Combined intrathecal and intravenous gene therapy for the treatment of infantile Batten disease

Abstract

A method for treating IBD or an IBD-related disorder in a subject in need thereof is provided, comprising a combination of intrathecal administration of a polynucleotide comprising a CLN1 open reading frame and intravenous administration of the polynucleotide. The polynucleotide comprising the CLN1 open reading frame is a wild-type CLN1 polynucleotide. In another embodiment, the polynucleotide comprising the CLN1 open reading frame comprises a codon-optimized polynucleotide sequence of the polynucleotide, or its complement is codon-optimized for expression in human cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 840,360, filed April 29, 2019, the contents of which are incorporated herein by reference in their entirety and for all purposes.

[0002] Incorporation of sequence listings The contents of the text file submitted electronically herewith are incorporated by reference in its entirety into this specification: A computer-readable copy of the Sequence Listing (File name: ABEO_004_01WO_SeqList_ST25.TXT, Creation date: April 27, 2020, File size: approximately 5.6 kilobytes). [Background technology]

[0003] Infantile neuronal lipofuscinosis (infantile Batten disease or "IBD"), or infantile neuronal ceroid lipofuscinosis (INCL), is an autosomal recessive disorder caused by mutations in the CLN1 gene. The CLN1 gene, located on 1p32, encodes a lysosomal enzyme called palmitoyl protein thioesterase 1 (PPT1). PPT1-deficient cells accumulate autofluorescent storage material and become dysfunctional, leading to neuroinflammation, neuronal loss, and neurodegeneration. Some children with mutations in CLN1 have later onset of symptoms and slower disease progression, which resembles juvenile-onset disease and is more typically associated with mutations in the CLN3 gene. Past treatments include enzyme replacement therapy, gene therapy, and administration of neural stem cells. Summary of the Invention

[0004] Provided herein is a method for treating IBD or an IBD-related disorder in a subject in need thereof, comprising, consisting essentially of, or consisting of, intrathecal administration of a polynucleotide comprising a CLN1 open reading frame followed by intravenous administration of the polynucleotide, thereby treating IBD or an IBD-related disorder. In one embodiment, the polynucleotide comprising the CLN1 open reading frame comprises a wild-type CLN1 polynucleotide. In another embodiment, the polynucleotide comprising the CLN1 open reading frame comprises a codon-optimized polynucleotide sequence of the polynucleotide, or its complement is codon-optimized for expression in human cells. In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence having at least about 90% identity to each of these, or their complements, and equivalents are identical in the codon-optimized nucleotides. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 shows a map of an exemplary CLN1 expression cassette. [Figure 2] Figure 2 shows serum enzyme activity of PPT1 in mice administered scAAV9 / CLN1 therapy. IT = intrathecal injection; IV = intravenous injection; Het = heterologous; KO = knockout. [Figure 3A] Figure 3A shows survival curves for cohorts of CLN1 knockout mice administered intrathecally with CLN1 AAV vectors at 4 or 12 weeks of age, and Figure 3B shows survival curves for cohorts of CLN1 knockout mice administered intrathecally with CLN1 AAV vectors at 20 or 26 weeks of age. [Figure 3B]Figure 3A shows survival curves for cohorts of CLN1 knockout mice administered intrathecally with CLN1 AAV vectors at 4 or 12 weeks of age, and Figure 3B shows survival curves for cohorts of CLN1 knockout mice administered intrathecally with CLN1 AAV vectors at 20 or 26 weeks of age. [Figure 4A] Figure 4A shows survival curves for cohorts of CLN1 knockout mice administered CLN1 AAV vectors intrathecally or intrathecally + intravenously at 4 weeks of age, and Figure 4B shows survival curves for cohorts of CLN1 knockout mice administered CLN1 AAV vectors intrathecally, intravenously, or intrathecally + intravenously at 20 weeks of age. [Figure 4B] Figure 4A shows survival curves for cohorts of CLN1 knockout mice administered CLN1 AAV vectors intrathecally or intrathecally + intravenously at 4 weeks of age, and Figure 4B shows survival curves for cohorts of CLN1 knockout mice administered CLN1 AAV vectors intrathecally, intravenously, or intrathecally + intravenously at 20 weeks of age. [Figure 5A] Figure 5A shows survival curves for CLN1 knockout mice administered CLN1 AAV vectors at different doses via different routes of administration before the onset of symptoms, and Figure 5B shows survival curves for CLN1 knockout mice administered CLN1 AAV vectors at different doses via different routes of administration after the onset of symptoms. [Figure 5B] Figure 5A shows survival curves for CLN1 knockout mice administered CLN1 AAV vectors at different doses via different routes of administration before the onset of symptoms, and Figure 5B shows survival curves for CLN1 knockout mice administered CLN1 AAV vectors at different doses via different routes of administration after the onset of symptoms. [Figure 6A] 6A-6B show swimming speed assessment in the Morris water maze. [Figure 6B] 6A-6B show swimming speed assessment in the Morris water maze. [Figure 7A]7A and 7B show the time to fall from the inverted wire hang. [Figure 7B] 7A and 7B show the time to fall from the inverted wire hang. [Figure 8] FIG. 8 shows normalized physical capacity score (PSC) versus relative survival time for the various mouse treatment groups. [Figure 9A] Figure 9A shows serum PPT1 levels in xenogeneic mice administered scAAV9 / CLN1 therapy as neonates, and Figure 9B shows swimming speed in xenogeneic mice administered scAAV9 / CLN1 therapy as neonates. [Figure 9B] Figure 9A shows serum PPT1 levels in xenogeneic mice administered scAAV9 / CLN1 therapy as neonates, and Figure 9B shows swimming speed in xenogeneic mice administered scAAV9 / CLN1 therapy as neonates. [Figure 10] FIG. 10 shows PPT1 enzyme activity measured in different tissues of rats treated with the scAAV9 / CLN1 vector. [Figure 11] FIG. 11 shows the levels of neutralizing antibodies against AAV9 in rats treated with the scAAV9 / CLN1 vector. [Figure 12] FIG. 12 shows a diagram of the development of IBD symptoms in mice treated with scAAV9 / CLN1 vectors at different time points. DETAILED DESCRIPTION OF THE INVENTION

[0006] definition Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying citation or by Arabic numerals. Full citations for publications identified by Arabic numerals are found immediately before the claims. The disclosures of these publications, patents, and published patent specifications are incorporated by reference in their entireties into this disclosure to more fully describe the state of the art to which this invention pertains.

[0007] The practice of this technique employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of those in the art. See, for example, Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor eds. (1995)), Harlow and Lane eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney ed. (1987)).

[0008] As used in describing the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0009] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted as including the recited materials or steps, and materials or steps that do not materially affect the basic and novel characteristics of the described embodiment. Thus, the term "consisting essentially of," as used herein, should not be interpreted as equivalent to "comprising." "Consisting of" means excluding more than trace amounts of other ingredients and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transitional phrases are within the scope of the present disclosure.

[0010] The term "about," as used herein when referring to a measurable value, such as an amount or concentration and the like, is meant to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0011] When the terms "acceptable," "effective," or "sufficient" are used to describe the selection of any ingredients, ranges, dosage forms, etc. disclosed herein, it is intended that said ingredients, ranges, dosage forms, etc. are suitable for the purpose disclosed.

