Adeno-associated virus delivery of CLN6 polynucleotide

Abstract

To provide an rAAV for CLN6 gene therapy of neuronal ceroid lipofuscinosis or CLN6-Batten disease, and a method of using rAAV.SOLUTION: Provided is a self-complementary recombinant adeno-associated virus 9 (scAAV9) encoding a CLN6 polypeptide, which includes, in the order from 5' to 3', a first AAV inverted terminal repeat, a CB promoter including a specific nucleotide sequence, a polynucleotide encoding the CLN6 polypeptide, and a scAAV9 genome including a second AAV inverted terminal repeat.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 800,915, filed Feb. 4, 2019; U.S. Provisional Patent Application No. 62 / 880,641, filed Jul. 30, 2019; U.S. Provisional Patent Application No. 62 / 881,151, filed Jul. 31, 2019; U.S. Provisional Patent Application No. 62 / 912,977, filed Oct. 9, 2019; and U.S. Provisional Patent Application No. 62 / 923,125, filed Oct. 18, 2019, all of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing This application includes, as a separate part of the disclosure, a computer-readable form sequence listing (filename: 53894_SeqListing.txt, created on Jan. 31, 2020, 24,923 bytes, ASCII text file), which is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to recombinant adeno-associated virus (rAAV) delivery of ceroid lipofuscinosis neuronal 6 (CLN6) polynucleotides. The present disclosure provides rAAVs and methods of using rAAVs for the gene therapy of neuronal ceroid lipofuscinosis (NCL) or CLN6-Batten disease.

Background Art

[0004] Neuronal ceroid lipofuscinosis (NCL) is a group of severe neurodegenerative disorders collectively referred to as Batten disease. These disorders affect the nervous system and typically worsen problems related to, for example, mobility and thinking abilities. Different NCLs are distinguished by their genetic causes.

[0005] CLN6 Batten disease can occur in two different forms, the more common late infantile variant (vLINCL), and the adult-onset form of NCL (also called type A Kufs disease) (Cannelii et al., Biochem Biophys Res Commun. 2009;379(4):892-7, Arsov et al., Am J Hum Genet. 2011;88(5):566-73). In vLINCL (referred to herein as CLN6 Batten disease), the age of onset is between 18 months and 6 years, and death usually occurs by 12 to 15 years of age. CLN6 Batten disease first presents as language impairment and delays in motor / cognitive development in infancy, and most patients are wheelchair-bound within 4 years of onset (Canafoglia et al., Neurology. 2015;85(4):316-24). The disease progresses to include vision loss, severe motor impairment, recurrent seizures, dementia and other neurodegenerative symptoms.

[0006] CLN6 is a 311-amino acid protein with seven putative transmembrane domains and is mainly localized in the endoplasmic reticulum. Similar to other CLN proteins, its exact function remains unknown, but it is involved in intracellular trafficking and lysosomal function. There are currently over 70 characterized mutations that cause disease in CLN6 (Warrier et al., Biochimica et Biophysica Acta. 2013;1832(11):1827-30), and most of these mutations result in either complete loss of the CLN6 protein or production of a truncated CLN6 protein product that is highly unstable and / or non-functional. Several natural animal models of CLN6 Batten disease have been described, including models in sheep, dogs, and mice. Cln6 nclf The natural mutation found in the mouse model (referred to herein as the "Cln6 nclf mouse") recapitulates many of the pathological and behavioral aspects of the disease (Morgan et al., PLoS One. 2013;8(11):e78694). Cln6 nclfThe mouse contains an insertion of an additional cytosine (c.307insC, frameshift after P102), resulting in a premature stop codon homologous to the mutations commonly seen in CLN6-Batten disease patients (Gao et al., Am J Hum Genet. 2002;70(2):324-35, Wheeler et al., Am J Hum Genet. 2002;70(2):537-42).

[0007] Currently, there is no treatment that can improve the symptoms of CLN6-Batten disease. Therefore, there remains a need in the art for a treatment for CLN6-Batten disease.

Summary of the Invention

[0008] Provided herein are methods and products for CLN6 gene therapy using recombinant AAV.

[0009] Provided herein is a recombinant adeno-associated virus 9 (rAAV9) encoding a CLN6 polypeptide, comprising a hybrid chicken β-actin (CB) promoter and a polynucleotide encoding a CLN6 polypeptide in a 5' to 3' order, and an rAAV9 genome. Optionally, the rAAV9 genome comprises a self-complementary genome. Alternatively, the rAAV9 genome comprises a single-stranded genome.

[0010] A self-complementary recombinant adeno-associated virus 9 (scAAV9) encoding the CLN6 polypeptide set forth in SEQ ID NO: 1 is provided, wherein the genome of the scAAV9 comprises, in a 5' to 3' order, a first AAV inverted terminal repeat, a hybrid chicken β-actin (CB) promoter comprising the sequence of SEQ ID NO: 3, a polynucleotide encoding the CLN6 polypeptide set forth in SEQ ID NO: 2, and a second AAV inverted terminal repeat. The polynucleotide encoding the CLN6 polypeptide can be at least 90% identical to SEQ ID NO: 2.

[0011] Also provided is an scAAV9 having a genome comprising, in 5' to 3' order, a first AAV inverted terminal repeat, a CMV enhancer, a hybrid chicken β-actin promoter (cb), an SV40 intron, a polynucleotide encoding the CLN6 polypeptide of SEQ ID NO: 1, and a second AAV inverted terminal repeat; an scAAV9 having a genome comprising, in 5' to 3' order, a first AAV inverted terminal repeat, a CB promoter comprising the sequence of SEQ ID NO: 3, a polynucleotide encoding the CLN6 polypeptide of SEQ ID NO: 1, a bovine growth hormone polyadenylation poly A sequence, and a second AAV inverted terminal repeat; and an scAAV9 having a genome comprising the gene cassette described by the nucleic acid sequence of SEQ ID NO: 4.

