaceutical composition will include sufficient genetic material to provide a therapeutically effective amount, that is, an amount sufficient to alleviate or improve the symptoms of the disease state in question, or an amount sufficient to impart the desired benefit. Pharmaceutical compositions typically include a pharmaceutically acceptable excipient. Such excipients include any medicament that itself does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids (e.g., water, saline, glycerol, and ethanol). Pharmaceutically acceptable salts may be included, such as inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances (e.g., wetting agents or emulsifiers, pH buffer substances, etc.) may be present in such a carrier.

[0115] As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit comprising a predetermined amount is optionally associated with a pharmaceutical carrier (excipient, diluent, vehicle or filler) that is calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. The unit dosage form can be in ampoules and vials that can contain, for example, a liquid composition or a composition in a freeze-dried or lyophilized state; for example, a sterile liquid carrier can be added prior to in vivo administration or delivery. A single unit dosage form can be contained in a multi-dose kit or container. Thus, for ease of administration and uniformity of dosage, for example, viral particles and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms.

[0116] The formulation containing AAV-TPP1 particles will contain an effective amount of rAAV particles in a carrier, which effective amount is readily determined by one skilled in the art. The AAV-TPP1 particles can typically be from about 1% to about 95% (w / w) of the composition, or even higher if appropriate. The amount to be administered depends on factors such as the age, weight, and physical condition of the mammal or human subject being treated. The effective dose can be determined by one of ordinary skill in the art through routine experiments establishing a dose-response curve.

[0117] VI. Definitions

[0118] "Promoter" refers to a nucleotide sequence that is typically located upstream (5') of a coding sequence and that directs and / or controls the expression of a coding sequence by providing recognition for RNA polymerase and other factors required for proper transcription. In some embodiments, the promoter comprises a sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the nucleic acid shown in SEQ ID NO: 1.

[0119] An "enhancer" is a DNA sequence that stimulates transcriptional activity and can be an intrinsic element of a promoter or a heterologous element that enhances expression levels or tissue specificity. It can operate in either orientation (5'->3' or 3'->5') and can function even when located upstream or downstream of a promoter.

[0120] Promoters and / or enhancers may be derived entirely from a native gene, or be composed of different elements derived from different elements found in nature, or even comprise synthetic DNA segments. Promoters or enhancers may contain DNA sequences that participate in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0121] As used herein, "transgene" conveniently refers to a nucleic acid sequence / polynucleotide that is intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene encoding an inhibitory RNA polypeptide or protein (e.g., TTP1), and is generally heterologous to the naturally occurring genomic sequence.

[0122] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by means of a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell by means of a viral particle can be referred to as the "transduction" of a cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism and further transmitted to the daughter cells or organism of the recipient cell or organism or inherited by it. Finally, the introduced transgene may be present outside the chromosome of the recipient cell or host organism, or only exist temporarily. Therefore, a "transduced cell" is a cell into which a transgene is introduced by means of transduction. Therefore, a "transduced" cell is a cell or its progeny into which a transgene has been introduced. The transduced cell can proliferate, transcribe the transgene and express the encoded protein. For gene therapy purposes and methods, the transduced cell can be in a mammal.

[0123] As used herein, the terms "modification" or "variant" and grammatical variations thereof refer to a deviation of a nucleic acid, polypeptide, or subsequence thereof from a reference sequence. Thus, modified and variant sequences can have substantially the same, higher, or lower expression, activity, or function as a reference sequence, while retaining at least some of the activity or function of the reference sequence. A specific type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, such as a missense or nonsense mutation.

[0124] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been 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, such as a variant TPP1 protein. A nucleic acid or polynucleotide variant can also refer to a combined sequence that has been codon-modified to encode a protein that still retains at least partial sequence identity with a reference sequence (e.g., a wild-type protein sequence) and has also been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without changing the amino acids of the TPP1 protein encoded thereby, and some codons of the nucleic acid variant will be changed, which in turn changes the amino acids of the protein encoded thereby.

