AAV vectors for treating cln2 disease

Abstract

This disclosure provides modified AAV vectors for expressing tripeptidyl peptidase 1 (TPP1) in objects. Some aspects of this disclosure provide modified adeno-associated virus 1 (AAV1) vectors comprising a capsid protein containing a targeting peptide and a nucleic acid molecule containing a sequence encoding a modified AAV genome, said modified AAV genome comprising a replication (rep) gene, a capsid (cap) gene, and a tripeptidyl peptidase 1 (TPP1) transgene.

Description

[0001] Priority Statement

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 592,318, filed October 23, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list XML, which has been submitted electronically and is incorporated herein by reference in its entirety. The XML sequence list was created on October 23, 2024, named CHOPP0072WO.xml, and has a size of 35,445 bytes. Technical Field

[0005] This disclosure generally pertains to the fields of medicine, virology, and neurology. More specifically, this disclosure relates to targeting peptides that deliver viral vectors to different structures in the brain, particularly in the treatment of TPP1 deficiency / CLN2 disease. Background Technology

[0006] TPP1 deficiency / CLN2 disease is a neurodegenerative disease in children caused by a deficiency of the soluble lysosomal enzyme TPP1 due to mutations in CLN2. Since patients with CLN2 cannot produce any functional TPP1 themselves, gene replacement is the optimal treatment option. TPP1 is a mannose-6-phosphate modified enzyme that can be used for cross-correction of defective cells through enzyme replacement or gene therapy. In terms of efficacy, gene therapy for TPP1 deficiency needs to be widely distributed throughout the brain. Transduction of ependymal cells lining the ventricles of dogs with CLN2 disease has shown therapeutic efficacy with improvements in neuropathology, increased lifespan, and symptom relief (Katz et al., 2013). However, transduction of the ependymal and deep brain structures in the brain of NHP patients is difficult to achieve with current AAV serotypes.

[0007] Adeno-associated virus (AAV) represents a potent therapeutic candidate for treating neurodegenerative diseases. AAV is a non-enveloped, single-stranded DNA virus that can infect both dividing and non-dividing cells. After infection, the virus does not exhibit robust integration into the host genome but persists as an episome in the cell nucleus. AAV carrier expression is spatially controlled by the level of the packaging capsid and the transgene promoter. Because treating diseases with AAV may require intervention in diseased tissues, problems may arise in target tissues containing gene expression profiles different from their healthy counterparts. Identifying the appropriate promoter sequences to drive therapeutic transgene expression is an important goal.

[0008] Different strategies have been developed to generate AAV vector variants, including rational design and directed evolution. Rational design methods utilize knowledge of the AAV capsid to make targeted changes to the capsid to alter 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 the capsid structure and are accomplished through random mutagenesis, capsid shuffling, or random peptide insertion. These strategies typically use in vitro systems or mice, which are ideal for cell- or mouse-based research but do not necessarily translate to clinical applications. In fact, no AAV variant currently specifically or effectively targets different brain structures. Therefore, there is a need for AAV variants capable of targeting different brain structures. Summary of the Invention

[0009] This disclosure provides a modified AAV vector for expressing tripeptidyl peptidase 1 (TPP1) in an object.

[0010] Some aspects of this disclosure provide a modified adeno-associated virus 1 (AAV1) vector containing a capsid protein, named EP+ capsid, containing the targeting peptide ERDRTRG (SEQ ID NO: 1). The capsid protein contains (i.e. encapsulates) a nucleic acid molecule containing a sequence encoding a modified AAV genome, said modified AAV genome containing a tripeptidyl peptidase 1 (TPP1) transgene and optionally containing a replication (rep) gene and a capsid (cap) gene.

[0011] In some aspects of this disclosure, the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. The flanking sequences of the targeting peptide may be adaptor sequences, wherein the adaptor sequences on each side of the targeting peptide are two or three amino acids long. The adaptor sequence on the N-terminal side of the targeting peptide may be SSA, and the adaptor sequence on the C-terminal side of the targeting peptide may be AS.

[0012] The sequence encoding the TPP1 transgene can be operatively linked to a polyadenylation signal. The sequence encoding the TPP1 transgene can also be operatively linked to the CMV early enhancer / chicken actin (CAG) promoter.

[0013] In an exemplary aspect of this disclosure, the adeno-associated virus may comprise a sequence having at least 95% identity with the sequences in Table 1, which collectively describe the AAV-EP+.iCAG.hTPP1 AAV vector:

[0014] Table 1: AAV-EP+.iCAG.hTPP1 AAV Vector

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[0034]

[0035] Some aspects of this disclosure include pharmaceutical compositions comprising AAV vectors of this disclosure. Advantageously, the vectors of this disclosure can be used to treat CLN2 disease in subjects. Therefore, this disclosure provides a method of treating a subject suffering from CLN2 disease, the method comprising administering to the subject a modified AAV1 vector comprising an EP+ capsid protein containing the targeting peptide ERDRTRG (SEQ ID NO: 1) and a nucleic acid molecule containing a sequence encoding a modified AAV genome comprising a tripeptidyl peptidase 1 (TPP1) transgene, optionally comprising a replication (rep) gene and a capsid (cap) gene.

[0036] When used in conjunction with the term "comprising / including" in the claims and / or description, the use of the word without a quantifier may mean "one / type," but it is also consistent with the meaning of "one / type or more / types," "at least one / type," and "one / type or more than one / type." The word "about" means 5% plus or minus the specified number.

[0037] It is anticipated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples indicate some specific embodiments of this disclosure, they are given by way of example only, as various changes and modifications to the spirit and scope of this disclosure will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0038] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these figures in conjunction with the detailed description of specific embodiments given herein.

[0039] Figure 1 These are a series of graphs showing the activity of AAV-EP+ enzyme in mouse tissues.

[0040] Figure 2 Cln2 is delivered after AAV-EP+. - / - Survival chart of mice.

[0041] Figure 3 This is a graph showing the tremor frequency after AAV-EP delivery in Cln2 knockout mice. Detailed Implementation

[0042] This disclosure provides a modified AAV vector for expressing tripeptidyl peptidase 1 (TPP1) in an object.

[0043] This disclosure provides modified AAV vectors for expressing tripeptidyl peptidase 1 (TPP1) in objects. Some aspects of this disclosure provide modified adeno-associated virus 1 (AAV1) vectors comprising a capsid protein containing a targeting peptide and a nucleic acid molecule containing a sequence encoding a modified AAV genome, said modified AAV genome comprising a replication (rep) gene, a capsid (cap) gene, and a tripeptidyl peptidase 1 (TPP1) transgene.

[0044] I. Batten disease / TPP1 deficiency

[0045] In some respects, this disclosure may relate to the treatment of neurological disorders, such as late-onset infantile neuronal ceroid lipofuscinose type 2 (CLN2) disease / Barten disease. Barten disease is a fatal neurological disorder that typically begins in childhood, usually between the ages of 5 and 10. It is generally an autosomal recessive inherited disease and is the common name for neuronal ceroid lipofuscinose (NCL). Although Barten disease is often considered to be juvenile NCL (or “type 3”), some physicians use the term Barten disease to describe all forms of NCL. Historically, NCL has been classified according to age of onset as infantile NCL (INCL), late-onset infantile NCL (LINCL), juvenile NCL (JNCL), or adult NCL (ANCL). At least 20 genes have been identified as associated with Barten disease, but the most common form, juvenile NCL, is associated with mutations in the CLN3 gene.