[0012] Also provided is an ssAAV9 having a genome comprising, in 5' to 3' order, a first AAV inverted terminal repeat, a CMV enhancer, a hybrid chicken β-actin promoter (CB), an SV40 intron, a polynucleotide encoding the CLN6 polypeptide of SEQ ID NO: 1, and a second AAV inverted terminal repeat; an ssAAV9 having a genome comprising, in 5' to 3' order, a first AAV inverted terminal repeat, a CB promoter comprising the sequence of SEQ ID NO: 3, a polynucleotide encoding the CLN6 polypeptide of SEQ ID NO: 1, a bovine growth hormone polyadenylation poly A sequence, and a second AAV inverted terminal repeat; or an ssAAV9 having a genome comprising the gene cassette described by the nucleic acid sequence of SEQ ID NO: 4.

[0013] The nucleic acid sequence set forth in SEQ ID NO: 4 is the gene cassette provided in FIG. 1A. Also provided is an rAAV9 comprising an ssAAV9 genome comprising a nucleic acid sequence that is at least 90% identical to the scAAV9 genome or the nucleic acid sequence of SEQ ID NO: 4, at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4, or at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4.

[0014] Further provided is a nucleic acid molecule comprising a first AAV inverted terminal repeat, a CB promoter comprising the nucleic acid sequence of SEQ ID NO: 3, a nucleic acid sequence encoding a CLN6 polypeptide of SEQ ID NO: 1, and a second AAV inverted terminal repeat. In some embodiments, the polynucleotide encoding the CLN6 polypeptide can be at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0015] Also provided is a nucleic acid molecule comprising a first AAV inverted terminal repeat, a CB promoter comprising the nucleotide sequence of SEQ ID NO: 3, an SV40 intron, a nucleic acid sequence encoding a CLN6 polypeptide of SEQ ID NO: 1, and a second AAV inverted terminal repeat. In addition, provided is a nucleic acid molecule comprising a first AAV inverted terminal repeat, a CB promoter comprising the nucleotide sequence of SEQ ID NO: 3, a nucleic acid encoding a CLN6 polypeptide of SEQ ID NO: 1, a BGH polyA sequence, and a second AAV inverted terminal repeat. In any of the provided polynucleotides, the CLN6 polypeptide can be encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0016] Provided is an rAAV having an scAAV genome or an ssAAV genome, wherein the genome comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4, or at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4, or at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4.

[0017] The provided rAAV can comprise any of the polynucleotides disclosed herein. In addition, provided are viral particles comprising any of the disclosed nucleic acids. Also provided are rAAVs having a self-complementary or single-stranded genome.

[0018] Also provided is a recombinant adeno-associated virus 9 (rAAV9) viral particle encoding a CLN6 polypeptide, comprising an rAAV9 genome comprising, in 5’ to 3’ order, a CMV enhancer comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 6, a CB promoter comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, and a polynucleotide encoding a CLN6 polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the provided rAAV9 viral particle comprises a self-complementary genome. Alternatively, the provided rAAV9 viral particle comprises a single-stranded genome.

[0019] Further provided is an rAAV9 viral particle, wherein the rAAV9 genome comprises, in 5’ to 3’ order, a first AAV inverted terminal repeat, a CMV enhancer comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 6, a CB promoter comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, a polynucleotide encoding a CLN6 polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a second AAV inverted terminal repeat. The provided rAAV9 particle comprises a polynucleotide encoding a CLN6 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. Any of the rAAV9 viral particles optionally further comprises an SV40 intron and / or a BGH polyA sequence.

[0020] In further embodiments, the rAAV9 viral particle comprises an AAV9 genome comprising a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4, at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4, or at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4.

[0021] In any of the provided rAAV, ssAAV, or scAAV, the AAV inverted terminal repeat can be an AAV2 inverted terminal repeat.

[0022] Also provided is a nucleic acid molecule comprising an rAAV9 genome that, in 5' to 3' order, comprises a first AAV inverted terminal repeat, a CMV enhancer comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 6, a CB promoter comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, and a polynucleotide encoding a CLN6 polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1. The provided nucleic acid molecule comprises a self-complementary genome and / or a single-stranded genome.

[0023] Further provided is a nucleic acid molecule comprising an rAAV9 genome that, in 5' to 3' order, comprises a first AAV inverted terminal repeat, a CMV enhancer comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 6, a CB promoter comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, a polynucleotide encoding a CLN6 polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a second AAV inverted terminal repeat. The provided nucleic acid molecule can comprise a polynucleotide encoding a CLN6 polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1. Additionally, the nucleic acid molecule can comprise an AAV9 genome comprising a nucleic acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4. Optionally, any of the provided nucleic acid molecules further comprises an SV40 intron and / or a BGH polyA sequence.

[0024] Further provided is a composition comprising the scAAV9 described herein, the nucleic acid molecule described herein, or the rAAV viral particle described herein, and at least one pharmaceutically acceptable excipient. Optionally, the pharmaceutically acceptable excipient includes a nonionic hypotonic compound, a buffer, a polymer, a salt, or a combination thereof. In some embodiments, the polymer is a copolymer. In some embodiments, the copolymer is a poloxamer. For example, the composition may at least include a pharmaceutically acceptable excipient comprising a nonionic hypotonic compound. For example, the pharmaceutically acceptable excipient may include about 20-40% nonionic hypotonic compound, or about 25% to about 35% nonionic hypotonic compound. An exemplary composition includes scAAV formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% nonionic hypotonic compound. Another exemplary composition includes scAAV formulated in 1×PBS containing 0.001% Pluronic F68.

[0025] Also further provided is a method for treating CLN6-Batten disease in a subject, comprising administering to the subject a therapeutically effective amount of any of the rAAV9 viral particles disclosed herein, any of the scAAV9 disclosed herein, any of the ssAAV disclosed herein, any of the nucleic acid molecules described herein, or any of the compositions described herein.

[0026] Also provided by the present disclosure is the use of a therapeutically effective amount of any of the rAAV9 viral particles disclosed herein, any of the scAAV9 disclosed herein, any of the ssAAV disclosed herein, any of the nucleic acid molecules described herein, or any of the compositions described herein for the preparation of a medicament for the treatment of CLN6-Batten disease.