[0012] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0013] The term "adeno-associated virus" or "AAV," as used herein, refers to a member of the class of viruses associated with this name, belonging to the genus Dependoparvovirus in the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes are capable of infecting cells from a variety of tissue types. At least 11 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAV PHP.B. AAV particles comprise, or alternatively consist essentially of, and / or consist of, three major viral proteins: VP1, VP2, and VP3. In one embodiment, AAV refers to an AAV of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. The AAV may be a self-complementary AAV (scAAV).

[0014] The term "cell," as used herein, can refer to a prokaryotic or eukaryotic cell, optionally obtained from a subject or commercially available source.

[0015] "Eukaryotic cells" include, or alternatively consist essentially of, and / or consist of, all of the kingdoms of life except Monera. They can be easily distinguished through their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotes, or organisms whose cells are organized into complex structures with internal membranes and cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include apes, cows, pigs, mice, rats, birds, reptiles, and humans, e.g., HEK293 and 293T cells.

[0016] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops, also called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary division. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella.

[0017] The term "encoding," when applied to a nucleic acid sequence, refers to a polynucleotide that is said to "encode" a polypeptide when, in its native state or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be derived therefrom.

[0018] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to specific molecular, biological, or cellular materials, and are intended to refer to those with minimal homology while still maintaining the desired structure or functionality. Non-limiting examples of equivalent polypeptides or polynucleotides include those that have at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity thereto or to a polypeptide or polynucleotide sequence, or polypeptides encoded by polynucleotides or their complements that hybridize under high stringency conditions to a polynucleotide encoding such a polypeptide sequence. High stringency conditions are described herein and are incorporated by reference. Alternatively, the equivalent is a polypeptide encoded by a polynucleotide or complement thereof having at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or at least 97% sequence identity to a reference polynucleotide, e.g., a wild-type polynucleotide. In embodiments relating to optimized polynucleotides or proteins, a biological equivalent has a nucleotide or amino acid sequence that is at a desired percent identity to the reference nucleotide or amino acid (e.g., at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity to it or to a polypeptide or polynucleotide sequence) while remaining unchanged, or a polypeptide encoded by a polynucleotide or complement thereof that hybridizes under high stringency conditions to a polynucleotide encoding such a polypeptide sequence.

[0019] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Alignment and homology or percent sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available at the web address: genome.jp / tools / clustalw / , last accessed January 13, 2017).

[0020] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively, less than 25% identity, with one of the sequences of the present disclosure.

[0021] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0022] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. A "gene product" or alternatively a "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0023] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on being operably linked to elements that contribute to or promote the initiation of transcription. "Operably linked" refers to polynucleotides arranged in a manner that allows them to function in a cell. In one embodiment, the present invention provides a promoter operably linked to a downstream sequence, such as a suicide gene, VEGF, 165A VEGF, a tet activator, etc.

[0024] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its native state or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be derived therefrom.

[0025] The term "isolated," as used herein, refers to a molecule or biological product or cellular material that is substantially free from other materials.

[0026] As used herein, the term "functional" may be used to mean modifying any molecular, biological, or cellular material so that it achieves a particular, specified effect.

[0027] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. As such, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, alternatively consisting essentially of, or consisting of, purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0028] The term "promoter," as used herein, refers to any sequence that regulates the expression of a coding sequence, e.g., a gene. A promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a regulatory sequence, a region of a polynucleotide sequence at which the initiation and rate of transcription are controlled. It may contain genetic elements that allow regulatory proteins and molecules to bind, such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, or the EF1 promoter. In some embodiments, the promoter is a chicken β-actin ("CBA") promoter.

[0029] Additional non-limiting exemplary promoters with certain target specificities are provided herein below, including, but not limited to, CMV, EF1a, SV40, PGK1 (human or mouse), P5, Ubc, human beta-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, and alpha-1-antitrypsin. Synthetic promoters may also be used for ubiquitous or tissue-specific expression. Additionally, viral promoters, some of which are listed above, may be useful in the methods disclosed herein, such as CMV, HIV, adenovirus, and AAV promoters. In some embodiments, the promoter is linked to an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include the RSV enhancer or the CMV enhancer.

[0030] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. An enhancer / promoter may be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition to a gene by means of gene manipulation (i.e., molecular biology techniques) such that transcription of that gene is directed by the linked enhancer / promoter.

[0031] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise, or alternatively consist essentially of, and / or consist of, the sequence of a protein or peptide. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0032] As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising, or alternatively consisting essentially of, and / or consisting of an intact replicon, such that the vector can be replicated when placed in a cell, for example, by the process of transformation. A vector may be a viral or non-viral vector. Viral vectors include retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papoviruses, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827. Exemplary non-viral vectors for delivering nucleic acid include: naked DNA; DNA complexed with cationic lipid, alone or in combination with cationic polymer; anionic and cationic liposomes; DNA-protein complexes, and particles that comprise, or alternatively essentially consist of, or further comprise, cationic polymers, such as heterogeneous polylysine, oligopeptides of defined length, and DNA aggregated with polyethyleneimine, and are sometimes contained in liposomes; and ternary complexes that comprise, or alternatively essentially consist of, or further comprise, virus and polylysine-DNA.Example of viral vector includes retroviral vector.

[0033] In an embodiment, the vector comprises an inducible promoter. In an embodiment, the inducible promoter is an inducible tetracycline promoter. The Tet-Off and Tet-On gene expression systems provide researchers with immediate access to the regulated high-level gene expression systems described by Gossen & Bujard (1992; Tet-Off) and Gossen et al. (1995; Tet-On). In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the culture medium. In contrast, expression is turned on by the addition of Dox in the Tet-On system. Both systems allow gene expression to be tightly regulated in response to various concentrations of Tc or Dox. The maximum expression level in the Tet system is very high and compares favorably with the maximum level obtainable from strong constitutive mammalian promoters, such as CMV (Yin et al., 1996). Unlike other inducible mammalian expression systems, gene regulation in the Tet system is highly specific, so interpretation of results is not complicated by pleiotropic effects or nonspecific induction. In E. coli, the Tet repressor protein (TetR) negatively regulates the genes of the tetracycline resistance operon on the Tn10 transposon. TetR blocks transcription of these genes by binding to the tet operator sequence (tetO) in the absence of Tc. TetR and tetO provide the basis for regulation and induction for use in mammalian experimental systems. In the Tet-On system, the regulatory protein is based on the "reverse" Tet repressor (rTetR), created by changing four amino acids in TetR (Hillen & Berens, 1994; Gossen et al., 1995). The resulting protein, rtTA (reverse tTA, also called tetracycline activator protein), is encoded by the pTet-On regulator plasmid. This gene may be in a separate vector as the therapeutic gene or may be encoded on the same gene.

[0034] In related embodiments, the vector further comprises, or alternatively consists essentially of, or further consists of, a nucleic acid encoding a tetracycline activator protein and a promoter that regulates expression of the tetracycline activator protein.

[0035] Other inducible systems useful in the vectors, isolated cells, viral packaging systems, and methods described herein include regulation by ecdysone, estrogen, progesterone, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG).