[0046] Barten disease is a terminal illness. Brineura is the first FDA-approved treatment to slow the loss of walking ability (walking) in symptomatic pediatric patients aged 3 years and older with late-onset infantile neuronal ceroid lipofuscin deposition type 2 (CLN2) (also known as tripeptidyl peptidase-1 (TPP1) deficiency). TPP1, also known as a lysosomal pepsin inhibitor-insensitive protease, is an enzyme encoded by the TPP1 gene in humans. Mutations in the TPP1 gene lead to late-onset infantile neuronal ceroid lipofuscin deposition. The human TPP1 gene encodes a member of the sedolisin family of serine proteases. The human gene has 13 exons and is located at the 11p15 band on chromosome 10.

[0047] Human TPP1 contains tripeptidyl peptidase I activity (TPP1 enzyme activity). TPP1 activity includes nonspecific lysosomal peptidase activity, which generates tripeptides from degradation products produced by lysosomal proteases. Substrate-specific studies have shown that TPP1 primarily cleaves tripeptides from the unsubstituted amino terminus of peptides and proteins. Endogenously expressed TPP1 is synthesized as a non-catalytically active enzyme. Upon targeting of the lysosome, TPP1 autocatalytically processes into a mature, active enzyme due to the acidic environment. TPP1 activity can be measured and / or quantified in vitro using known methods. See, for example, Junaid et al., 1999.

[0048] Human TPP1 is 61 kDa in size and consists of 563 amino acids. A 34.5 kDa isoform 1 with 320 amino acids is generated through selective splicing, with the peptide fragment lacking amino acids 1 through 243. TPP1 contains a globular structure with a subtilisin-like folding Ser 475-Glu 272-Asp 360 catalytic triplet. It also contains octahedral coordinated Ca2+. + The binding site is a characteristic feature of the S53 sedolisin peptidase family. Unlike other S53 peptidases, it has a sterically restricted P4 substrate pocket, which may help it preferentially cleave tripeptides from the unsubstituted N-terminus of proteins. Two alternative conformations of catalytic Asp276 are associated with the activation state of TPP1.

[0049] High expression of TPP1 was found in bone marrow, placenta, lung, pineal gland, and lymphocytes. This protease functions in lysosomes to cleave N-terminal tripeptides from substrates and exhibits weak endopeptidase activity. It is synthesized as a non-catalytically active enzyme, activated upon acidification, and undergoes autoproteolytic cleavage.

[0050] Neuronal ceroid lipofuscinosis (NCL) is a group of inherited neurodegenerative diseases characterized by the presence of autofluorescent lipopigments in neurons and other cell types. Over the past two decades, mounting evidence suggests that NCL is caused by mutations in eight different genes, including those encoding several soluble proteins (cathepsin D, PPT1, and TPP1). Mutations in the TPP1 gene lead to late-onset infantile neuronal ceroid lipofuscinosis, a disease associated with the inability to degrade specific neuropeptides and ATP synthase subunits in lysosomes. Mutations in the TPP1 gene result in late-onset infantile neuronal ceroid lipofuscinosis, a fatal childhood neurodegenerative disease. It has been demonstrated that a single intravitreal injection of autologous bone marrow-derived stem cells transduced with a TPP1-expressing construct significantly inhibits disease-related retinal functional defects and structural changes in the early stages of disease progression. These results suggest that ex vivo gene therapy using autologous stem cells could be an effective means of achieving sustained delivery of therapeutic compounds to tissues such as the retina, for which systemic administration would be ineffective.

[0051] In some implementations, TPP1 can be controlled by a promoter (such as the CAG promoter). The CAG promoter is a strongly synthetic promoter, commonly used to drive high levels of gene expression in mammalian expression vectors. The CAG promoter is constructed from the following sequence:

[0052] (C) Early enhancer element of cytomegalovirus (CMV)

[0053] (A) The promoter, first exon, and first intron of the chicken β-actin gene.

[0054] (G) Splice acceptor of rabbit β-globin gene.

[0055] The resulting synthetic elements were used in the pCAGGS expression vector. The start codon located in the proximal region of the second exon was disrupted by digestion with the NcoI restriction endonuclease and replacement of the site with a HindIII linker. Although the entire construct is commonly referred to as the “CAG promoter,” it comprises a portion of the transcriptional sequence (the first exon and first intron of the chicken β-actin gene) and enhancer elements. In addition to the CMV immediate early enhancer, the introns of the chicken β-actin gene also contain enhancer elements that are highly conserved in vertebrates. The 3' portion of this promoter has a high GC content and is therefore difficult to amplify by PCR.

[0056] II. Adeno-associated virus (AAV) vector

[0057] Adeno-associated virus (AAV) is a small, non-pathogenic virus belonging to the parvoviridae family. To date, many serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAVs differ from other members of this family in that they rely on helper viruses for replication.

[0058] The AAV genome can exist in an extrachromosomal state without integrating into the host cell genome; it has a broad host range; it can transduce both dividing and non-dividing cells in vitro and in vivo, maintaining high levels of transduced gene expression. AAV viral particles are thermostable; resistant to solvents, detergents, pH changes, and temperature; and can be column purified and / or concentrated using CsCl gradients or other methods. The AAV genome contains positive or negative sense single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7 kb AAV genome consists of a segment of positive or negative sense single-stranded DNA. The genome ends with short inverted terminal repeats (ITRs), which fold into hairpin structures and serve as the starting point for viral DNA replication.

[0059] The AAV “genome” refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form AAV particles. AAV particles typically contain the AAV genome packaged with AAV capsid proteins. In the case of constructing or preparing recombinant vectors using recombinant plasmids, 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 the “plasmid backbone,” which is important for plasmid cloning and amplification (the processes required for plasmid proliferation and production), but it is not itself packaged or encapsulated within the viral particle. Therefore, the AAV vector “genome” refers to the nucleic acid packaged or encapsulated by AAV capsid proteins.

[0060] AAV virions (particles) are non-enveloped icosahedral particles approximately 25 nm in diameter containing an AAV capsid. AAV particles exhibit icosahedral symmetry and are composed of three associated capsid proteins, VP1, VP2, and VP3, which interact to form the capsid. The genome of most native AAVs typically contains two open reading frames (ORFs), sometimes referred to as the left and right ORFs. The right ORF usually encodes the capsid proteins VP1, VP2, and VP3. These proteins are typically present in a 1:1:10 ratio, but can be present in different ratios and are all derived from the right ORF. The VP1, VP2, and VP3 capsid proteins are distinguished from each other through alternative splicing and unique start codons. Deletion analysis has shown that removal or alteration of VP1 translated from information from alternative splicing results in reduced yield of infectious particles. Mutations within the VP3 coding region result in the inability to produce any single-stranded daughter DNA or infectious particles. In some embodiments, the genome of the AAV particle encodes one, two, or all three VP1, VP2, and VP3 polypeptides.

[0061] The left ORF typically encodes non-structural 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 associated with the preferential integration of the AAV genome into the q-arm of human chromosome 19. Rep68 and Rep78 have been shown to possess NTP-binding activity as well as DNA and RNA helicase activity. Some Rep proteins possess nuclear localization signals and several potential phosphorylation sites. In some embodiments, the AAV (e.g., rAAV) genome encodes some or all of the Rep proteins. In some embodiments, the AAV (e.g., rAAV) genome does not encode Rep proteins. In some embodiments, one or more of the Rep proteins may be delivered trans- and therefore not included in the AAV particles containing nucleic acids encoding polypeptides.

[0062] The AAV genome contains short inverted terminal repeats (ITRs) at its ends, which have the potential to fold into T-hairpin structures that serve as the starting point for viral DNA replication. Therefore, the AAV genome contains one or more ITR sequences (e.g., ITR sequence pairs) located on the flanks of the single-stranded viral DNA genome. The length of each ITR sequence is typically about 145 bases. Within the ITR region, two elements considered central to ITR function have been described: the GAGC repeat motif and the terminal resolution site (trs). When the ITR is in a linear or hairpin conformation, the repeat motif exhibits binding of Rep. This binding is thought to cleave Rep68 / 78 at the trs, occurring in a site-specific and strand-specific manner. In addition to their role in replication, these two elements have also shown to be central to viral integration. Rep binding sites with adjacent trs are contained within the integration locus on chromosome 19. These elements have been shown to be functional and essential for locus-specific integration.