[0125] 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, such as naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments), and sequence variants thereof, as long as the polypeptide (e.g., TPP1) retains a certain degree of function or activity. Therefore, in the methods and uses of the present disclosure, such a polypeptide encoded by a nucleic acid sequence need not be identical to the endogenous protein that is defective or whose activity, function or expression is insufficient, missing, or absent in the mammal being treated.

[0126] Some non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (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).

[0127] An example of amino acid modification is conservative amino acid replacement or deletion. In some specific embodiments, the modified sequence 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 a targeting peptide into the capsid protein of a viral particle. Peptides that target recombinant viral vectors to the central nervous system (e.g., different brain regions) have been identified.

[0128] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a native molecule. For nucleotide sequences, variants include those that encode the same amino acid sequence as the native protein due to the degeneracy of the genetic code. For example, these naturally occurring allelic variants can be determined using molecular biology techniques (such as, for example, using polymerase chain reaction (PCR) and hybridization techniques). Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as those produced using site-directed mutagenesis (which encodes the native protein), and those encoding polypeptides with amino acid substitutions. In general, the nucleotide sequence variants of the present disclosure will have at least 40%, 50%, 60% to 70%, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% to 79%, usually at least 80%, such as 81% to 84%, at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% to 98% sequence identity with 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 activity or function of the wild-type).

[0129] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence that has 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 compared to a reference sequence using one of the alignment programs described using standard parameters. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. Substantial identity of amino acid sequences for these purposes generally means sequence identity of at least 70%, at least 80%, 90% or even at least 95%.

[0130] In the context of polypeptides, the term "substantially identical" means that the polypeptide comprises 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 to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunoreactive with antibodies raised against the second polypeptide. Thus, for example, a polypeptide is identical to a second polypeptide where the two peptides differ only in conservative substitutions.

[0131] The term "treatment" and its variations refer to both therapeutic treatments and preventative or precautionary measures, wherein the purpose is to prevent, inhibit, alleviate or reduce undesirable physiological changes or disorders, such as the occurrence, progression or worsening of disorders. For the purposes of this disclosure, useful or desired clinical outcomes include, but are not limited to, alleviation of symptoms, weakening of disease extent, stabilization of symptoms or adverse effects of the disease (i.e., no worsening or progression), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonged survival compared to the expected survival without treatment. Those in need of treatment include those already suffering from the disease or disorder and those susceptible to the disease (e.g., as determined by genetic assays).

[0132] VII.Medicine Box

[0133] The present disclosure provides a kit having packaging material and containing one or more components therein. The kit typically includes a label or package insert containing a description of the components or instructions for use of the components therein in vitro, in vivo, or ex vivo. The kit may include a collection of such components (e.g., nucleic acids, recombinant vectors, and / or viral particles).

[0134] A kit refers to a physical structure that houses one or more components of the kit. The packaging material can maintain the sterility of the components and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0135] The label or insert may include information on one or more components, dosage amounts, clinical pharmacology (including mechanism of action, pharmacokinetics and pharmacodynamics) of the active ingredient. The label or insert may include information indicating the manufacturer, batch number, place of manufacture and date of manufacture, and expiration date. The label or insert may include information indicating manufacturer information, batch number, manufacturer location and date. The label or insert may include information on the disease for which the kit components may be used. The label or insert may include instructions for clinicians or subjects to use one or more of the kit components in a method, use, treatment regimen or therapeutic regimen. The instructions may include dosage amounts, frequency or duration, and instructions for implementing any method, use, treatment regimen or prevention or therapeutic regimen described herein.