[0036] As used herein, the term "recombinant expression system" or "recombinant vector" refers to one or more genetic constructs for the expression of certain genetic material formed by recombination.

[0037] A population of cells contemplates a collection of one or more cells that are phenotypically and / or genotypically identical (clonal) or non-identical. A substantially homogeneous population of cells is one that has at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical phenotype as measured by preselected markers.

[0038] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria, or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vehicles, which are typically used in the art and have been described for expression in various eukaryotic and prokaryotic hosts, and can be used for simple protein expression in addition to gene therapy.

[0039] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "introducing," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., via viral infection / transfection or various other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used for introducing polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contain a replication origin compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, numerous vectors are known that can mediate the transfer of genes into mammalian cells.

[0040] As used herein, the term "codon-optimized" refers to a coding sequence that has been optimized compared to a wild-type coding sequence (e.g., the coding sequence of PPT1) to increase expression of the coding sequence by replacing one or more codons normally present in the coding sequence with codons for the same (synonymous) amino acid. In some embodiments, the substitutions minimize rare codons (e.g., human codons), increase the total GC content, reduce the CpG content, remove cryptic splice donor or acceptor sites, and / or add or remove ribosome entry sites, e.g., Kozak sequences. PCT International Application Publication No. WO 2017 / 218450, published December 21, 2017 (incorporated herein by reference), discloses a codon-optimized CLN1 gene sequence, methods for producing the codon-optimized CLN1 gene, and general methods for delivering monotherapy using the same.

[0041] A "plasmid" is an extrachromosomal DNA molecule separate from chromosomal DNA that can replicate independently of chromosomal DNA. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within populations of microorganisms and typically confer a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar circumstances.

[0042] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites that allows for easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of protein. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. The bacteria can be induced to manufacture large amounts of protein from the inserted gene only if they produce the protein that confers antibiotic resistance.

[0043] In embodiments in which gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising, or alternatively consisting essentially of, a viral genome or portion thereof, and a transgene. Adenoviruses (Ad) are a relatively well-characterized, historically homologous group of viruses, including over 50 serotypes. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. Such vectors are commercially available from suppliers such as Takara Bio USA (Mountain View, CA), Vector Biolabs (Philadelphia, PA), and Creative Biogene (Shirley, NY). Wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Wold and Toth (2013) Curr. Gene. Ther. 13(6):421-433, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470, and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0044] Vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of in vitro or in vivo RNA transcription and are commercially available from suppliers such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate redundant, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0045] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also comprise, or alternatively consist essentially of, and / or consist of targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be achieved by, but is not limited to, protein transfection techniques; alternatively, culture conditions that can enhance expression and / or promote the activity of the proteins disclosed herein are other, but not limited to, techniques.

[0046] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminus of newly synthesized secretory or membrane polypeptides or proteins. It acts to direct the polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965.

[0047] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids generally comprise oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid.

[0048] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid used to provide additional components necessary for the replication and packaging of a viral particle or recombinant viral particle, such as a modified AAV disclosed herein. The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus may encode enzymes necessary for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0049] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus.Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells that are co-infected with a helper virus to provide certain functions.General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Because the various serotypes are well known to be quite closely related, both structurally and functionally, even at the genetic level, it is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication date of the reviews (see, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes appear to exhibit very similar replication properties mediated by homologous rep genes, and all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes across the length of the genome; and by the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0050] As used herein, "AAV vector" refers to a vector that comprises, consists essentially of, or consists of one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeat (ITR) sequences. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that provides the functionality of the rep and cap gene products, for example, by transfection of the host cell. In embodiments, the AAV vector contains a promoter, at least one nucleic acid capable of encoding at least one protein or RNA, and / or an enhancer and / or terminator within the flanking ITRs that are packaged into infectious AAV particles. The encapsidated nucleic acid portion is sometimes referred to as the AAV vector genome. A plasmid containing an AAV vector may also contain elements for manufacturing purposes, such as antibiotic resistance genes, but these are not encapsidated and therefore do not form part of the AAV particle.

[0051] An "AAV virion" or "AAV viral particle" or "AAV viral vector" or "AAV vector particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. Thus, production of an AAV vector particle necessarily includes production of an AAV vector, which is contained within the AAV vector particle.

[0052] In some embodiments, AAV is a replication-deficient parvovirus, and its single-stranded DNA genome is approximately 4.7 kb in length, including two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank accession number NC_002077, the complete genome of AAV-2 is provided in GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is provided in GenBank accession number NC_1829, the complete genome of AAV-4 is provided in GenBank accession number NC_001829, the AAV-5 genome is provided in GenBank accession number AF085716, the complete genome of AAV-6 is provided in GenBank accession number NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively, and the AAV-9 genome is provided in Gao et al., J. Virol., 78: The AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928 (incorporated herein by reference). U.S. Pat. No. 9,434,928 also provides the sequences of capsid proteins and self-complementary genomes. In one embodiment, the genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), capsid encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs.Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p9) couple with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) to result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties that ultimately contribute to viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and a non-consensus translation initiation site result in the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0053] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytotoxic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, enabling the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slow-dividing and non-dividing cells and persist essentially for the lifespan of those cells as transcriptionally active intranuclear episomes (extrachromosomal elements). The AAV proviral genome can be inserted as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another significant feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making refrigerated storage of AAV less important. AAV can even be lyophilized. And AAV-infected cells are not resistant to superinfection.

[0054] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of the transgene product in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94: 13921-13926 (1997). Furthermore, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. The AAV DNA in the rAAV genome can be from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B, and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).The nucleotide sequences of the genomes of various AAV serotypes are known in the art. Other suitable AAV particles are described in International Application PCT / US2019 / 064396, filed December 4, 2019, which is incorporated herein by reference in its entirety, particularly with respect to AAV particles and AAV capsid proteins.

[0055] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to a composition to be detected, e.g., a polynucleotide or protein, such as an antibody, to produce a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP) and the like. The label may be detectable alone (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A simply detected response generally comprises, or alternatively consists essentially of, and / or consists of a response whose presence is merely confirmed, while a quantified response generally comprises, or alternatively consists essentially of, and / or consists of a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response may be generated directly using a luminophore or fluorophore associated with the assay component actually responsible for binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0056] Examples of luminescent labels that generate signals include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally comprises, or alternatively consists essentially of, or further consists of, a change in or in the presence of a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0057] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0058] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of cells or tissues, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule.The choice of functional group of the fluorescent label depends on the site of attachment to either a linker, agent, marker, or second labeling agent.

[0059] Attachment of the fluorescent label may be directly to the cellular component or compound, or alternatively via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to an intermediate include, but are not limited to, antigen / antibody, e.g., rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0060] A "composition" is intended to mean a combination of an active polypeptide, polynucleotide, or antibody with another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0061] A "pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide or antibody with an inert or active carrier, e.g., a solid support, which makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0062] As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0063] A "subject" of diagnosis or treatment is a cell or an animal, such as a mammal, or a human. The subject is not limited to a specific species, and includes non-human animals that are subject to diagnosis or treatment, such as those that are subject to infection, or animal models, such as apes, murines, such as rats, mice, chinchillas, canines, such as dogs, lagomorphs, such as rabbits, livestock, sports animals, and pets. Human patients are also included in the term.