[0063] The term “recombinant” as a modifier for vectors (e.g., recombinant viral vectors, such as lentiviruses or parvoviruses (e.g., AAV) vectors) and sequences (e.g., recombinant nucleic acid sequences and polypeptides) means that the composition has been manipulated (i.e., modified) in a way that would not normally occur in nature. A specific example of a recombinant vector (e.g., AAV, retroviral, or lentiviral vector) is the insertion of a nucleic acid sequence that is not normally present in the wild-type viral genome into the viral genome. An example of a recombinant nucleic acid sequence is a nucleic acid (e.g., a gene) encoding a repressive RNA cloned into a vector, which may or may not have the 5', 3', and / or intron regions that the gene would normally be associated with within the viral genome. While the term “recombinant” is not always used herein to refer to vectors (e.g., viral vectors) and sequences (e.g., polynucleotides), the term “recombinant” encompasses nucleic acid sequences, polynucleotides, transgenes, etc., without any such omissions.

[0064] 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 and replace it with a non-natural nucleic acid (e.g., a nucleic acid sequence). Typically, for example, in the case of AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are preserved in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) differs from a viral (e.g., AAV) genome because a portion of the viral genome has been replaced with a non-natural sequence targeting the viral genome nucleic acid (e.g., a nucleic acid encoding a transactivator, a repressive RNA, or a therapeutic protein). Therefore, the incorporation of such a non-natural nucleic acid sequence defines a viral vector as a "recombinant" vector, which in the case of AAV may be referred to as an "rAAV vector."

[0065] In some embodiments, the AAV (e.g., rAAV) comprises two ITRs. In some embodiments, the AAV (e.g., rAAV) comprises an ITR pair. In some embodiments, the AAV (e.g., rAAV) comprises an ITR pair located on the flanks (i.e., at each 5' and 3' end) of a nucleic acid sequence that at least encodes a functional or active polypeptide.

[0066] AAV vectors (e.g., rAAV vectors) may be packaged and referred to herein as “AAV particles” for subsequent infection (transduction) of cells in vitro, in vitro, or in vivo. Where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle may also be referred to as an “rAAV particle.” In some embodiments, the AAV particle is an rAAV particle. An rAAV particle typically contains an rAAV vector or a portion thereof. There may be one or more rAAV particles (e.g., multiple AAV particles). An rAAV particle typically contains proteins (e.g., capsid proteins) that encapsulate or package the genome of the rAAV vector. It should be noted that references to rAAV vectors may also be used to refer to rAAV particles.

[0067] Any suitable AAV particle (e.g., rAAV particle) may be used in the methods or uses described herein. The rAAV particle and / or the genome contained therein may be derived from any suitable AAV serotype or strain. The rAAV particle and / or the genome contained therein may be derived from two or more AAV serotypes or strains. Thus, rAAV may contain proteins and / or nucleic acids or portions thereof from any AAV serotype or strain, wherein the AAV particle is suitable for infection and / or transduction in 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.

[0068] In some embodiments, multiple rAAV particles comprise particles of the same strain or serotype (or subgroup or variant), or are derived from the same strain or serotype (or subgroup or variant). In some embodiments, multiple rAAV particles comprise a mixture of two or more different rAAV particles (e.g., different serotypes and / or strains).

[0069] As used herein, the term "serotype" refers to a distinguishing manner of AAVs having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies against one AAV and antibodies against another. Such differences in cross-reactivity 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 AAV serotypes). Although it is possible for AAV variants (including capsid variants) to be serologically indistinguishable from reference AAVs or other AAV serotypes, they differ from reference AAVs or other AAV serotypes by at least one nucleotide or amino acid residue.

[0070] In some embodiments, the rAAV vector based on a first serotype genome corresponds to a serotype of one or more capsid proteins that package the vector. For example, a serotype containing one or more AAV nucleic acids (e.g., ITRs) of the AAV vector genome corresponds to a serotype containing a capsid of rAAV particles.

[0071] In some embodiments, the rAAV vector genome may be based on an AAV (e.g., AAV2) serotype genome that is different from the serotype of one or more AAV capsid proteins that package the vector. For example, the rAAV vector genome may contain nucleic acids derived from AAV1 (e.g., ITR), while at least one or more of the three capsid proteins are derived from different serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotypes or variants thereof.

[0072] In some embodiments, the rAAV particle or its vector genome associated with the reference serotype has a polynucleotide, polypeptide, or subsequence comprising or composed of the following sequences: having at least 60% or higher (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identity with the polynucleotide, polypeptide, or subsequence of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle. In some specific embodiments, the rAAV particle or its vector genome associated with the reference serotype has a capsid or ITR sequence comprising or composed of the following sequences: at least 60% or higher (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identity with the capsid or ITR sequence of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0073] In some embodiments, the methods described herein include using, applying, or delivering rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0074] In some embodiments, the methods described herein include using, applying, or delivering rAAV1 particles. In some embodiments, the rAAV1 particles comprise an AAV1 capsid. In some embodiments, the rAAV1 particles comprise one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that have at least 60%, 65%, 70%, 75%, or higher identity with 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% identity. In some embodiments, the rAAV2 particle contains VP1, VP2, and VP3 capsid proteins that have at least 75% or higher identity with the corresponding capsid proteins of the natural 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.

[0075] In some embodiments, rAAV2 particles are variants of natural or wild-type AAV1 particles. In some aspects, one or more capsid proteins of the 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 natural or wild-type AAV1 particles.

[0076] In some embodiments, the rAAV particle contains one or two ITRs (e.g., an ITR pair) that have at least 75% or higher identity with the corresponding ITRs of natural or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, or AAV-rh10, for example, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity, as long as it retains one or more desired ITR functions (e.g., the ability to form hairpins, which allows DNA replication; the ability to transport AAV particles to the wild). DNA integration into the host cell genome; and / or packaging, if desired.

[0077] In some embodiments, the rAAV1 particle contains one or two ITRs (e.g., ITR pairs) that have at least 75% or higher identity with the corresponding ITRs of the natural or wild-type AAV1 particle, 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% identity, provided that it retains one or more desired ITR functions (e.g., the ability to form hairpins, which allows DNA replication; integration of AAV DNA into the host cell genome; and / or packaging, if desired).

[0078] The rAAV particle may contain an ITR with any suitable number of "GAGC" repeats. In some embodiments, the ITR of the AAV2 particle contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In some embodiments, the rAAV2 particle contains an ITR containing three "GAGC" repeats. In some embodiments, the rAAV2 particle contains an ITR with fewer than four "GAGC" repeats. In some embodiments, the rAAV2 particle contains an ITR with more than four "GAGC" repeats. In some embodiments, the ITR of the rAAV2 particle contains a Rep binding site, wherein the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0079] 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 specific embodiments, the DNA length 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.

[0080] rAAV vectors containing nucleic acid sequences that direct the expression of RNAi or peptides can be generated using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and propagated using an AAV viral packaging system. Transducible AAV particles are able to bind to and enter mammalian cells, subsequently delivering nucleic acid payloads (e.g., heterologous genes) to the cell nucleus. Thus, intact rAAV particles with transducible capabilities are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells typically lack replication capacity and require additional protein mechanisms for self-replication. Therefore, rAAV particles configured to transduce mammalian cells are modified to bind to and enter mammalian cells and deliver nucleic acids to the cells, wherein the nucleic acids for delivery are typically located between AAV ITR pairs in the rAAV genome.