[0136] The label or insert may include information about any benefits (e.g., preventive or therapeutic benefits) that the components may provide. The label or insert may include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which a particular composition is not suitable for use. Adverse side effects or complications may also occur when a subject has been, will be, or is currently taking one or more other medications that may be incompatible with the composition or with another treatment regimen or therapeutic regimen that the subject has been, will be, or is currently experiencing that is incompatible with the composition, and therefore, the instructions may include information about such incompatibilities.

[0137] The label or insert includes "printed matter", for example, paper or cardboard, either alone or affixed to a component, kit or packaging material (e.g., box), or attached to an ampoule, tube or vial containing a kit component. The label or insert may additionally include computer-readable media, such as a barcode printed label, a magnetic disk, an optical disk (e.g., CD- or DVD-ROM / RAM, DVD), an MP3, or an electronic storage medium (e.g., RAM and ROM) or a mixture of these, such as magnetic / optical storage media, flash memory, hybrid, and memory type cards.

[0138] VIII. Examples

[0139] The following examples are included to illustrate some preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the technology disclosed in the following examples represents that the inventors have found that the technology plays a good role in the practice of the present disclosure, and therefore can be considered to constitute a preferred mode for the practice of the present invention. However, it will be appreciated by those skilled in the art that many changes may be made to some disclosed specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0140] Example 1—Evolved AAV capsids for gene therapy of CLN2 disease

[0141] New AAV capsids were developed that preferentially transduce ependymal cells as well as neurons in NHPs (described in PCT / US2020 / 061464). Subsequently, a gene therapy for CLN2 disease (previously described in a prior patent, packaged in AAV2) was designed using an AAV capsid that drives expression of tripeptidyl peptidase 1 (TPP1) in neurons throughout the brain and a ubiquitous neuronal promoter sequence (CAG). As shown in Appendix 1, hTPP1 secreted by the transduced ependyma was widely and stably expressed in the brain, was well tolerated, and produced a dose-dependent therapeutic benefit in TPP1-deficient mice. In mice, peptide-modified AAV resulted in a significant improvement in the biodistribution of hTPP1 and improved the phenotype at a dose significantly lower (5E10 vg) than the conventional serotype (1E11 vg).

[0142] ***

[0143] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present disclosure have been described according to some preferred embodiments, it will be apparent to those skilled in the art that, without departing from the concept, spirit and scope of the present disclosure, changes can be applied to the step or sequence of steps of the method described herein and the method described herein. More specifically, it will be apparent that certain medicaments all related in both chemical and physiological aspects can be used to replace the medicament described herein, while achieving identical or similar results. All such similar substitutions and changes apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0144] IX. References

[0145] The following references, to the extent they provide exemplary procedural or other details supplementary to those described herein, are specifically incorporated herein by reference.

[0146] U.S. Patent No. 8,299,215

[0147] U.S. Patent No. 8,691,948

[0148] U.S. Patent Application Publication No. 2018 / 0142259

[0149] Chen et al.,Molecular signatures of disease brain endothelia providenew sites for CNS-directed enzyme therapy.Nat Med,2009.15(10):p.1215-8.

[0150] Chen et al.,Overcoming Limitations Inherent in Sulfamidase to ImproveMucopolysaccharidosis IIIA Gene Therapy.Mol Ther,2018.26(4):p.1118-1126.

[0151] Deverman et al.,Cre-dependent selection yields AAV variants forwidespread gene transfer to the adult brain.Nat Biotechnol,2016.34(2):p.204-9.

[0152] Hartz et al.,Isolation of Cerebral Capillaries from Fresh Human BrainTissue.J Vis Exp,2018(139).

[0153] Hordeaux et al.,The Neurotropic Properties of AAV-PHP.B Are Limitedto C57BL / 6J Mice.Mol Ther,2018.26(3):p.664-668.

[0154] Junaid et al.,A Novel Assay for Lysosomal Pepstatin-InsensitiveProteinase and its Application for the Diagnosis of Late-Infantile NeuronalCeroid Lipofuscinosis.Clin Chim Act,1999.281:p.169-176.