[0064] The term "tissue" is used herein to refer to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, dead, and / or may have genetic mutations. Biological tissues may include any single tissue (e.g., a collection of cells, which may be interconnected) or a group of tissues that make up an organ, part, or region of an organism's body. Tissues may comprise, or alternatively consist essentially of, or consist of, a homogenous cellular material, or they may be composite structures, such as those found in regions of the body, including the thorax, which may include, by way of example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination of the following.

[0065] The term "IBD-related disorder," as used herein, refers to a disease, disorder, syndrome, or condition caused by or symptomatic of a decrease or alteration in the expression of the CLN1 gene in a subject compared to the expression level or activity in normal subjects, asymptomatic subjects, or the population.

[0066] As used herein, "treating" a disease in a subject or "treatment" thereof refers to (1) preventing the occurrence of symptoms or disease in a subject who is predisposed to or has not yet exhibited symptoms of the disease, (2) inhibiting or halting the development of the disease, or (3) causing the disease or symptoms of the disease to ameliorate or regress. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results can include, but are not limited to, one or more of the following, whether detectable or undetectable: alleviation or amelioration of one or more symptoms, reduction in the severity of a condition (including a disease), stabilization (i.e., not worsening) of the condition (including a disease), delay or slowing of the progression of a condition (including a disease), remission or palliation of a condition (including a disease), and relief (whether partial or total).

[0067] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question and the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to pharmaceutical compositions. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to result in partial or full function restoration of a defective gene in a subject. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to result in gene expression in a subject. Those skilled in the art can determine the appropriate amount depending on these and other factors.

[0068] The term "vg" refers to the virus unit provided for gene therapy, and is typically described as vg per kg of subject.The experiments described herein are the amounts used to treat mice, and if the subject being treated is not a mouse, it is assumed that the amount will be converted to the appropriate amount for the subject being treated, such as a human, infant or newborn.In one embodiment, the amount administered is in vg / kg, thus taking into account the difference in size and weight of the subject being treated.

[0069] In embodiments, the effective amount depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the target subject and the method of use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount may comprise, alternatively consist essentially of, and / or consist of one or more administrations of the composition, depending on the embodiment.

[0070] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be effected in one dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health, or sex of the subject being treated. Single or multiple administrations can be carried out, with the dose level and pattern selected by the treating physician, or in the case of companion animals and animals, the treating veterinarian. Suitable dosage formulations and methods for administering agents are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will vary with the composition used for therapy, the purpose of the therapy, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.

[0071] As used herein, the term "modified," when applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from the wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0072] Modes for Carrying Out the Disclosure Provided herein is a method of treating IBD or an IBD-related disorder in a subject in need thereof, comprising, consisting essentially of, or consisting of intrathecal administration of a polynucleotide comprising a CLN1 open reading frame followed by intravenous administration of the polynucleotide, thereby treating IBD or an IBD-related disorder. Optionally, intravenous administration may precede the intrathecal administration.

[0073] In one embodiment, the polynucleotide comprising the CLN1 open reading frame is part of a vector genome, and delivery of the polynucleotide is achieved by administering viral particles comprising the vector genome. Accordingly, in one embodiment, provided herein is a method of treating infantile Batten disease (IBD) or an IBD-related disorder in a subject, comprising a combination of intrathecal and intravenous administration of an effective amount of viral particles comprising the polynucleotide.

[0074] Also provided are AAV vector particles, AAV vectors, and capsid proteins that have use in delivering polynucleotides. The viral particle may be an AAV viral particle, such as an AAV9 viral particle.

[0075] In one embodiment, the polynucleotide comprising the CLN1 open reading frame comprises a wild-type CLN1 polynucleotide. In another embodiment, the polynucleotide comprising the CLN1 open reading frame comprises a codon-optimized polynucleotide sequence of CLN1, or its complement is codon-optimized for expression in human cells. In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity thereto, respectively, or their complements, where equivalents are identical in the codon-optimized nucleotides. In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 3. SEQ ID NO: 1: Human codon-optimized CLN1 open reading frame (additional stop codon is underlined) ATGGCTTCTCCGGGGTGTCTGTGGCTGCTGGCAGTGGCACTCCTTCCCTGG ACTTGCGCCAGCCGGGCTCTGCAGCACCTCGACCCTCCAGCCCCTCTTCCA CTGGTGATTTGGCACGGAATGGGTGATTCCTGCTGTAATCCCCTGTCAATG GGAGCCATCAAGAAGATGGTGGAGAAGAAGATCCCTGGAATCTACGTGCT GTCACTGGAGATTGGAAAGACCCTGATGGAGGACGTCGAGAACTCCTTCT TCCTCAATGTCAACTCTCAAGTGACCACCGTCTGCCAGGCCCTGGCCAAG GACCCGAAGCTGCAGCAGGGGTATAATGCTATGGGGTTCAGCCAGGGAG GACAGTTCCTTCGGGCTGTGGCCCAACGCTGCCCTAGCCCACCCATGATCA ACCTGATCTCAGTGGGTGGCCAGCATCAGGGCGTGTTCGGACTTCCCCG TGTCCCGGGGAATCCTCTCATATCTGCGACTTCATCCGCAAAACTCTCAAT GCAGGCGCTTATTCAAAGGTCGTCCAAGAGAGGCTGGTGCAAGCCGAGTA CTGGCACGATCCCATTAAGGAGGACGTGTACAGAAATCACTCAATCTTTC TGGCCGACATTAACCAGGAGAGGGGAATTAACGAATCATATAAGAAGAA TCTCATGGCCCTCAAAAAGTTCGTCATGGTGAAGTTCCTTAACGATAGCAT TGTGGACCCAGTGGACAGCGAATGGTTCGGATTTTACCGCTCAGGCCAGG CAAAAGAAACCATCCCTCTCCAAGAGACTTCTCTTTACACCCAAGACAGA CTTGGGCTTAAGGAATGGATAACGCTGGTCAGCTGGTGTTCCTCGCCAC CGAAGGTGACCATCTGCAGCTCAGCGAAGAGTGGTTCTACGCTCATATCA TCCCGTTTCTTGGTTGA TAA