[0081] Suitable host cells for generating transducible AAV particles include, but are not limited to, microbial, yeast, insect, and mammalian cells that can or have been used as acceptors of heterologous rAAV vectors. Cells from a stable human cell line, HEK293 (easily obtained through, for example, the American Type Culture Collection, accession number ATCC CRL1573), can be used. In some embodiments, modified human embryonic kidney cell lines (e.g., HEK293) transformed with adenovirus type 5 DNA fragments and expressing the adenovirus Ela and Elb genes are used to generate recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform for generating rAAV particles therein. Methods for generating high-titer AAV particles capable of transducing mammalian cells are known in the art.

[0082] In some implementations, AAV helper functions are introduced into host cells by transfecting them with an AAV helper construct before or simultaneously with transfection of the AAV expression vector. Therefore, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the missing AAV function necessary for productive AAV transduction. AAV helper constructs typically lack AAV ITRs and cannot replicate or package themselves. These constructs can be in the form of plasmids, phages, transposons, granules, viruses, or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and PIM29+45, which encode both the Rep and Cap expression products. Many other vectors encoding the Rep and / or Cap expression products are known.

[0083] III. TPP1 as a therapeutic agent

[0084] In some embodiments, viral gene transfer methods may be used to introduce nucleic acids into mammalian cells. Such methods can be used to administer nucleic acids encoding therapeutic proteins to cells in culture or within a host organism. In some embodiments, the therapeutic protein may be used to treat late-onset infantile neuronal cerebrolipofuscin deposition type 2 (CLN2) disease / Barten disease. Some embodiments may involve the expression of peptides containing tripeptidyl peptidase 1 (TPP1) activity.

[0085] A polypeptide containing TPP1 activity refers to a mammalian TPP1 protein or a portion thereof, said TPP1 protein or a portion thereof exhibiting 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, as determined using a suitable peptide substrate, for example, by the method of Junaid et al., 1999, or another comparable method. In some embodiments, a polypeptide containing TPP1 activity refers to a mammalian TPP1 protein or its subsequences or variants, said TPP1 protein or its subsequences or variants exhibiting 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.

[0086] The polypeptide containing TPP1 activity may comprise a truncated, mutated, chimeric, or modified form of TPP1 polypeptide that retains at least a portion of TPP1 activity. The polypeptide containing TPP1 activity may comprise a portion of TPP1 protein derived from any suitable organism (e.g., derived from mammals, derived from humans, derived from non-human mammals, such as dogs, pigs, cattle, etc.). In some embodiments, the polypeptide containing TPP1 activity has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with TPP1.

[0087] In some embodiments, the AAV particle comprises an AAV capsid protein and a nucleic acid encoding a polypeptide containing TPP1 activity. In some embodiments, the AAV particle comprises an AAV capsid protein and a nucleic acid directing the expression and / or secretion of a polypeptide containing TPP1 activity. In some embodiments, the AAV particle comprises an AAV capsid protein and a nucleic acid encoding a TPP1 polypeptide or its enzymatically active portion. In some embodiments, the AAV particle comprises an AAV capsid protein and a nucleic acid directing the expression and / or secretion of a TPP1 polypeptide or its enzymatically active portion. In some embodiments, the applied nucleic acid encodes TPP1, TPP1 with significant identity to wild-type TPP1, and / or variants, mutants, or fragments of TPP1.

[0088] Recombinant TTP1 peptides may have amino acid deletions and / or substitutions; therefore, proteins with deletions, proteins with substitutions, and proteins with both deletions and substitutions are modified proteins. In some embodiments, these proteins may also include intercalations or added amino acids, such as proteins with fusion proteins or proteins with linkers. A “modified deletion protein” lacks one or more residues of the native protein but may have the specificity and / or activity of the native protein.

[0089] Substitution or alternative variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be engineered to regulate one or more properties of the polypeptide, particularly its effector function and / or bioavailability. Substitutions can be conserved or non-conserved, meaning that one amino acid is replaced by an amino acid of similar shape and charge. Conserved 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 10 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. In addition to deletions or substitutions, modified proteins may have residue insertions, which typically involve adding at least one residue to a polypeptide. This can include inserting a targeted peptide or polypeptide or simply a single residue.

[0090] The term "biologically equivalent" is well known in the art and is further defined in detail herein. Therefore, it includes sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to those of a control polypeptide, provided that the protein's biological activity is maintained. In some respects, recombinant proteins may be biologically equivalent to their natural counterparts.

[0091] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, and as long as the sequence meets the criteria set forth above (including maintaining biological protein activity in the case of protein expression), it remains essentially as shown in the sequence disclosed herein. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, contain multiple non-coding sequences flanking the 5' or 3' portion of the coding region, or may contain multiple internal sequences known to exist within genes, i.e., introns.

[0092] IV. Application Method

[0093] In some respects, viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro or ex vivo and subsequently administered to patients. The term "vector" refers to a small vector nucleic acid molecule, plasmid, virus (e.g., AAV vector), or other carrier that can be manipulated by insertion into or incorporation of nucleic acids. Vectors (e.g., viral vectors) can be used to introduce / transfer nucleic acid sequences into / from cells, whereby the nucleic acid sequences are transcribed and, if encoding proteins, subsequently translated by the cells.

[0094] Any suitable cell or mammal may be administered or treated by the methods or uses described herein. Generally, mammals requiring the methods described herein are suspected of having or expressing abnormal or anomalous proteins associated with a disease state. Alternatively, the mammalian recipient may have a condition suitable for gene replacement therapy. As used herein, “gene replacement therapy” refers to the administration of exogenous genetic material encoding a therapeutic agent to the recipient, followed by in situ expression of the administered genetic material. Therefore, the phrase “condition suitable for gene replacement therapy” covers conditions such as hereditary diseases (i.e., disease conditions attributable to one or more gene defects), acquired conditions (i.e., pathological conditions not attributable to congenital defects), cancer, and preventative processes (i.e., prevention of disease or undesirable medical conditions). Therefore, as used herein, the term “therapeutic agent” refers to any agent or substance that has a beneficial effect on the mammalian recipient. Thus, “therapeutic agent” encompasses both therapeutic and preventative molecules having nucleic acid or protein components.

[0095] Some non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domesticated animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, etc.). In some embodiments, the non-rodent mammal is a human. A mammal can be of any age or at any developmental stage (e.g., an adult, adolescent, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a mammal can be an animal disease model, for example, an animal model having or expressing abnormal or anomalous proteins associated with a disease state, or an animal model that inadequately expresses proteins leading to a disease state.

[0096] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years of age or older) and children (under 18 years of age). Adults include the elderly. A representative adult is 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.

[0097] In some embodiments, the method includes administering multiple viral particles to a mammal as illustrated herein, wherein the severity, frequency, progression, or onset time of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, alleviated, prevented, suppressed, or delayed. In some embodiments, the method includes administering multiple viral particles to a mammal to treat unpleasant symptoms of a disease state (e.g., a neurodegenerative disease). In some embodiments, the method includes administering multiple viral particles to a mammal to stabilize, delay, or prevent the worsening or progression of a disease state (e.g., a neurodegenerative disease), or to reverse unpleasant symptoms of a disease state (e.g., a neurodegenerative disease).

[0098] In some embodiments, the method includes administering multiple viral particles to the central nervous system or a portion thereof of a mammal, as illustrated herein, and reducing, alleviating, preventing, suppressing, or delaying the severity, frequency, progression, or onset time of one or more symptoms of a disease state (e.g., a neurodegenerative disease) 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.