[0155] Katz et al.,AAV gene transfer delays disease onset in a TPP 1-deficient canine model of the late infantile form of Batten disease.SciTransl Med,2015.7(313):p.313r180.

[0156] Keiser et al.,Broad distribution of ataxin 1silencing in rhesuscerebella for spinocerebellar ataxia type 1 therapy.Brain,2015.138(Pt 12):p.3555-66.

[0157] Koerber et al.,DNA shuffling of adeno-associated virus yieldsfunctionally diverse viral progeny.Mol Ther,2008.16(10):p.1703-9.

[0158] Matsuzaki et al.,Intravenous administration of the adeno-associatedvirus-PHP.B capsid fails to upregulate transduction efficiency in themarmoset brain.Neurosci Iett,2018.665:p.182-188.

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Claims

1. A method of 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 operable in the neurons or ependymal cells, e.g., central nervous system neurons, wherein the AAV comprises a modified capsid protein comprising a targeting sequence ERDRTRG (SEQ ID NO: 3), and wherein the modified AAV does not target dorsal root ganglia.

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

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

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

9. 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 the TPP1 comprises the sequence of SEQ ID NO:

4.

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

4.

12. The method of any one of claims 1 to 11, wherein the targeting sequence is flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three amino acids long.

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

5.

15. 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. The method of any one of claims 1 to 15, wherein administration is direct intracerebroventricular or intracisternal or intrathecal delivery.

17. The method of any one of claims 1 to 16, wherein the modified AAV administration occurs more than once.

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

19. The method of claim 17, wherein the modified AAV administration occurs monthly, every other month, every two months, every three months, every four months, every six months, annually, every other year, every three years, every four years, or every five years.

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

21. The method of claim 20, wherein administering comprises administering at about 1×10 6 to about 1×10 14 Vector genomes / kilogram (vg / kg) were administered.

22. The method of claim 21, wherein administering comprises administering at about 1×10 7 to 1×10 14 , about 1×10 8 to 1×10 14 , about 1×10 9 to 1×10 14 , about 1×10 10 to 1×10 14 , about 1×10 10 to 1×10 13 , about 1×10 10 to 1×10 13 , about 1×10 10 to 1×10 11 , about 1×10 11 to 1×10 12 or about 1×10 12 to 1×10 13 Vector genomes / kilogram (vg / kg) of patient were administered.

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

24. The method of any one of claims 1 to 23, wherein the subject has Late Infantile Neuronal Ceroid Lipofuscinosis Type 2 (CLN2) Disease / Batten Disease.

25. The method of any one of claims 1 to 24, wherein the subject is less than 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 operable in neurons or ependymal cells, 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 dorsal root ganglia.

27. The modified AAV of claim 26, wherein the promoter can 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 the 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, wherein the modified AAV 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 the TPP1 comprises the sequence of SEQ ID NO:

4.

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

4.

37. The modified AAV of any one of claims 26 to 36, wherein the targeting sequence is flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence are two or three 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. Use of a modified adeno-associated virus (AAV) encoding tripeptidyl peptidase 1 (TPP1) under the control of a promoter operable in neurons for expressing TPP1 in neurons or ependymal cells, wherein the AAV comprises a modified capsid protein comprising the targeting sequence ERDRTRG (SEQ ID NO: 3), wherein 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 operable in neurons or ependymal cells for treating late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease in a subject suffering from the disease, wherein the modified adeno-associated virus (AAV) comprises a modified capsid protein comprising the targeting sequence ERDRTRG (SEQ ID NO: 3), wherein 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 operable in neurons or ependymal cells in the preparation of a medicament for treating late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease / Batten disease in a subject suffering from the disease, wherein the modified adeno-associated virus (AAV) comprises a modified capsid protein comprising the targeting sequence ERDRTRG (SEQ ID NO: 3), wherein the modified AAV does not target the dorsal root ganglion.

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