[0076] SEQ ID NO:2 (wild-type CLN1) is known in the art, as disclosed, for example, by the information provided at genenames.org / data / gene-symbol-report / #! / hgnc_id / HGNC:9325 (last accessed April 29, 2019), and is shown below: ATGGCGTCGCCCGGCTGCCTGTGGCTCTTGGCTGTGGCTCTCCTGCCATGGACCTGCGCTTCTCGGGCCTGCAGCATCTGGACCCGCGGCGCCGCCTGCCGTTGGTGATCTGGCATGGGATGGGAGACAGCTGTTGCAATCCCTTAAGCATGGGTGCTTATTAAAAAAATGGTGGAGAAGAAAATACCTGGAATTTACGTCTTATCTTTTAGAGATTGGGAAGACCCTGAT GGAGGACGTGGAGAACAGCTTCTTCTTGAATGTCAATTCCCAAGTAACAACAGTGTGTCAGGCACTTGCTAAGGATCCTAAATTGCAGCAAGGCTACAATGCTATGGGATTCTCCCAGGGAGGCCAATTTCTGAGGGCAGTGGCTCAGAGATGCCCTTCACCTCCCATGATCAATCTGATCTCGGTTGGGGGACAACATCAAGGTGTTTTTGACTCCCTCGATGCCCAG GAGAGAGCTCTCACATCTGTGACTTCCATCGAAAAACACTGAATGCTGGGGCGTACTCCCAAAGTTGTTCAGGAACGCCTCGTGCAAGCCGAATACTGGCATGACCCCATAAAGGGATGTGTATCGCAACCACAGCATCTTCTTGGCAGATATAAATCAGGAGCGGGGTATCAATGAGTCCTACAAGAAAAACCTGATGGCCCTGAAAGAATTTGTGATGGTGAAATTC CTCAATGATTCCATTGTGGACCCTGTAGATTCGGAGTGGTTTGGATTTTCAGAAGTGGCCAAGCCAAGGAAACCATTCCCTTACAGAGACCTCCTGTACACACAGGCCCTGGGCTAAAGGAAATGGACAATGCAGGACAGCTAGTGTTTCTGGCTACAGAAGGGACCATCTTCAGTTGTCTGAAGGAATGGTTTTATGCCCACATCATACCATTCCTTGGATGA

[0077] suitable CLN1 gene, sequence number 3: MASPGCLWLLAVALLPWTCASRALQHLDPPAPLPLVIWHGMGDSCCNPLSMGAIKKMVEKKIPGIYVLSLEIGKTLMEDVENSFFLNVNSQVTTVCQALAKDPKLQQGYNAMGFSQGGQFLRAVAQRCPSPPMINLISVGGQHQGVFGLPRCP GESSHICDFIRKTLNAGAYSKVVQERLVQAEYWHDPIKEDVYRNHSIFLADINQERGINESYKKNLMALKKFVMVKFLNDSIVDPVDSEWFGFYRSGQAKETIPLQETSLYTQDRLGLKEMDNAGQLVFLATEGDHLQLSEEWFYAHIIPFLG The PPT1 protein may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that differ from SEQ ID NO: 3. The PPT1 protein may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from SEQ ID NO: 3.

[0078] The subject may be a mammal, for example, a human. The human may be a human infant, for example, between about 2 months and about 24 months, between about 2 years and 12 years, between about 2 years and 5 years, or between 2 years and 5 years.

[0079] The polynucleotide can be operably linked to additional elements, for example, the polynucleotide is operably linked to a promoter, and / or the promoter is a chicken beta actin promoter, and / or the polynucleotide is operably linked to an enhancer, and / or the enhancer is a cytomegalovirus enhancer, and / or the polynucleotide is operably linked to an intron, and / or the intron is a hybrid / modified MVM intron, and / or the polynucleotide is operably linked to a polyadenylation signal, and / or the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0080] The polynucleotide may be part of a vector genome for delivery of the polynucleotide, and optionally the vector genome comprising the polynucleotide is packaged into viral particles comprising one or more of a wild-type capsid protein, a mutant capsid protein, a tissue-tropic capsid protein, and a modified capsid protein having altered tropism compared to the wild-type capsid protein, which modified capsid protein is optionally liver-detargeted.

[0081] The vector may comprise at least one adeno-associated virus (AAV) inverted terminal repeat (ITR), for example, the vector comprises two AAV ITRs, and / or the two AAV ITRs have the same nucleotide sequence, and / or the two AAV ITRs have different nucleotide sequences, and / or the AAV ITRs are AAV2 ITRs, and / or the viral vector is a self-complementary AAV genome.

[0082] In one embodiment, the vector genome comprising the polynucleotide comprises an enhancer, a promoter, an intron, a human CLN1 open reading frame, and a polyadenylation site. In a further embodiment, the polynucleotide comprises an AAV ITR, an enhancer, a promoter, an intron, a human CLN1 open reading frame, a polyadenylation site, and an AAV ITR. In an even further embodiment, the polynucleotide comprises a CMV enhancer, a chicken beta-actin promoter, a hybrid / modified MVM intron, a human CLN1 open reading frame, and a bovine growth hormone polyadenylation site. In an even further embodiment, the polynucleotide comprises a mutant AAV ITR, a CMV enhancer, a chicken beta-actin promoter, a hybrid / modified MVM intron, a human CLN1 open reading frame, a bovine growth hormone polyadenylation site, and a wild-type AAV ITR. In an even further embodiment, the polynucleotide comprises SEQ ID NO: 1 or 2, or their respective equivalents.

[0083] The polynucleotide or the viral particle comprising the polynucleotide is administered in an amount to express functional CLN1 (i.e., PPT1 protein) in the subject. The amount administered can be for transient or long-term expression of functional CLN1 (i.e., PPT1 protein) in the subject.

[0084] In one embodiment, the amount administered intrathecally is the same as or different from the amount delivered intravenously.

[0085] In further embodiments, the polynucleotide or viral particle comprising the polynucleotide is administered intrathecally and intravenously to a subject before or after the onset of symptoms. Children with IBD generally develop symptoms between 2 and 24 months of age, often by about 18 months of age.

[0086] The amount to be delivered will vary with the subject and disease being treated. In one embodiment, the amount is considered a medium or high dose. For illustrative purposes only (using mice as an example), the amount to be delivered is approximately 2.0 x 10 11 vg~approx. 8.0×10 11 vg or 2.0×10 11 vg / kg ~ approx. 8.0×10 11 In a further embodiment, the polynucleotide is about 7.0 x 10 10 vg~approx. 8.0×10 11 vg or 7.0 × 10 10 vg / kg ~ approx. 8.0×10 11 In still further embodiments, administration is at about 7.0 x 10 vg / kg before the onset of symptoms. 10 vg (or vg / kg) ~ approx. 8.0 × 10 11 In still further embodiments, the polynucleotide is delivered in an amount of about 7.0 x 10 vg (or vg / kg). 11 vg (or vg / kg) ~ approx. 8.0 × 10 11 vg (or vg / kg).

[0087] In one embodiment, the amount of viral particles for intrathecal administration in humans is about 1.0 x 10 12 vg~approx. 1.0×10 17 In embodiments, the amount for intrathecal administration in humans is about 1.0 x 10 12 vg~approx. 1.0×10 14 vg, approx. 1.0×10 13 vg~approx. 1.0×10 15 vg, approx. 1.0×10 14 vg~approx. 1.0×10 16 vg, approx. 1.0×10 15 vg~approx. 1.0×10 17 vg, approx. 1.0×10 13 vg~approx. 1.0×10 14 vg, approx. 1.0×10 14 vg~approx. 1.0×10 15 vg, or approximately 1.0 × 10 15 vg~approx. 1.0×10 16 vg.

[0088] In one embodiment, the amount of viral particles for intravenous administration in humans is about 1.0 x 10 11 vg / kg ~ approx. 2.0×10 16 In embodiments, the amount for intravenous administration in humans is about 1.0 x 10 11 vg / kg ~ approx. 2.0×10 13 vg / kg, approximately 1.0×10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 2.0×10 15 vg / kg, approximately 1.0×10 14 vg / kg ~ approx. 2.0×10 16 vg / kg, approximately 1.0×10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, approximately 1.0×10 14 vg / kg ~ approx. 2.0×10 15 vg / kg.