[0099] In some implementations, symptoms or adverse effects include early, intermediate, or late symptoms; behavioral, personality, or language symptoms; swallowing, motor, seizure, tremor, or restlessness symptoms; ataxia; and / or cognitive symptoms such as memory or organizational abilities.

[0100] In some embodiments, the method includes administering or delivering AAV-TPP1 particles to a mammal and administering one or more immunosuppressants to the mammal. In some embodiments, the method includes administering or delivering AAV-TPP1 particles to a mammal and administering two, three, four, or more immunosuppressants to the mammal. In some embodiments, the method includes administering or delivering AAV-TPP1 particles to a mammal and administering two immunosuppressants to the mammal. In a representative embodiment, a method of treating a mammal includes administering or delivering AAV-TPP1 particles to the mammal and administering first and second immunosuppressants to the mammal.

[0101] In cases where two or more immunosuppressants are administered, 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 some embodiments, the immunosuppressant is an anti-inflammatory agent. In some embodiments, the immunosuppressant is a mycophenolate ester or a derivative thereof. An example of such a mycophenolate ester derivative is mycophenolate mofetil (MMF). In some embodiments, the immunosuppressant is cyclosporine or a derivative thereof. In some embodiments, the first immunosuppressant comprises cyclosporine, and the second immunosuppressant comprises a mycophenolate ester or a derivative thereof (e.g., MMF). In some embodiments, the first immunosuppressant comprises cyclosporine, and the second immunosuppressant comprises MMF.

[0102] In some embodiments, an immunosuppressant is administered before, during, and / or after administration of AAV-TPP1 particles to a mammal. In some embodiments, an immunosuppressant is administered concurrently with administration of AAV-TPP1 particles to a mammal. In some embodiments, an immunosuppressant is administered after administration of AAV-TPP1 particles to a mammal. In some embodiments, an immunosuppressant is administered approximately 1 to approximately 60 minutes after administration of AAV-TPP1 particles to a mammal, approximately 1 to approximately 24 hours after administration, approximately 1 to approximately 100 days after administration, approximately 1 to approximately 12 months after administration, or approximately 1 to approximately 5 years after administration. In some embodiments, an immunosuppressant is administered before administration of AAV-TPP1 particles to a mammal. In some embodiments, an immunosuppressant is administered approximately 1 to approximately 60 minutes before administration, approximately 1 to approximately 24 hours before administration, approximately 1 to approximately 100 days before administration, or approximately 1 to approximately 3 months before administration of AAV-TPP1 particles to a mammal. In some embodiments, an immunosuppressant is administered approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days prior to administration of AAV-TPP1 particles to the mammal. In some embodiments, an immunosuppressant is administered at predetermined intervals (e.g., once daily, twice daily, three times daily, every other day, once weekly, once every two weeks, once every two months, or combinations thereof) before, during, and / or after administration of AAV-TPP1 particles to the mammal.

[0103] In some implementations, a first immunosuppressant is administered to the mammal at least 1 to 7 days before administration of AAV-TPP1 particles, or about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to administration of AAV-TPP1 particles; and a second immunosuppressant is administered to the mammal during administration of AAV-TPP1 particles and / or about 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 200 days, 300 days, 350 days, 400 days, or 500 days after administration of AAV-TPP1 particles. In some embodiments, cyclosporine is administered to mammals at least 1 to 7 days before, or about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to, administration of AAV-TPP1 particles to mammals; and mycophenolate mofetil or its derivatives (e.g., MMF) are administered to mammals during and / or about 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 200 days, 300 days, 350 days, 400 days, or 500 days after, administration of AAV-TPP1 particles to mammals. In some embodiments, cyclosporine is administered at fixed intervals approximately 1 to 7 days before, or approximately 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to, and after treatment with AAV-TPP1 particles; and mycophenolate mofetil or a derivative thereof (e.g., MMF) is administered once approximately 1 to 7 days before, or approximately 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to, during, and / or approximately 10 to 40 days after, the administration of AAV-TPP1 particles to mammals.

[0104] Immunosuppressants can be administered at any suitable dose. In some embodiments, cyclosporine is administered once, twice, or three times daily, or 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 some embodiments, cyclosporine is administered twice daily at about 10 mg / kg. In some embodiments, cyclosporine is administered twice daily at about 10 mg / kg for a period of at least about 1 month, about 2 months, about 3 months, about 4 months, or about 5 months. In some embodiments, after about 1 to 2 months following administration of AAV-TPP1 particles to a mammal, the dose of cyclosporine is gradually reduced to less than about 5 mg / kg or less than about 2 mg / kg.

[0105] In some embodiments, mycophenolate mofetil or its derivatives (e.g., MMF) are administered once, twice, or three times daily to 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 some embodiments, mycophenolate mofetil or its derivatives (e.g., MMF) are administered once daily at a dose of about 10 to about 20 mg / kg. In some embodiments, about 1 to 2 months after administration of AAV-TPP1 particles to a mammal, the dose of mycophenolate mofetil or its derivatives (e.g., MMF) is reduced to a dose of less than about 5 mg / kg or less than about 2 mg / kg. Immunosuppressants can be formulated into any suitable formulation appropriate for a particular route of administration. A variety of pharmaceutically acceptable formulations of immunosuppressants are commercially available and readily accessible to medical practitioners.

[0106] Immunosuppressants can be administered via any suitable route. In some embodiments, immunosuppressants are administered orally. In some embodiments, mycophenolate mofetil or its derivatives (e.g., mycophenolate mofetil (MMF)) are administered orally. In some embodiments, cyclosporine is administered orally. Immunosuppressants can 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).

[0107] In some 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 with LSD). In some embodiments, the central nervous system includes the brain, spinal cord, and cerebrospinal fluid (CSF). In some embodiments, the method includes administering one or more AAV-TPP1 particles to the brain or spinal cord or CSF of a mammal. In some embodiments, the AAV-TPP1 particles are administered to a portion of the brain or spinal cord. In some 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, the AAV-TPP1 particles may be delivered directly to the cerebellomedullary cistern, intraventricular space, ventricles, subarachnoid space, intrathecal space, or ependyma. In some embodiments, the method includes administering one or more AAV-TPP1 particles to the ependyma of a mammal.

[0108] In some embodiments, AAV-TPP1 particles are applied to one or more types of cells in mammals that are exposed to CSF, for example, by contacting the cells with the AAV-TPP1 particles. Some non-limiting examples of cells exposed to CSF ​​include ependymal cells, piacular cells, endothelial cells, and / or meningeal cells. In some embodiments, AAV-TPP1 particles are applied to ependymal cells. In some embodiments, AAV-TPP1 particles are delivered to ependymal cells, for example, by contacting the ependymal cells with the AAV-TPP1 particles.

[0109] In some embodiments, AAV-TPP1 particles are delivered locally. "Local delivery" means delivering the active agent directly to a target site within a mammal (e.g., directly to a tissue or fluid). For example, the agent can be delivered locally to an organ, tissue, or specific anatomical location by direct injection. In some embodiments, one or more AAV-TPP1 particles are delivered or administered to the brain, spinal cord, or their tissues or fluids (e.g., CSF, such as ependymal cells, pia mater cells, endothelial cells, and / or meningeal cells) by direct injection. For example, AAV-TPP1 particles can be delivered directly to the CSF, cerebellomedullary cistern, intraventricular space, ventricles, subarachnoid space, and / or ependyma by direct injection. In some embodiments, AAV-TPP1 particles are brought into contact with the tissues, fluids, or cells of the brain or spinal cord by direct injection into the tissues or fluids of the brain or spinal cord. In some embodiments, AAV-TPP1 particles are not delivered systemically by, for example, intravenous, subcutaneous, or intramuscular injection or intravenous infusion. In some implementations, AAV-TPP1 particles are delivered to tissues or fluids of the brain or spinal cord via stereotactic injection.