[0027] Also further provided is a composition for treating CLN6-Batten disease comprising a therapeutically effective amount of any of the rAAV9s disclosed herein, any of the scAAV9s disclosed herein, any of the ssAAVs disclosed herein, any of the nucleic acid molecules described herein, or any of the compositions described herein.

[0028] In any of the provided methods, uses, or compositions for treating CLN6-Batten disease, the composition, rAAV9, scAAV9, or ssAAV and / or nucleic acid molecule is administered via a route selected from the group consisting of intrathecal, intraventricular, intracerebral, intravenous, and combinations thereof.

[0029] Exemplary dosages of scAAV9, ssAAV, or rAAV9 administered by the intrathecal route are from about 1×10 11 vg of scAAV, ssAAV, or rAAV9 virus particles to about 1×10 15 vg of scAAV or AAV9 virus particles, or from about 1×10 12 vg of scAAV, ssAAV, or rAAV9 virus particles to about 1×10 14 vg of scAAV, ssAAV, or AAV9 virus particles. For example, about 1×10 13 vg of scAAV, ssAAV, or rAAV9 virus particles may be administered to a subject, or about 1.5×10 13 of scAAV, ssAAV, or rAAV9 virus particles may be administered to a subject, or about 6×10 13 vg of scAAV, ssAAV, or rAAV9 virus particles may be administered to a subject.

[0030] The methods, uses, or compositions for treating CLN6-Batten disease disclosed herein result in one or more of (a) a decrease or delay in the lysosomal accumulation of autofluorescent storage material, (b) a decrease or delay in the lysosomal accumulation of ATP synthase subunit C, (c) a decrease or delay in glial activation (astrocyte and / or microglia) activation, (d) a decrease or delay in astrocytosis, (e) a decrease or delay in brain volume loss as measured by MRI, (f) a decrease or delay in the onset of seizures, and (g) stabilization, a decrease in progression, or improvement of one or more of the scales used to assess the progression and / or improvement of CLN6 Batten disease, such as the Unified Batten Disease Rating Scale (UBDRS) rating scale, the Hamburg motor and language scale, or the Mullen Scales of Early Learning (MSEL), compared to a subject prior to treatment or an untreated patient with CLN6-Batten disease. The subject can be maintained in the Trendelenberg position after administration of the rAAV9, ssAAV9 viral particles, scAAV, or nucleic acid molecule disclosed herein.

[0031] Also provided is a method of treating a patient in need of treatment for CLN6 disease, comprising delivering to the brain or spinal cord of a patient in need thereof a composition comprising any one of the disclosed rAAV viral particles provided herein, any one of the scAAV9 disclosed herein, any one of the ssAAV9 disclosed herein, any one of the nucleic acid molecules described herein, or any one of the compositions described herein.

[0032] In addition, the present disclosure provides the use of any one of the disclosed rAAV viral particles provided herein, any one of the scAAV9 disclosed herein, any one of the ssAAV9 disclosed herein, any one of the nucleic acid molecules described herein, or any one of the compositions described herein for the preparation of a medicament for delivering the ssAAV9, nucleic acid molecule, or composition to the brain or spinal cord of a patient in need thereof.

[0033] Further provided is a composition comprising any one of the disclosed rAAV viral particles provided herein, any of the scAAV9 disclosed herein, any of the ssAAV9 disclosed herein, any of the nucleic acid molecules described herein, or any of the compositions described herein, for delivering the ssAAV9, nucleic acid molecule, or composition to the brain or spinal cord of a patient in need thereof.

[0034] In any of the provided methods, uses or compositions, the composition can be delivered by intrathecal, intraventricular, intracerebral, or intravenous injection, or combinations thereof. Any of the provided methods further comprises placing the patient in the Trendelenburg position after intrathecal injection of the composition, rAAV9, ssAAV9 or scAAV, or nucleic acid molecule disclosed herein.

[0035] In any of the provided methods or uses, the composition or agent can comprise a non-ionic low-osmolar contrast agent. For example, the composition can comprise a non-ionic low-osmolar contrast agent selected from the group consisting of iopamidol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol,ioxilan, and combinations thereof.

[0036] The administered composition or agent can comprise a pharmaceutically acceptable excipient. For example, the pharmaceutically acceptable excipient can comprise about 20-40% non-ionic low-osmolar compound, or about 25%-about 35% non-ionic low-osmolar compound. Exemplary compositions comprise scAAV formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and about 25%-about 35% non-ionic low-osmolar compound. Another exemplary composition comprises scAAV formulated in 1× PBS and 0.001% pluronic F68.

[0037] In any of the methods, uses, or compositions provided, the composition or agent may be delivered to the brain or spinal cord, the composition or agent may be delivered to the brainstem, or may be delivered to the cerebellum, may be delivered to the visual cortex, or may be delivered to the motor cortex. Further, in any of the methods provided, the composition or agent may be delivered to the brain or spinal cord, and the composition may be delivered to neurons, glial cells, or both. For example, delivery to the brain or spinal cord includes delivery to cells of the nervous system such as neurons, lower motor neurons, microglial cells, oligodendrocytes, astrocytes, Schwann cells, or combinations thereof.

[0038] The methods, uses, and compositions disclosed herein result in one or more of (a) a decrease or delay in lysosomal accumulation of autofluorescent storage material, (b) a decrease or delay in lysosomal accumulation of ATP synthase subunit C, (c) a decrease or delay in glial activation (astrocyte and / or microglia) activation, (d) a decrease or delay in astrocytosis, (e) a decrease or delay in brain volume loss as measured by MRI, (f) a decrease or delay in the onset of seizures, and (g) stabilization, decrease in progression, or improvement of one or more of the measures used to assess the progression and / or improvement of CLN6 Batten disease, such as the Unified Batten Disease Rating Scale (UBDRS) rating scale, the Hamburg motor and language scale, or the Mullen Scales of Early Learning (MSEL), compared to a subject before treatment or an untreated CLN6-Batten disease patient.