[0089] In addition to treating IBD, the methods may be used to treat any disorder associated with expression of the CLN1 gene, such as infantile, late-onset infantile, juvenile, or adult-onset neuronal ceroid lipofuscinosis. They may also be used for other IBD-related disorders resulting from low or abnormal expression of CLN1, some of which are described in WO 2017 / 218450 (incorporated herein by reference).

[0090] Also provided are kits comprising a pharmaceutical composition comprising a CLN1 polynucleotide in a pharmaceutically acceptable carrier and instructions for use in the methods as disclosed herein.

[0091] Methods for producing AAV viral particles Various approaches can be used to produce AAV viral vectors.In some embodiments, packaging is achieved by using helper virus or helper plasmid and cell line.Helper virus or helper plasmid contains the elements and sequences that promote the production of viral vectors.In another embodiment, helper plasmid is stably integrated into the genome of packaging cell line, so that packaging cell line does not require additional transfection with helper plasmid.

[0092] In embodiments, the cells are packaging or helper cell lines. In embodiments, the helper cell lines are eukaryotic cells, such as HEK293 cells or 293T cells. In embodiments, the helper cells are yeast cells or insect cells.

[0093] In one embodiment, the cell comprises a nucleic acid encoding a tetracycline activator protein and a promoter that regulates the expression of the tetracycline activator protein. In one embodiment, the promoter that regulates the expression of the tetracycline activator protein is a constitutive promoter. In one embodiment, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0094] The helper plasmid may comprise, for example, at least one viral helper DNA sequence derived from a replication-incompetent viral genome that encodes in trans all the virion proteins required for packaging replication-incompetent AAV and for producing virion proteins capable of packaging replication-incompetent AAV at high titers without producing replication-competent AAV.

[0095] Helper plasmids for packaging AAV are known in the art; see, for example, U.S. Patent Application Publication No. 2004 / 0235174 A1 (incorporated herein by reference). As described therein, AAV helper plasmids may contain, as helper virus DNA sequences, non-limiting examples of which include the Ad5 genes E2A, E4, and VA, controlled by their respective native promoters or heterologous promoters. AAV helper plasmids may additionally contain an expression cassette for expressing a marker protein, such as a fluorescent protein, to enable simple detection of transfection of desired target cells.

[0096] The present disclosure provides a method for producing AAV particles, comprising transfecting any one of the AAV helper plasmids disclosed herein and any one of the AAV vectors disclosed herein into a packaging cell line.In an embodiment, the AAV helper plasmid and the AAV vector are co-transfected into the packaging cell line.In an embodiment, the cell line is a mammalian cell line, for example, a human embryonic kidney (HEK) 293 cell line.The present disclosure provides a cell comprising any one of the AAV vectors and / or AAV particles disclosed herein.

[0097] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising any one of the AAV vectors, AAV capsids, and / or AAV particles described herein. Typically, the AAV particles are administered for therapy.

[0098] Pharmaceutical compositions as described herein may be formulated by any method known or developed in the art of pharmacology, including, but not limited to, contacting the active ingredient (e.g., a viral particle or a recombinant vector) with an excipient or other accessory ingredient, dividing the product into dosage units, or packaging it. The viral particles of the present disclosure may be formulated to have desirable characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or tropism, modulated or enhanced translation of the encoded protein in vivo, and the release profile of the encoded protein in vivo.

[0099] As such, the pharmaceutical composition may further comprise saline, lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, core-shell nanoparticle, peptide, protein, cell transfected with a viral vector (e.g., for transplantation into a subject), nanoparticle mimic, or a combination thereof. In embodiments, the pharmaceutical composition is formulated as nanoparticles. In embodiments, the nanoparticles are self-assembled nucleic acid nanoparticles.

[0100] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses. The amount of active ingredient generally equals the dosage of the active ingredient administered to a subject and / or a convenient ratio of such a dosage, such as half or one-third of such a dosage. Formulations of the present invention may include one or more excipients in amounts that, taken together, increase the stability of the viral vector, increase cell transfection or transduction by the viral vector, increase the expression of the protein encoded by the viral vector, and / or modify the release profile of the protein encoded by the viral vector. In embodiments, the pharmaceutical composition comprises an excipient. Non-limiting examples of excipients include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, or combinations thereof.

[0101] In an embodiment, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0102] Further Numbered Embodiments Further numbered embodiments of the present disclosure are provided as follows:

[0103] Embodiment 1. A method of treating infantile Batten disease (IBD) or an IBD-related disorder in a subject in need thereof, comprising intrathecal administration of a first polynucleotide comprising a CLN1 open reading frame and intravenous administration of a second polynucleotide comprising a CLN1 open reading frame, thereby treating IBD or an IBD-related disorder.

[0104] Embodiment 2. The method of embodiment 1, wherein said intrathecal administration precedes said intravenous administration.

[0105] Embodiment 3. The method of embodiment 1 or 2, wherein the CLN1 open reading frame comprises a wild-type CLN1 polynucleotide, a codon-optimized sequence, or a nucleotide sequence having at least about 90% identity to each of the foregoing.

[0106] Embodiment 4. The method of embodiment 3, wherein the codon-optimized sequence is SEQ ID NO:1.

[0107] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the CLN1 open reading frame encodes a polypeptide sequence having at least about 90% identity to SEQ ID NO:3.

[0108] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the subject is a human patient.

[0109] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the CLN1 open reading frame is operably linked to a promoter.

[0110] Embodiment 8. The method of embodiment 7, wherein the promoter is a chicken beta-actin promoter.

[0111] Embodiment 9 The method of any one of embodiments 1 to 8, wherein the CLN1 open reading frame is operably linked to an enhancer.

[0112] Embodiment 10. The method of embodiment 9, wherein the enhancer is a cytomegalovirus enhancer.

[0113] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the CLN1 open reading frame is operably linked to an intron.

[0114] Embodiment 12. The method of embodiment 11, wherein the intron is a hybrid / modified MVM intron.

[0115] Embodiment 13 The method of any one of embodiments 1 to 12, wherein the CLN1 open reading frame is operably linked to a polyadenylation signal.

[0116] Embodiment 14 The method of embodiment 13, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0117] Embodiment 15. The method of any one of embodiments 1 to 14, wherein administering the polynucleotide comprises administering a vector comprising the polynucleotide.

[0118] Embodiment 16. The method of embodiment 15, wherein the vector is packaged into a viral particle comprising one or more of a wild-type capsid protein, a mutant capsid protein, a tissue-tropic capsid protein, or a modified capsid protein, wherein the modified capsid protein has altered tropism compared to the wild-type capsid protein.

[0119] Embodiment 17. The method of embodiment 15, wherein the vector further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0120] Embodiment 18. The method of embodiment 17, wherein the vector comprises two AAV ITRs.

[0121] Embodiment 19. The method of embodiment 18, wherein the two AAV ITRs have the same nucleotide sequence.

[0122] Embodiment 20. The method of embodiment 18, wherein the two AAV ITRs have different nucleotide sequences.

[0123] Embodiment 21 The method of embodiments 19 and 20, wherein the AAV ITRs are AAV2 ITRs.

[0124] Embodiment 22. The method of any one of embodiments 16 to 21, wherein the viral vector is a self-complementary AAV genome.