[0110] In some embodiments, one or more AAV-TPP1 particles are delivered or administered by directly injecting AAV-TPP1 particles into the brain, spinal cord, or their tissues or fluids (e.g., CSF such as the ependyma). In one specific aspect, AAV-TPP particles transduce ependymal cells, pia mater cells, endothelial cells, and / or meningeal cells.

[0111] Given the teachings herein, such as the dosage ranges provided, it will be apparent to those skilled in the art that the effective amount of AAV-TPP1 particles can be determined empirically. Administration may be continuous or intermittent at a single dose throughout the treatment process. The effective dose administered can be determined by those skilled in the art and may vary depending on the AAV serotype, viral titer, and the weight, condition, and species of the mammal being treated. Single and multiple administrations may be performed at dose levels, targets, and times selected by the treating physician.

[0112] In some implementations, multiple AAV-TPP1 particles are applied. Multiple AAV particles, as used herein, refer to approximately 1 × 10⁻⁶ particles. 5 To approximately 1×10 8 Each particle.

[0113] In some embodiments, AAV-TPP1 particles are distributed in a concentration of about 1 × 10⁻⁶ in about 1 to about 5 ml. 5 To approximately 1×10 16 Dosage: approximately 1 to approximately 3 ml of 1×10⁻⁶ vg / ml. 7 To approximately 1×10 14 A dose of vg / ml; or approximately 1 to approximately 2 ml of 1×10 8 To approximately 1×10 13 Administered at a dose of vg / ml. In some embodiments, AAV-TPP1 particles are administered at approximately 1 × 10⁻⁶. 8 To approximately 1×10 15 Vg / kg is the dose administered to the body weight of the mammal being treated. For example, AAV-TPP1 granules can be administered at a dose of approximately 1 × 10⁻⁶. 8 vg / kg, approximately 5×10 8 vg / kg, approximately 1×10 9 vg / kg, approximately 5×10 9 vg / kg, approximately 1×10 10 vg / kg, approximately 5×10 10 vg / kg, approximately 1×10 11 vg / kg, approximately 5×10 11 vg / kg, approximately 1×10 12 vg / kg, approximately 5×10 12 vg / kg, approximately 1×10 13 vg / kg, approximately 5×10 13 vg / kg, approximately 1×10 14 vg / kg, approximately 5×10 14 vg / kg or approximately 1×10 15 vg / kg is the weight of the treated mammal.

[0114] AAV-TPP1 particles can be administered in one or more doses. For example, multiple doses can be administered as needed to maintain sufficient enzyme activity.

[0115] V. Pharmaceutical Composition

[0116] As used herein, the terms "medicinal" and "physiologically acceptable" mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A "medicinal" or "physiologically acceptable" composition is not a biologically or otherwise undesirable substance; for example, the substance can be administered to a subject without causing significant undesirable biological effects. Such compositions, "medicinal" and "physiologically acceptable" formulations and compositions may be sterile. Such pharmaceutical formulations and compositions can be used, for example, to administer viral particles to a subject.

[0117] 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, isotonics, and absorption enhancers or absorption delayers that are compatible with drug administration, in vivo contact, or in vivo delivery.

[0118] Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickeners. Complementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into formulations and compositions.

[0119] Pharmaceutical compositions typically contain pharmaceutically acceptable excipients. Such excipients include any agent that does not induce 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 contained therein, such as inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Additionally, such carriers may contain auxiliary substances such as surfactants, wetting agents or emulsifiers, pH buffers, etc.

[0120] Pharmaceutical compositions may be formulated to be compatible with specific routes of administration or delivery as shown herein or known to those skilled in the art. Therefore, pharmaceutical compositions may contain carriers, diluents, or excipients suitable for administration or delivery via multiple routes.

[0121] Suitable pharmaceutical forms for injection or infusion of AAV-TPP1 particles may include sterile aqueous solutions or dispersions suitable for the ad hoc preparation of sterile injectable or infusionable solutions or dispersions optionally encapsulated in liposomes. In all cases, the final form should be a sterile fluid and stable under the conditions of preparation, use, and storage. The liquid carrier or loading agent may be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by forming liposomes, by maintaining the desired particle size in the case of dispersions, or by using surfactants. Isotonic agents may be included, such as sugars, buffers, or salts (e.g., sodium chloride). Prolonged absorption of the injectable composition may be achieved by using a delayed absorption agent, such as aluminum monostearate and gelatin, in the composition.

[0122] The solution or suspension of AAV-TPP1 particles may optionally contain the following components: sterile diluents such as water for injection, saline solutions (e.g., phosphate buffered saline, PBS), artificial CSF, non-volatile oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), 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 regulating tension such as sodium chloride or dextrose.

[0123] Pharmaceutical formulations, compositions, and delivery systems suitable for the compositions, methods, and uses described herein are known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2003) 20th edition, Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th edition, Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th edition, Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklasa, Pharmaceutical Calculations (2001) 11th edition, Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., 2004).

[0124] To facilitate application and ensure 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 the individual to be treated; each unit contains a predetermined amount of active compound calculated to associate with the desired drug carrier to produce the desired therapeutic effect. The dosage unit form depends on the amount of AAV-TPP1 particles necessary to produce the desired effect.

[0125] The required dosage can be formulated as a single dose or in multiple dose units. The dosage can be adjusted to the appropriate AAV-TPP1 particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0126] In one embodiment, the pharmaceutical composition will contain sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to alleviate or improve the symptoms of the disease state in question, or an amount sufficient to impart the desired benefit. The pharmaceutical composition typically contains pharmaceutically acceptable excipients. Such excipients include any agent that does not induce 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 contained therein, such as inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Additionally, such carriers may contain excipients such as wetting agents or emulsifiers, pH buffers, etc.

[0127] 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 drug carrier (excipient, diluent, load, or filler) and, when administered in one or more doses, is calculated to produce the desired effect (e.g., preventative or therapeutic effect). Unit dosage forms may be in, for example, ampoules and vials, which may contain a liquid composition or a composition in a lyophilized or freeze-dried state; for example, a sterile liquid carrier may be added prior to in vivo administration or delivery.

[0128] Single-unit dosage forms can be contained in multi-dose kits or containers. Therefore, for ease of administration and dosage uniformity, viral particles and their pharmaceutical compositions can be packaged as single or multiple unit dosage forms.

[0129] Formulations containing AAV-TPP1 particles will contain an effective amount of rAAV particles in the carrier, which can be readily determined by those skilled in the art. The AAV-TPP1 particles are typically 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 mammalian or human subject to be treated. The effective dose can be determined by those skilled in the art through routine experiments to establish dose-response curves.

[0130] VI. Other vector sequences

[0131] A “promoter” is a nucleotide sequence typically located upstream (5') of a coding sequence that directs and / or controls the expression of the coding sequence by providing recognition of RNA polymerase and other factors required for proper transcription. In some embodiments, the promoter comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity with the nucleic acid shown in SEQ ID NO: 8.

[0132] An enhancer is a DNA sequence that can stimulate transcriptional activity and can be an intrinsic element of a promoter or a heterologous element that enhances expression at a level or tissue specific. It can operate in either direction (5'->3' or 3'->5') and can function even if it is located upstream or downstream of a promoter.

[0133] Promoters and / or enhancers may be derived entirely from natural genes, or consist of different elements derived from different elements found in nature, or even contain synthetic DNA segments. Promoters or enhancers may contain DNA sequences involved in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological, or developmental conditions.

[0134] As used herein, “transgenic” conveniently refers to a nucleic acid sequence / polynucleotide intended or introduced into a cell or organism. Transgenics include any nucleic acid, such as a gene encoding a repressive RNA polypeptide or protein (e.g., TTP1), and are typically heterologous relative to naturally occurring genomic sequences.