[0039] In any of the methods, compositions, and uses described herein, the treatment, composition, or agent stabilizes or delays the disease progression of CLN-6 Batten disease. In particular, disease progression is evaluated using the UBDRS scale, the Hamburg motor and language scale, the impact of treatment on quality of life using the Pediatric Quality of Life (PEDSQOL) scale, the Mullen Scales of Early Learning (MSEL), the likelihood of long-term survival, or combinations thereof.

[0040] In any of the methods, uses, or compositions described herein, the treatment, composition, or agent reduces or delays one or more symptoms of CLN-6 Batten disease selected from the following, compared to untreated CLN6-Batten disease patients: (a) brain volume loss, (b) loss of cognitive function, and (c) language delay. In particular, the treatment stabilizes or delays the disease progression of CLN-6 Batten disease. For example, disease progression is evaluated using the UBDRS scale, the Hamburg motor and language scale, the impact of treatment on quality of life using the Pediatric Quality of Life (PEDSQOL) scale, the Mullen Early Learning Scale (MSEL), the likelihood of long-term survival, or a combination thereof.

[0041] In any of the methods, uses, or compositions described herein, the age of the subject is 80 months or less, 75 months or less, 70 months or less, 65 months or less, 62 months or less, 60 months or less, 55 months or less, 50 months or less, or 40 months or less.

[0042] In view of the lack of effective treatments for CLN6 Batten disease, Cln6 nclf mouse models were used to test the efficacy of introducing functional human CLN6 by adeno-associated virus (AAV)-mediated gene therapy. The preclinical results provided herein suggest that the use of AAV serotype 9 enables efficient expression of human CLN6 protein throughout the CNS where the most affected cells are located. To evaluate the safety of the treatment in a large animal model, three 4-year-old cynomolgus monkeys were administered scAAV9.CB.CLN6 by intrathecal lumbar CSF injection and monitored for up to 6 months after injection. High levels of transgene expression were seen throughout the brains and spinal cords of all animals, and no adverse effects or pathology were observed. Postnatal intracerebroventricular (ICV) injection of scAAV9.CB.CLN6 into the CSF of mice induced persistent transgene expression in vivo in the mice. Administration of scAAV9.CB.CLN6 reduced classical features of this disease, including the accumulation of autofluorescent storage material and ATP synthase subunit C, reactive gliosis, and loss of dendritic spines. Importantly, this gene therapy, since it is Cln6 nclf induced persistent transgene expression in vivo in mice.nclf It prevents many of the motor, memory and learning, and survival impairments in mice, thus providing broad functional benefits. These results strongly emphasize the therapeutic potential of CSF-delivered scAAV9.CB.CLN6 for the treatment of CLN6-Batten disease.

[0043] The headings in this specification are for the convenience of the reader and are not intended to be limiting.

[0044] The use of "may" and "can" in this specification is for the purpose of describing the various embodiments included in the claims and is not for the purpose of indicating uncertainty about the scope of the claims.

Brief Description of the Drawings

[0045]

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Mode for Carrying Out the Invention

[0046] The present disclosure provides methods and products for treating CLN6-Batten disease. The method involves delivering a CLN6 polynucleotide to a subject using an rAAV as a gene delivery vector.

[0047] Adeno-associated virus (AAV) is a replication-defective parvovirus, and its single-stranded DNA genome is approximately 4.7 kb in length, including two 145-nucleotide inverted terminal repeats (ITRs), and can be used to refer to the virus itself or its derivatives. This term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise specified. There are multiple serotypes of AAV. Each serotype of AAV is associated with a specific clade, and its members share serological and functional similarities. Thus, AAV may be referred to by its clade. For example, the AAV9 sequence is called a "clade F" sequence (Gao et al., J. Virol., 78:6381-6388 (2004)). The present disclosure contemplates the use of any sequence within a specific clade, such as clade F. 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 numbers 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 a part of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively, the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), the AAV-11 genome is provided in Virology, 330(2):375-383 (2004), a part of the AAV-12 genome is provided in GenBank accession number DQ813647, and a part of the AAV-13 genome is provided in GenBank accession number EU285562. The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. The sequence of the AAV-B1 genome is provided in Choudhury et al., Mol. The., 24(7):1247-1257 (2016). Cis-acting sequences that direct viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 relative to their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.In combination with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), four rep proteins (rep78, rep68, rep52, and rep40) are produced from the rep gene by two rep promoters (p5 and p19). The rep proteins have multiple enzymatic properties that ultimately participate in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved 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).

[0048] AAV has unique characteristics that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells and enables the possibility of targeting many different tissues in vivo. Additionally, AAV can transduce both slowly dividing and non-dividing cells and can persist essentially throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The native AAV proviral genome is infectious as cloned DNA in a plasmid that enables the construction of recombinant genomes. Further, since signals that direct AAV replication, genome capsid formation, and integration are contained within the ITRs of the AAV genome, it can be contacted with foreign DNA such as a promoter, DNA of interest, and a gene cassette containing a polyadenylation signal that can replace some or all of the approximately 4.3 kb internal of the genome (encoding replication and structural capsid proteins, rep-cap). In some cases, the rep and cap proteins are provided in trans. Another important feature of AAV is that it is a very stable and robust virus. This readily withstands the conditions (several hours at 56 °C to 65 °C) used to inactivate adenovirus, reducing the importance of cryopreservation of AAV. AAV can be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0049] As used herein, the term "AAV" refers to wild-type AAV virus or viral particles. The terms "AAV", "AAV virus", and "AAV viral particles" are used interchangeably herein. The term "rAAV" refers to recombinant AAV virus or recombinant infectious encapsulated viral particles. The terms "rAAV", "rAAV virus", and "rAAV viral particles" are used interchangeably herein.