[0125] Embodiment 23. The method of any one of embodiments 15 to 22, wherein the vector comprises a polynucleotide comprising an enhancer, a promoter, an intron, a human CLN1 open reading frame, and a polyadenylation site.

[0126] Embodiment 24. The method of any one of embodiments 15 to 22, wherein the vector comprises a polynucleotide comprising an AAV ITR, an enhancer, a promoter, an intron, a human CLN1 open reading frame, a polyadenylation site, and an AAV ITR.

[0127] Embodiment 25. The method of any one of embodiments 15 to 22, wherein the vector comprises a polynucleotide comprising a CMV enhancer, a chicken beta actin promoter, a hybrid / modified MVM intron, a human CLN1 open reading frame, and a bovine growth hormone polyadenylation site.

[0128] Embodiment 26. The method of any one of embodiments 15 to 22, wherein the vector comprises a polynucleotide comprising a mutant AAV ITR, a CMV enhancer, a chicken beta-actin promoter, a hybrid / modified MVM intron, a human CLN1 open reading frame, a bovine growth hormone polyadenylation site, and a wild-type AAV ITR.

[0129] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the polynucleotide is administered in an amount sufficient to express functional CLN1 in the subject.

[0130] Embodiment 28 The method of embodiment 27, wherein the polynucleotide is administered in an amount for long-term expression of the functional CLN1 in the subject.

[0131] Embodiment 29. The method of any one of embodiments 1 to 28, wherein the amount administered intrathecally is the same as or different from the amount delivered intravenously.

[0132] Embodiment 30. The method of any one of embodiments 1-29, wherein the polynucleotide is administered intrathecally to the subject before symptoms occur.

[0133] Embodiment 31. The amount of vector for intrathecal administration is about 1.0 x 10 14 vg~approx. 1.0×10 15 31. The method of any one of embodiments 15 to 30, wherein vg.

[0134] Embodiment 32. The amount of vector for intravenous administration is about 1.0 x 10 13 vg / kg ~ approx. 2.0×10 14 32. The method of any one of embodiments 15 to 31, wherein the saturation dose is 100 mg / kg.

[0135] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the intrathecal administration and the intravenous administration are performed after symptom onset.

[0136] Embodiment 34. The method of any one of embodiments 1 to 32, wherein the intrathecal administration and the intravenous administration are administered before the onset of symptoms.

[0137] Embodiment 35. The method of any one of embodiments 1-34, wherein the disorder is infantile, late-infantile, juvenile, or adult-onset neuronal ceroid lipofuscinosis.

[0138] Embodiment 36. The method of any one of embodiments 15 to 35, wherein the vector is an AAV vector.

[0139] Embodiment 37. The method of embodiment 37, wherein the AAV vector is an AAV9 vector.

[0140] Embodiment 38 The method of embodiment 36 or 37, wherein the AAV vector is encapsidated in a wild-type capsid protein.

[0141] Embodiment 39. The vector of embodiment 36 or 37, wherein the AAV vector is encapsidated in a modified capsid protein having altered tropism compared to the wild-type capsid protein.

[0142] Embodiment 40 The method of embodiment 49, wherein the modified capsid protein is liver-detargeted.

[0143] Embodiment 41. A kit comprising a pharmaceutical composition comprising the CLN polynucleotide in a pharmaceutically acceptable carrier and instructions for use in the method of any one of embodiments 1 to 40. [Example]

[0144] Example 1 Intrathecal and intravenous administration of AAV expressing PPT1 protein We developed an AAV vector genome cassette to express the PPT1 protein encoded by the CLN1 ORF. This cassette was designed to provide maximum expression from a self-complementary AAV genome packaged within multiple AAV capsids. From 5' to 3', the cassette contains a mutant AAV2 ITR, a CMV enhancer, a chicken beta-actin promoter, a hybrid / modified MVM intron, a codon-optimized human CLN1 ORF, a bovine growth hormone polyadenylation site, and wild-type (WT) AAV2 ITR (Figure 1). CLN1 expression was verified by transfecting HEK293 cells with the expression cassette, and the expressed protein was detected in the cells and culture medium by Western blot.

[0145] A CLN1 expression cassette was packaged into wild-type AAV9 capsids, and the resulting AAV viral particles were used to intrathecally and / or intravenously dose CLN1 knockout mice.

[0146] Figure 2 shows serum enzyme activity of PPT1 in mice administered scAAV9 / CLN1 therapy. The vector was administered intrathecally to wild-type, xenogeneic, and CLN1 knockout mice at 7 × 10 10 or 7×10 11 at a vector genome dose of 7 × 10 or intravenously 11 The vector genome was injected at a dose of 100 mg / kg / day. The vector was administered at week 20. PPT1 serum enzyme activity was measured at 4, 8, and 17–37 weeks after treatment. Supraphysiological PPT1 serum enzyme activity levels were observed at all time points and dosages.

[0147] Example 2 Combined intrathecal and intravenous administration of AAV expressing PPT1 protein improves the lifespan of CLN1 knockout mice AAV viral particles expressing PPT1 protein were prepared as described in Example 1 and tested for their effect on the lifespan of CLN1 knockout mice when administered intrathecally and / or intravenously.

[0148] Figures 3A-3B show the lifespan of CLN1 knockout mice administered scAAV9 / CLN1 intrathecally. The shaded areas indicate the survival range relative to untreated xenograft mice. In Figure 3A, various doses of vector genome were administered at 1, 4, and 12 weeks prior to the onset of symptoms. The results showed that intrathecal administration of scAAV9 / CLN1 prolonged survival in a dose-dependent manner when given early. In Figure 3B, the vector was administered intrathecally to CLN1 knockout mice at 7 x 10 10 or 7×10 11The vector genome was injected at 20 or 26 weeks after symptom onset. Results showed that higher doses of scAAV9 / CLN1 administered after symptom onset prolonged survival of CLN1 knockout mice, but the survival benefit was much smaller than that of scAAV9 / CLN1 administered before symptom onset.

[0149] Figures 4A-5B show the lifespan of CLN1 knockout mice given various doses of scAAV9 / CLN1 via intrathecal, intravenous, or a combination of intrathecal and intravenous administration. The shaded areas indicate the survival range relative to untreated xenograft mice. Notably, Figures 4B and 5B show that for CLN1 knockout mice given scAAV9 / CLN1 at 20 weeks (post-symptom onset), the combination of intrathecal and intravenous administration conferred a significantly greater survival benefit compared to intrathecal or intravenous administration alone.

[0150] Example 3 Combined intrathecal and intravenous administration of AAV expressing PPT1 protein improves the performance of CLN1 knockout mice in behavioral assays Behavioral assays were performed on treated mice to detect behavioral improvements after intrathecal and / or intravenous administration of AAV viral particles expressing PPT1 protein. AAV viral particles were prepared as described in Example 1.