[0135] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism via a vector (e.g., viral particles). Therefore, the introduction of a transgene into a cell via viral particles can be termed "transduction" of the 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 that cell or organism and further passed on to the recipient cell or organism's daughter cells or organisms, or inherited by them. Finally, the introduced transgene may exist outside the chromosomes of the recipient cell or host organism, or only temporarily. Therefore, a "transduced cell" is a cell into which a transgene has been introduced via transduction. Thus, a "transduced" cell is a cell in which a transgene has been introduced, or its daughters. Transduced cells can proliferate, transcribe the transgene, and express the encoded protein. For gene therapy purposes and methods, transduced cells can be used in mammals.

[0136] As used herein, the terms “modification” or “variant” and their grammatical variations refer to nucleic acids, polypeptides, or their subsequences that deviate from a reference sequence. Therefore, modified and variant sequences may have substantially the same expression, activity, or function as the reference sequence, or higher or lower expression, but retain at least some of the activity or function of the reference sequence. A specific type of variant is a mutant protein, which is a gene-encoded protein with a mutation, such as a missense or nonsense mutation.

[0137] A “nucleic acid” or “polynucleotide” variant refers to a modified sequence that has been genetically altered compared to the wild type. The sequence can be genetically modified without changing the protein sequence it encodes. Alternatively, the sequence can be genetically modified to encode a variant protein, such as the variant TPP1 protein. A nucleic acid or polynucleotide variant can also refer to a combinatorial sequence that has been codon-modified to encode a protein that retains at least partial sequence identity with a reference sequence (e.g., the 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 can be changed without changing the amino acids of the TPP1 protein it encodes, and some codons of the nucleic acid variant can be changed, which in turn changes the amino acids of the protein it encodes.

[0138] The terms “protein” and “peptide” are used interchangeably herein. The “peptide” encoded by a “nucleic acid”, “polynucleotide”, or “transgenic” disclosed herein includes partial or full-length natural sequences, such as naturally occurring wild-type and functional polymorphic proteins, their functional subsequences (fragments), and their sequence variants, provided that the peptide (e.g., TPP1) retains a degree of function or activity. Therefore, in the methods and uses of this disclosure, such peptides encoded by nucleic acid sequences need not be identical to endogenous proteins that are defective or have insufficient activity, function, or expression, are absent, or are missing in the mammal being treated.

[0139] 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).

[0140] One example of amino acid modification is conserved amino acid substitution or deletion. In some specific embodiments, the modified or variant sequence retains at least part of the function or activity of the unmodified sequence (e.g., wild-type sequence). Another example of amino acid modification is the introduction of targeting peptides into the capsid proteins of viral particles. Peptides that target recombinant viral vectors to the central nervous system (e.g., different brain regions) have been identified.

[0141] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a natural molecule. For nucleotide sequences, variants include those sequences that encode the same amino acid sequence as the natural protein due to the degeneracy of the genetic code. These naturally occurring allelic variants, for example, can be identified using molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those produced by site-directed mutagenesis (which encode natural proteins), and those encoding polypeptides with amino acid substitutions. Generally, the nucleotide sequence variants of this disclosure will have at least 40%, 50%, 60% to 70%, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% to 79%, typically at least 80%, such as 81% to 84%, and at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% to 98% sequence identity with the natural (endogenous) nucleotide sequence. In some implementations, the variants are biologically functional (i.e., retaining 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).

[0142] The term "significant identity" of a polynucleotide sequence means, using one of the alignment procedures and standard parameters, that the polynucleotide contains 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. Those 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. Significant identity of amino acid sequences for these purposes generally means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

[0143] In the case of peptides, the term "significant identity" means that within a specified comparison window, the peptide contains a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence. The indication that two peptide sequences are identical is that one peptide is immunoreactive with an antibody against the second peptide. Therefore, for example, in the case where the two peptides differ only in conserved substitutions, the peptide is identical to the second peptide.

[0144] The term "treatment" and its variations refer to both therapeutic treatment and preventative or preventative measures aimed at preventing, suppressing, mitigating, or reducing undesirable physiological changes or disorders, such as the onset, progression, or worsening of a disorder. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, attenuation of disease severity, stabilization of symptoms or adverse effects of the disease (i.e., no worsening or progression), delay or slowing of disease progression, improvement or reduction of disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonged survival compared to expected survival without treatment. Those requiring treatment include those already suffering from the condition or disorder and those with a predisposition to the disease (e.g., as determined by genetic testing).

[0145] VII. Medicine Box

[0146] This disclosure provides a pharmaceutical kit having packaging material and containing one or more components. The kit typically includes a label or packaging insert containing a description of the components or instructions for the use of the components in vitro, in vivo, or ex vivo. The kit may contain a collection of such components (e.g., nucleic acids, recombinant vectors, and / or viral particles).

[0147] A medicine box is a physical structure that contains one or more components of the medicine box. The packaging material keeps the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0148] The label or insert may contain information on the clinical pharmacology (including mechanism of action, pharmacokinetics, and pharmacodynamics) of one or more of the components, dosage, and active ingredient. The label or insert may contain information on the manufacturer, batch number, place of manufacture, date of manufacture, and expiration date. The label or insert may contain information on the disease to which the kit components are intended. The label or insert may contain instructions for clinicians or subjects to use one or more of the kit components in a method, use, or treatment regimen. These instructions may include dosage, frequency, or duration, and instructions for carrying out any of the methods, uses, treatment regimens, or prevention or treatment regimens described herein.

[0149] Labels or inserts may contain information about any benefits the components may provide, such as preventative or therapeutic benefits. Labels or inserts may also contain information about potential adverse side effects, complications, or reactions, such as warnings to subjects or clinicians about situations where the particular composition is unsuitable. Adverse side effects or complications may also occur if the subject is taking, will take, or is currently taking one or more other medications incompatible with the composition, or if the subject is experiencing, will take, or is currently undergoing another treatment or regimen incompatible with the composition; therefore, the instructions may contain information about such incompatibilities.

[0150] Labels or inserts include “printed material,” such as paper or cardboard, or individually or affixed to components, pillboxes, or packaging materials (e.g., boxes), or attached to ampoules, tubes, or vials containing pillbox components. Labels or inserts may also include computer-readable media, such as barcode printed labels, disks, optical discs (e.g., CD-ROM or DVD-ROM / RAM, DVD), MP3s, or electronic storage media (e.g., RAM and ROM) or mixtures of these, such as magnetic / optical storage media, flash memory, hybrid media, and memory type cards.

[0151] VIII. Examples

[0152] The following embodiments are included to illustrate preferred embodiments. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the embodiments, and therefore can be considered to constitute preferred modes for their practice. However, those skilled in the art will understand from this disclosure that many changes can be made to some of the specific embodiments disclosed without departing from the spirit and scope of this disclosure and still obtaining the same or similar results.

[0153] Example 1 - Proof-of-concept study of the efficacy of TPP1 delivery via AAV-Ep+ in CLN2- / - mice

[0154] AAV-Ep+ was loaded with TPP1 transgene and administered intracerebral (ICV) in 7-week-old CLN- / - mice at a total dose of 5E+10 vg.

[0155] The study observed TPP1 activity levels in different CNS substructures (including the striatum, cerebellum, and prefrontal cortex), TPP1 activity levels in non-CNS structures (including the heart and spleen), and survival tremor.