[0050] The term "rAAV genome" refers to a polynucleotide sequence derived from a native AAV genome that has been modified. In some embodiments, the rAAV genome has been modified to remove the native cap and rep genes. In some embodiments, the rAAV genome includes endogenous 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV genome includes ITRs from an AAV serotype different from the AAV serotype from which the AAV genome is derived. In some embodiments, the rAAV genome includes a transgene of interest (e.g., a polynucleotide encoding CLN6) flanked at the 5' and 3' ends by inverted terminal repeats (ITRs). In some embodiments, the rAAV genome includes a "gene cassette". An exemplary gene cassette is depicted in FIG. 1A and the nucleic acid sequence is set forth in SEQ ID NO: 4. The rAAV genome can be a self-complementary (sc) genome, referred to herein as an "scAAV genome". Alternatively, the rAAV genome can be a single-stranded (ss) genome, referred to herein as an "ssAAV genome".

[0051] The term "scAAV" refers to an rAAV virus or rAAV viral particle that includes a self-complementary genome. The term "ssAAV" refers to an rAAV virus or rAAV viral particle that includes a single-stranded genome.

[0052] The rAAV genomes provided herein can include a polynucleotide encoding a CLN6 polypeptide. The CLN6 polypeptide includes the amino acid sequence set forth in SEQ ID NO: 1 or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 and encodes a polypeptide having CLN6 activity (e.g., an increase in the clearance of lysosomal autofluorescent storage material, a decrease in the lysosomal accumulation of ATP synthase subunit C, and at least one of a decrease in the activation of astrocytes and microglia in a patient when treated compared to the patient prior to treatment).

[0053] The rAAV genomes provided herein optionally include a polynucleotide encoding a CLN6 polypeptide, which polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO:2, or a polynucleotide that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO:2, and encodes a polypeptide having CLN6 activity (e.g., an increase in clearance of lysosomal autofluorescent storage material, a decrease in lysosomal accumulation of ATP synthase subunit C, and at least one of a decrease in activation of astrocytes and microglia in a patient when treated as compared to the patient prior to treatment).

[0054] In some embodiments, the rAAV genomes provided herein encode a polypeptide having CLN6 activity and include a polynucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:2, or its complement. The term "stringent" is used to refer to conditions generally understood in the art as being stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68°C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42°C. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

[0055] The rAAV genomes provided herein, in some embodiments, include one or more AAV ITRs adjacent to a polynucleotide encoding a CLN6 polypeptide. The CLN6 polynucleotide is operably linked to a transcriptional control element (including, but not limited to, a promoter, an enhancer, and / or a polyadenylation signal sequence) that is functional in a target cell to form a gene cassette. Examples of promoters are the chicken β-actin promoter and the P546 promoter. Additional promoters contemplated herein include, but are not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, and human gene promoters (e.g., but not limited to, the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter). Further provided herein is the CB promoter sequence set forth in SEQ ID NO: 3, a promoter having CB transcriptional promoting activity, and a promoter sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 3. Other examples of transcriptional control elements are tissue-specific control elements, such as promoters that enable specific expression within neurons or that enable specific expression within astrocytes. Examples include the neuron-specific enolase and glial fibrillary acidic protein promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline-regulated promoter.The gene cassette may also contain intron sequences that facilitate the processing of the CLN6 RNA transcript when expressed in mammalian cells. An example of such an intron is the SV40 intron.

[0056] "Packaging" refers to the series of intracellular events that result in the assembly and capsid formation of AAV particles. The term "production" refers to the process of producing rAAV (infectious, encapsulated rAAV particles) by packaging cells.

[0057] The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication protein and capsid-forming protein of adeno-associated virus, respectively. AAV rep and cap are referred to herein as AAV "packaging genes".

[0058] A "helper virus" for AAV refers to a virus that enables AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of such helper viruses for AAV, including adenoviruses, herpesviruses, and poxviruses such as vaccinia virus, are known in the art. Adenoviruses can include several different subgroups, but adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and are available from depository institutions such as the ATCC. Herpesviridae viruses include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), and these are also available from depository institutions such as the ATCC.

[0059] The "helper virus function" refers to a function encoded by a helper virus genome that enables AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, the "helper virus function" can be provided in many ways, including by providing a helper virus or by providing, for example, a polynucleotide sequence encoding the necessary function in trans to producer cells.

[0060] The rAAV genomes provided herein lack AAV rep and cap DNA. The AAV DNA within the rAAV genomes contemplated herein (e.g., ITRs) can be from any AAV serotype suitable for deriving recombinant viruses, 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 rh.74, and AAV-B1. As described above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. rAAV with capsid mutations are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Modified capsids herein are also contemplated and include capsids with various post-translational modifications such as glycosylation and deamidation. Deamidation of asparagine or glutamine side chains to convert asparagine residues to aspartic acid or isoaspartic acid residues and conversion of glutamine to glutamic acid or isoglutamic acid are contemplated in the rAAV capsids provided herein. See, for example, Giles et al. Molecular Therapy, 26(12):2848-2862 (2018). The modified capsids herein are also contemplated to include targeting sequences that direct rAAV to diseased tissues and organs in need of treatment.

[0061] The DNA plasmids provided herein contain the rAAV genomes described herein. The DNA plasmids are introduced into cells that are permissive to infection by an AAV helper virus (e.g., an adenovirus, an E1-deleted adenovirus, or a herpes virus) for assembling the rAAV genome into infectious virus particles using the AAV9 capsid protein. Techniques for producing rAAV in which the rAAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cells are standard in the art. Production of rAAV particles requires that the following components, the rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not present therein), and the helper virus functions, be present within a single cell (referred to herein as a packaging cell). The rep and cap genes of AAV may be derived from any AAV serotype from which the recombinant virus may be derived, and may be derived from an AAV serotype different from the rAAV genome ITR. Generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety. In various embodiments, the AAV capsid protein may be modified to enhance delivery of the recombinant rAAV. Modifications to the capsid protein are generally known in the art. See, for example, US2005 / 0053922 and US2009 / 0202490, the entire disclosures of which are incorporated herein by reference.