[0151] To test swimming ability, mice (untreated xenografts, untreated knockouts, and knockouts treated with various doses of vector at 4 or 20 weeks of age) were placed in a Morris water maze, consisting of a 122 cm diameter pool filled with 45 cm of water located in a room with numerous visual cues. Each mouse was given four trials per day over 2–3 days to swim to a platform using patterned cylinders extending above the water surface as a cue. For each trial, the mouse was placed in one of four possible positions in the pool (randomized order) and then given 60 s to find the visible platform. If the mouse found the platform, the trial ended, and the animal remained on the platform for 10 s before the next trial began. If the platform was not found, the mouse was placed on the platform for 10 s before the next trial. Tests were performed at different ages, and swimming speed was measured. Figures 6A–6B show the results. Remarkably, among knockout mice treated with vector at 20 weeks, those given a combination of intrathecal and intravenous vector administration showed significantly slower disease progression and generally maintained swimming speed beyond 52 weeks of age, at which point knockout mice given only intrathecal or only intravenous vector died (Figure 6B).

[0152] In the grip strength test, mice (untreated xenogeneic mice, untreated knockout mice, and knockout mice treated with various doses of vector at 4 or 20 weeks of age) were placed on the lid of a large metal cage. The lid was gently shaken to induce the mice to grasp the metal grid. The cage top was then turned over, and the latency for the mice to fall off the lid was recorded. The maximum trial length was 60 seconds. The test was performed at different ages. The latency to fall was measured. Figures 7A-7B show the results. Notably, among knockout mice treated with vector at 20 weeks of age, those receiving a combination of intrathecal and intravenous vector administration showed significantly slower strength loss and performed better in these tasks than those receiving only intrathecal or only intravenous vector administration (Figure 7B).

[0153] Figure 8 shows the normalized physical performance score (PSC) versus relative survival time for various mouse treatment groups. PSC was derived from the area under the curve (spline fit) for the within-treatment average over time, with the different mice normalized to 1. PSC was combined with data from the weight-accelerated rotarod and wire hang. Median survival for the different mice was set at 712 days. Results showed a strong correlation between the relative survival time and relative physical performance of the mice.

[0154] Example 4 Analysis of PPT1 levels and its physiological effects in CLN1 knockout mice administered AAV viral particle-expressed PPT1 protein The efficacy of scAAV9 / CLN1 therapy in neonatal mice was examined. The vector (2.8 × 10 11 vg). Serum PPT1 levels (Figure 9A) and swimming speed (Figure 9B) were examined at different ages. The results show no adverse effects on vector-treated heterozygous mice, despite the long-term expression of supraphysiological levels of serum PPT1 enzyme activity.

[0155] PPT1 enzyme activity was also measured in different tissues of rats treated with the scAAV9 / CLN1 vector. Rats were treated with either vehicle control or the scAAV9 / CLN1 vector according to the doses and routes of administration indicated in Figure 10. The results show that rats treated with the scAAV9 / CLN1 vector exhibited sustained supraphysiological levels of PPT1 enzyme activity across tissues.

[0156] The generation of neutralizing antibodies against AAV9 was also examined in rats treated with the scAAV9 / CLN1 vector. Rats were treated with either vehicle control or the scAAV9 / CLN1 vector according to the doses and routes of administration indicated in Figure 11, and anti-AAV9 neutralizing antibody titers were measured at 4 and 12 weeks. The results show that rats developed neutralizing antibodies against AAV9 regardless of the route of vector administration (intravenous, intrathecal, or a combination of intravenous and intrathecal).

[0157] Example 5 Combined intrathecal and intravenous administration of AAV viral particles delays symptom onset and improves lifespan in CLN1 knockout mice Figure 12 shows a diagram of symptom onset in mice treated with scAAV9 / CLN1 vectors via different administration routes (intrathecal or intrathecal + intravenous combo) at different time points (1, 4, 12, 20, and 26 weeks). The shaded area indicates the survival range relative to untreated xenograft mice. Overall, early treatment with scAAV9 / CLN1 vectors provided greater benefit, but the combination of intrathecal and intravenous administration provided significantly greater benefit than intrathecal administration alone when treatment was administered at later time points (e.g., 20 weeks).

Claims

1. 1. A pharmaceutical composition for treating Infantile Batten Disease (IBD) or an IBD-related disorder in a subject in need thereof, the pharmaceutical composition comprising: first AAV viral particles comprising a polynucleotide comprising a CLN1 gene; and second AAV viral particles comprising a polynucleotide comprising a CLN1 gene, wherein the first AAV viral particles comprise 1.0 x 10 １３ vg / kg~1.0x10 １６ vg / kg is administered intrathecally, followed by administration of 1.0 x 10 of a second AAV viral particle comprising a polynucleotide comprising the CLN1 gene. １２ vg / kg~2.0x10 １５ vg / kg is administered intravenously to treat IBD or an IBD-related disorder, wherein said IBD-related disorder is infantile, late-infantile, juvenile, or adult-onset neuronal ceroid lipofuscinosis.

2. 2. The pharmaceutical composition of claim 1, wherein the polynucleotide of the first AAV viral particle and / or the polynucleotide of the second AAV viral particle comprises a wild-type CLN1 gene sequence.

3. 2. The pharmaceutical composition of claim 1, wherein the polynucleotide of the first AAV viral particle and / or the polynucleotide of the second AAV viral particle comprises a codon-optimized CLN1 gene sequence.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the polynucleotide of the first viral particle and / or the polynucleotide of the second viral particle comprises a codon-optimized CLN1 gene sequence having at least 90% identity to SEQ ID NO:1 or at least 90% identity to SEQ ID NO:

3.

5. the polynucleotide of the first viral particle and / or the polynucleotide of the second viral particle is a) a codon-optimized CLN1 gene sequence comprising the sequence of SEQ ID NO: 1, or b) a nucleotide sequence encoding a polynucleotide sequence comprising the sequence of SEQ ID NO:3 The pharmaceutical composition according to any one of claims 1 to 3, comprising:

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is a human patient.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the intrathecal administration and the intravenous administration are performed after the onset of symptoms or before the onset of symptoms.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the first and second viral particles are independently selected from AAV2, AAV8, AAV6, AAV8, and AAV9 viral particles.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the AAV viral particles comprise one or more of a wild-type capsid protein, a mutant capsid protein, a tissue-tropic capsid protein, or a modified capsid protein having altered tropism compared to the wild-type capsid protein.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the AAV viral particles are AAV9 viral particles.

11. 1.0 x 10 of first AAV viral particles １４ vg / kg~1.0×10 １５ The pharmaceutical composition of any one of claims 1 to 10, wherein vg / kg is administered intrathecally.

12. 1.0 x 10 of the second AAV viral particle １３ vg / kg~2.0×10 １４ The pharmaceutical composition according to any one of claims 1 to 10, wherein vg / kg is administered intravenously.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the polynucleotide of the first viral particle and / or the polynucleotide of the second viral particle is operably linked to a promoter, an enhancer, an intron, and / or a polyadenylation signal.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the first viral particle and / or the second viral particle comprises, in a 5' to 3' direction, an AAV ITR, an enhancer, a promoter, an intron, a polynucleotide comprising a human CLN1 gene, a polyadenylation site, and a vector genome comprising an AAV ITR.

15. 15. The pharmaceutical composition of claim 14, wherein the enhancer is a CMV enhancer, the promoter is a chicken beta actin promoter, the intron is a hybrid / modified MVM intron, and / or the polyadenylation site is a bovine growth hormone polyadenylation site.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the first viral particle and the second viral particle comprise the same vector genome.

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