[0156] Figure 1 Details of AAV-EP+ enzyme activity in mouse tissues collected 5 weeks after injection. This figure shows TPP1 activity in untreated or unilaterally injected Cln2+ / - and Cln- / - mice treated with 5.0 E + 10 vg AAV-Ep+. Mice injected with AAV-Ep+ showed increased TPP1 activity in different analyzed brain regions compared to the endogenous TPP1 activity of untreated Cln2- / - mice, and also significantly increased relative to normal mice. The heart and spleen of injected Cln2- / - mice, although not the direct site of treatment, showed minimal increases in TPP1 activity levels compared to untreated Cln2- / - mice.

[0157] Figure 2 Cln2 is delivered after AAV-EP+. - / -A graph showing mouse survival. This graph illustrates the percentage of survival over time in Cln2+ / +, Cln2- / -, and Cln2- / - + 5.0E + 10 vg epAAV mice. Overall, the lowest survival rate was observed in Cln2- / - mice (n=10), with a mean of 17.1 weeks and a maximum of 19.7 weeks. Gene therapy treatment with 5.0E+ 10 vg AAV-Ep+ significantly improved the half-life of Cln2- / - (n=10) to a mean of 18.6 weeks, with a maximum of 28.6 weeks. 100% survival was observed in Cln2+ / - mice (n = 9). According to the Mantel-Cox test, .

[0158] Figure 3 This is a graph showing the frequency of tremor after AAV-EP delivery in Cln2 knockout mice. AAV-Ep+ delivery of TPP1 rescued tremor in Cln2 knockout mice to near ClN2+ / + levels. The graph illustrates the assessment of resting tremor in Clnr1+, Cln2-1-, and Cln2-1- + 5.0E + 10 vg epAAV mice. A resting tremor phenotype was detectable in untreated 13-week-old Cln2- / - mice relative to Cln2+ / +, with severity increasing dramatically over time. Cln2- / - mice treated with 5.0E + 10 vg AAV-Ep+ showed a significantly delayed onset of resting tremor until week 16, with a significantly reduced phenotype and slower progression over time.

[0159]

[0160] According to this disclosure, all compositions and methods disclosed and claimed herein can be performed and implemented without excessive experimentation. Although the compositions and methods of this disclosure have been described according to some preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the compositions and methods described herein, as well as the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of this disclosure. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein, while achieving the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0161] In addition to what is shown and described herein, numerous modifications and many other embodiments of this disclosure will be apparent to those skilled in the art from the entirety of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, examples, and guidance suitable for the practice of this disclosure with its various embodiments and equivalents.

[0162] By incorporating references

[0163] Throughout this disclosure, references and citations have been made to other documents (such as patents, patent applications, patent publications, journals, books, papers, online content, etc.). All such documents are incorporated herein by reference in their entirety for all purposes.

Claims

1. A modified adeno-associated virus 1 (AAV1) vector comprising: EP+ capsid protein containing the targeting peptide ERDRTRG (SEQ ID NO: 1), and A nucleic acid molecule containing a modified AAV genome, wherein the modified AAV genome comprises: Tripeptidyl peptidase-1 (TPP1) transgene.

2. The modified AAV vector of claim 1, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

3. The modified AAV vector of claim 1, wherein the targeting peptide is inserted between residues 590 and 600 of the AAV1 capsid protein.

4. The modified AAV vector of claim 1, wherein the flanking sides of the targeting peptide are adapter sequences, wherein the adapter sequences on each side of the targeting peptide are two or three amino acids long.

5. The modified AAV vector of claim 4, wherein the adapter sequence on the N-terminal side of the targeting peptide is SSA, and the adapter sequence on the C-terminal side of the targeting peptide is AS.

6. The modified AAV vector of claim 1, wherein the sequence encoding the TPP1 transgene is operatively linked to a polyadenylation signal.

7. The modified AAV vector of claim 1, wherein the sequence encoding the TPP1 transgene is operatively linked to a promoter and / or enhancer.

8. The modified AAV vector of claim 1, wherein the sequence encoding the TPP1 transgene is operatively linked to the CMV early enhancer / chicken actin (CAG) promoter.

9. The modified AAV vector of claim 1, wherein the EP+ capsid protein comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:

3.

10. The modified AAV vector of claim 1, wherein the EP+ capsid protein comprises the sequence of SEQ ID NO:

3.

11. The modified AAV vector of claim 1, wherein the sequence encoding the TPP1 transgene comprises a sequence having at least 95% identity with the sequence of SEQ ID NO:

5.

12. The modified AAV vector of claim 11, wherein the sequence encoding the TPP1 transgene is flanked by inverted terminal repeats (ITRs).

13. The modified AAV vector of claim 11, wherein the sequence encoding the TPP1 transgene comprises a sequence having at least 95% identity with the sequence of SEQ ID NO:

6.

14. The modified AAV vector of claim 1, wherein the sequence encoding the TPP1 transgene encodes an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:

7.

15. The modified AAV vector of claim 8, wherein the CAG promoter comprises a sequence having at least 95% identity with the sequence of SEQ ID NO:

8.

16. A pharmaceutical composition comprising the AAV carrier of claim 1.

17. A method of treating a subject with CLN2 disease, the method comprising administering a modified AAV1 vector to the subject, the modified AAV1 vector comprising: EP+ capsid protein containing the targeting peptide ERDRTRG (SEQ ID NO: 1), and A nucleic acid molecule containing a modified AAV genome, wherein the modified AAV genome comprises: Tripeptidyl peptidase-1 (TPP1) transgene.

18. The method of claim 17, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

19. The method of claim 17, wherein the targeting peptide is inserted between residues 590 and 600 of the AAV1 capsid protein.

20. The method of claim 17, wherein the flanking sides of the targeting peptide are linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

21. The method of claim 20, wherein the N-terminal linker sequence of the targeting peptide is SSA, and the C-terminal linker sequence of the targeting peptide is AS.

22. The method of claim 17, wherein the sequence encoding the TPP1 transgene is operatively linked to a polyadenylation signal.

23. The method of claim 17, wherein the sequence encoding the TPP1 transgene is operatively linked to a promoter and / or enhancer.

24. The method of claim 17, wherein the sequence encoding the TPP1 transgene is operatively linked to the CAG promoter.

25. The method of claim 17, wherein the EP+ capsid protein comprises an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:

3.

26. The method of claim 17, wherein the EP+ capsid protein comprises the sequence of SEQ ID NO:

3.

27. The method of claim 17, wherein the sequence encoding the TPP1 transgene comprises a sequence having at least 95% identity with the sequence of SEQ ID NO:

5.

28. The method of claim 27, wherein the flanking part of the sequence encoding the TPP1 transgene is an inverted terminal repeat (ITR).

29. The method of claim 27, wherein the sequence encoding the TPP1 transgene comprises a sequence having at least 95% identity with the sequence of SEQ ID NO:

6.

30. The method of claim 27, wherein the sequence encoding the TPP1 transgene encodes an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO:

7.

31. The method of claim 24, wherein the CAG promoter comprises a sequence having at least 95% identity with the sequence of SEQ ID NO:

8.

32. The method of claim 17, wherein the modified AAV1 carrier is administered as a pharmaceutical composition.

33. A plasmid containing a cap gene encoding an EP+ capsid protein, said EP+ capsid protein containing the targeting peptide ERDRTRG (SEQ ID NO: 1).

34. The plasmid of claim 33, further comprising a replication (rep) gene.

35. A host cell containing a cap gene encoding an EP+ capsid protein, wherein the EP+ capsid protein contains the targeting peptide ERDRTRG (SEQ ID NO: 1).

36. The host cell of claim 35, further comprising a nucleic acid molecule containing a modified AAV genome containing a tripeptidyl peptidase 1 (TPP1) transgene.

37. The host cell of claim 35 or 36, further comprising a replication (rep) gene.

38. A method for producing a modified AAV1 vector, the method comprising culturing the host cell of claim 37 under conditions that allow for the production of the modified AAV1 vector.

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