[0062] The method of generating packaging cells is to create a cell line that stably expresses all the components necessary for the production of rAAV. For example, a plasmid (or plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as the neomycin resistance gene may be integrated into the cell's genome. The rAAV genome may be introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Subsequently, the packaging cell line can be infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method is to use an adenovirus or baculovirus instead of a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0063] The general principles of rAAV particle production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mo1. Cell. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988), Samulski et al. (1989, J. Virol., 63:3822-3828), U.S. Patent No. 5,173,414, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, and the portions of the documents regarding rAAV particle production are particularly emphasized.

[0064] Also provided herein are packaging cells that produce infectious rAAV particles. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (a 293-derived cell line). In another embodiment, the packaging cells can be cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (African green monkey fetal lung cells).

[0065] Also provided herein are rAAVs (e.g., infectious encapsidated rAAV particles) that contain the rAAV genome of the present disclosure. The genome of the rAAV lacks AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the rAAV genome. The rAAV genome can be a self-complementary (sc) genome. An rAAV having an sc genome is referred to herein as scAAV. The rAAV genome can be a single-stranded (ss) genome. An rAAV having a single-stranded genome is referred to herein as ssAAV.

[0066] An exemplary rAAV provided herein is an scAAV designated "scAAV9.CB.CLN6". The scAAV9.CB.CLN6 scAAV contains an scAAV genome that includes the human CLN6 cDNA under the control of the hybrid chicken β-actin (CB) promoter (SEQ ID NO: 3). The scAAV genome also includes an SV40 intron (upstream of the human CLN6 cDNA) and a bovine growth hormone polyadenylation (BGH polyA) terminator sequence (downstream of the human CLN6 cDNA). The sequence of this scAAV9.CB.CLN6 gene cassette is set forth in SEQ ID NO: 4. The scAAV genome is packaged in an AAV9 capsid and includes AAV2 ITRs (one ITR is upstream of the CB promoter and the other ITR is downstream of the BGH polyA terminator sequence).

[0067] rAAV can be purified by standard methods in the art, for example, by column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper virus are known in the art and can include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0068] Compositions comprising rAAV are also provided. The compositions comprise rAAV encoding a CLN6 polypeptide. The compositions can comprise two or more rAAV encoding different polypeptides of interest. In some embodiments, the rAAV is scAAV or ssAAV.

[0069] The compositions provided herein comprise rAAV and one or more pharmaceutically acceptable excipients. Acceptable excipients are non-toxic to the recipient, and preferably are inert at the dosages and concentrations employed, and include buffers such as phosphates [e.g., phosphate buffered saline (PBS)], citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, copolymers such as poloxamer 188, pluronics (e.g., pluronic F68) or polyethylene glycol (PEG), but are not limited thereto. The compositions provided herein can include a pharmaceutically acceptable aqueous excipient containing a nonionic hypoosmotic compound such as iodixanol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, and the aqueous excipient containing the nonionic hypoosmotic compound can have one or more of the following properties: a weight osmolality by vapor pressure osmometry of about 180 mgl / mL, about 322 mOsm / kg water, a volume osmolality of about 273 mOsm / L, an absolute viscosity of about 2.3 cp at 20° C. and about 1.5 cp at 37° C., and a specific gravity of about 1.164 at 37° C. Exemplary compositions include about 20-40% nonionic hypoosmotic compound, or about 25%-about 35% nonionic hypoosmotic compound. Exemplary compositions include scAAV or rAAV viral particles formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and about 25%-about 35% nonionic hypoosmotic compound. Another exemplary composition includes scAAV formulated in 1× PBS and 0.001% pluronic F68.

[0070] The dosage of rAAV administered by the methods of the present disclosure will vary, for example, according to the particular rAAV, mode of administration, timing of administration, treatment goal, individual, and cell type targeted, and can be determined by standard methods in the art. The dosage may be expressed in units of viral genome (vg). Dosages contemplated herein are about 1×10 11 about 1×10 12 about 1×10 13 about 1.1×10 13 about 1.2×10 13 about 1.3× 13 about 1.5×10 13 about 2×10 13 about 2.5×10 13 about 3×10 13 about 3.5×10 13 about 4×10 13 about 4.5×10 13 about 5×10 13 about 6×10 13 about 1×10 14 about 2×10 14 about 3×10 14 about 4×10 14 about 5×10 14 about 1×10 15 about 1×10 16 up to, or more than, including a total viral genome. About 1×10 11 to about 1×10 15 vg, about 1×10 12 to about 1×10 15 vg, about 1×10 12 to about 1×10 14 vg, about 1×10 13 to about 6×10 14 vg, and dosages of about 6×10 13 to about 1.0×10 14 vg are also contemplated. One dosage exemplified herein is 6×10 13 vg. Other dosages exemplified herein are 1.5×10 13 .

[0071] A method of transducing a target cell (including, but not limited to, cells of the nervous system, neurons or glial cells) with rAAV is provided. Cells of the nervous system include neurons, lower motor neurons, microglial cells, oligodendrocytes, astrocytes, Schwann cells, or combinations thereof.

[0072] The term "transduction" is used to refer to the administration / delivery of a CLN6 polynucleotide to a target cell, either in vivo or in vitro, via a replication-deficient rAAV of the present disclosure that results in the expression of a functional polypeptide by the recipient cell. Transduction of cells with the rAAV of the present disclosure results in the persistent expression of a polypeptide or RNA encoded by the rAAV. Accordingly, the present disclosure provides a method of administering / delivering an rAAV encoding a CLN6 polypeptide to a subject by an intrathecal, intraventricular, intracerebral, or intravenous route, or any combination thereof. Intrathecal delivery refers to delivery into the space under the arachnoid membrane of the brain or spinal cord. In some embodiments, intrathecal administration is by cisternal administration.

[0073] Intrathecal administration is exemplified herein. These methods include transducing one or more of the rAAVs described herein into a target cell (including, but not limited to, neurons and / or glial cells). In some embodiments, rAAV viral particles containing a polynucleotide encoding a CLN6 polypeptide are administered or delivered to a patient's brain and / or spinal cord. In some embodiments, the polynucleotide is delivered to the brain. Regions of the brain where delivery is contemplated include, but are not limited to, the motor cortex, visual cortex, cerebellum, and brainstem. In some embodiments, the polynucleotide is delivered to the spinal cord. In some embodiments, the polynucleotide is delivered to lower motor neurons. The polynucleotide can be delivered to neurons and glial cells. Glial cells are microglial cells, oligodendrocytes, or astrocytes. In some embodiments, the polynucleotide is delivered to Schwann cells.

[0074] In some embodiments of the methods provided herein, the patient is maintained in the Trendelenburg position (head-down position) for a period of time (e.g., about 5, about 10, about 15, or about 20 minutes) after administration of rAAV. For example, the patient may be tilted at a position with the head lowered by about 1 degree to about 30 degrees, about 15 to about 30 degrees, about 30 to about 60 degrees, about 60 to about 90 degrees, or about 90 to about 180 degrees.

[0075] The methods provided herein include administering an effective dose or effective multiple doses of a composition comprising the rAAV provided herein to a subject in need thereof (e.g., an animal including, but not limited to, a human patient). When the dose is administered prior to the onset of CLN6-Batten disease, the administration is prophylactic. When the dose is administered after the onset of CLN6-Batten disease, the administration is therapeutic. An effective dose is a dose that alleviates (eliminates, stabilizes, or reduces) at least one symptom associated with the disease, delays or prevents the progression of the disease, reduces the scope of the disease, brings about remission (partial or complete) of the disease, and / or extends survival. Compared to a subject prior to treatment or compared to an untreated subject, the methods provided herein result in stabilization, decreased progression, or improvement of one or more of the measures used to assess the progression and / or improvement of CLN6 Batten disease, such as the Unified Batten Disease Rating System (UBDRS) or the Hamburg Motor and Language Scale, or the Mullen Scales of Early Learning (MSEL). The UBDRS assessment scales (Marshall et al., Neurology. 2005 65(2):275-279) [including the UBDRS Physical Assessment Scale, the UBDRS Seizure Assessment Scale, the UBDRS Behavior Assessment Scale, the UBDRS Ability Assessment Scale, the UBDRS Symptom Onset Order, and the UBDRS Clinical Global Impression (CGI)], the Pediatric Quality of Life Scale (PEDSQOL) measure motor function, language function, cognitive function, and survival. Compared to a subject prior to treatment or compared to an untreated subject, the methods provided herein result in one or more of the following: a decrease or delay in the lysosomal accumulation of autofluorescent storage material, a decrease or delay in the lysosomal accumulation of ATP synthase subunit C, a decrease or delay in glial activation (astrocyte and / or microglia) activation, a decrease or delay in astrocytosis, and a decrease or delay in brain volume loss as measured by MRI.

[0076] Combination therapies are also provided. As used herein, combination includes either concurrent or sequential treatment. Combinations of the methods described herein with standard drug therapies are specifically contemplated. Further specifically contemplated are combinations of compositions for use in accordance with the invention, either concurrently or sequentially (e.g., a combination of scAAV9.P546.CLN6 with a contrast agent disclosed herein).

[0077] Delivery to subjects that require postnatal delivery is contemplated, but intrauterine delivery to the fetus is also contemplated.

Examples

[0078] The following examples illustrate specific embodiments, it being understood that modifications and variations will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be imposed on the invention.

[0079] In the example, self-complementary AAV carrying CLN6 cDNA (designated scAAV9.CB.CLN6) was produced under the control of the hybrid chicken β-actin (CB) promoter. Intraventricular injection (6×10 13 vg / animal) into the CSF of day 1 postnatal mice was sufficient to induce stable and robust expression of CLN6 protein throughout the CNS for up to 18 months. Progression of CLN6-Batten disease is associated with accumulation of ASM, aggregation of ATP synthase subunit C, decrease in synapse spine density, increase in GFAP reactivity in astrocytes, and increase in CD68 staining in microglia. Cln6 nclf mice show increases in ASM and ATP synthase subunit C and decrease in dendritic spines at 2 months, and increases in GFAP and CD68 reactivity up to 6 months of age. CLN6 nclf Injection of scAAV9.CB.CLN6 into mice decreased the accumulation of ASM and ATP synthase subunit C, increased the dendritic spine density, and decreased the levels of CD68+ microglia and GFAP+ astrocyte reactivity.

[0080] Example 1 Production of scAAV9.CB.CLN6 The human CLN6 cDNA clone was obtained from Origene, Rockville, MD. The hCLN6 cDNA was further subcloned into the AAV9 genome under the hybrid chicken β-actin promoter (CB) and tested in vitro and in vivo. A self-complementary adeno-associated virus (scAAV) serotype 9 viral genome containing the human CLN6 (hCLN6) gene under the control of the chicken β-actin (CB) hybrid promoter was generated. A schematic diagram of the plasmid construct showing the CLN6 cDNA inserted between the AAV2 ITRs is provided in FIG. 1A. The plasmid construct also contains the CP promoter, simian virus 40 (SV40) chimeric intron, and bovine growth hormone (BGH) polyadenylation signal (BGH polyA).

[0081] scAAV9.CB.CLN6 was produced under cGMP conditions by transient triple plasmid transfection using the double-stranded AAV2-ITR-based CB-CLN6 vector, a plasmid encoding the Rep2Cap9 sequence, together with the adenovirus helper plasmid pHelper (Stratagene, Santa Clara, CA) in HEK293 cells (36) as described previously (Gao et al., J. Virol., 78:6381-6388 (2004)). The purity and titer of the vector were evaluated by 4-12% sodium dodecyl sulfate-acrylamide gel electrophoresis and silver staining as well as qPCR analysis. After cloning, the expression of the transgene was confirmed in vitro in HEK293 cells and in vivo via uterine ICV electroporation on embryonic day 15.5 (see FIGS. 1B and 1C). This analysis confirmed neuronal targeting and the expression of human CLN6 protein in vivo.

[0082] Example 2 CLN6 nclf Expression analysis of CSF-delivered scAAV9.CB.CLN6 in mice Cell targeting and expression To confirm the expression and in vivo distribution of virus-introduced human CLN6 in mice, scAAV9.CB.CLN6 was administered to CLN6 mice within 24 hours after birth via a single intracerebroventricular (ICV) injection, and the expression was monitored at various time points for 2 months. Wild-type mice and CLN6 mice injected with an equal volume of PBS were used as controls. The effective dose was 5×10 nclf vg / mouse using the NCH virus vector core titer. scAAV9.CB.CLN6 was formulated in 1×PBS and 0.001% Pluronic F68, or in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188. nclf Examination of hCLN6 expression by RT-PCR at 2, 6, and 18 months after injection showed persistent and robust hCLN6 expression in the cortex of scAAV9.CB.CLN6-injected Cln6 mice compared to PBS-injected controls (Figures 2A, 3A). These results were similar to previously reported scAAV9-CB-GFP expression levels (see, for example, Foust et al. Mol Ther. 2013;21(12):2148-59, Foust et al. Nat Biotechnol. 2010:28(3):271-4, Meyer et al. Mol Ther. 2015;23(3):477-87). In Figure 2A, the upper gel and graph are representative RT-PCR gels and densitometry (normalized to GAPDH). These data demonstrated an increase in gene expression after scAAV9.CB.CLN6 delivery compared to PBS-injected Cln6 mice. The lower gel and graph show CLN6 protein expression measured by Western blotting. ICV delivery of the scAAV9.CB.CLN6 vector showed a significant increase in hCLN6 protein expression in the cerebral cortex of CLN6 mice. 10

[0083] nclf nclf nclf

[0084] ​​​​​To examine the local distribution of transgene expression, hCLN6 transcripts were visualized using a modified in situ hybridization method called RNAScope (copyright). scAAV9.CB.CLN6 injected Cln6 nclf mice maintained widespread transduction of hCLN6 over all regions of the brain at 2, 6, and 18 months, including the somatosensory cortex and the VPM / VPL nuclei of the thalamus (Cln6 nclf two regions that have been shown to be affected earliest in the disease progression of mice) (Figure 2B, left panel; Figure 3B; upper panel, Figure 4A).

[0085] To examine the expression of hCLN6 protein in the CNS, immunoblotting of cortical brain lysates collected from scAAV9.CB.CLN6 injected Cln6 nclf and PBS injected controls was performed using an anti-hCLN6 antibody. Consistent with what was seen with RNA expression, robust hCLN6 protein expression was seen throughout the CNS at 2, 6, and 18 months of age (Figure 2B, right panel, Figure 3B, lower panel). Furthermore, immunolabeling of brain tissue using an anti-hCLN6 antibody confirmed expression throughout the brains of scAAV9.CB.CLN6 treated Cln6 nclf mice (Figure 4B). Collectively, these findings demonstrated that CSF delivery of scAAV9.CB.CLN6 by ICV injection can stably produce hCLN6 transcripts and protein in disease-relevant regions of the CNS.

[0086] Improvement of pathology after delivery of scAAV9.CB.CLN6 Accumulation of autofluorescent storage material (ASM) The accumulation of the autofluorescent storage material (ASM) is a prominent histological marker for the progression of Batten disease (Mole et al., Biochim Biophys Acta - Mol Basis Dis. 2015;1852(10):2237 - 2241, Cotman et al., Clin Lipidol. 2012 Feb;7(1):79 - 91, Seehafer et al., Neurobiol Aging. 2006;27:576 - 588). The accumulation of ASM is a powerful indicator of disease progression for many forms of Batten disease (Bosch et al., J Neurosci. 2016;36(37):9669 - 9682, Morgan et al., PloS One. 2013;8(11):e78694). It is contemplated herein that a decrease in ASM be used as an indicator of treatment success.

[0087] At 2, 6, and 18 months after treatment, Cln6 mice injected with scAAV9.CB.CLN6 nclf showed a decrease in the accumulation of ASM in the VPM / VPL nuclei of the thalamus and in the somatosensory cortex of the brain compared to PBS - injected mice (Figure 5, Figure 3C). Since PBS - treated Cln6nclf mice die by 15 months of age (Figure 9D), terminally ill 12 - to 14 - month - old PBS - treated Cln6 nclf mice were used for comparison with 18 - month - old scAAV9.CB.CLN6 - treated Cln6 nclf mice. In particular, the amount of ASM accumulation in these 18 - month - old scAAV9.CB.CLN6 - injected Cln6 nclf mice was comparable to that of age - matched untreated wild - type mice. Figure 9E shows that male (left panel) and female (right panel) scAAV9.CB.CLN6 - treated mice have weights similar to wild - type mice by age, but untreated Cln6 nclf mice were shown to lose weight during the course of the study. Cln6 mice injected with PBS nclf (Cln6 nclf PBS) began to lose weight at approximately 10 - 11 months of age (male) and 13 - 14 months of age (female).

[0088] Accumulation of mitochondrial protein ATP synthase subunit C The accumulation of ATP synthase subunit C was analyzed in the brain tissues from wild-type, PBS-injected CLN6 nclf mice or scAAV9.CB.CLN6-injected Cln6 nlcf mice. In healthy individuals, this protein is part of the respiratory chain in the mitochondrial membrane, but in patients with Batten disease, this protein abnormally accumulates in lysosomes (Palmer et al., Am J Med Genet. 1992;42(4):561-567). In Cln6 nclf mice, compared with wild-type animals, the accumulation of subunit C became apparent by 2 months of age in the posterior medial ventral nucleus and posterior lateral ventral nucleus (VPM / VPL region) of the thalamus, which is a brain region that is often affected early in the NCL mouse model (Morgan et al., PLoS One. 2013;8(11):e78694, Pontikis et al., Neurobiol Dis. 2005;20(3):823-836). At 2 months, 6 months, and 18 months of age, Cln6 nclf mice treated with scAAV9.CB.CLN6 had a significantly decreased level of accumulation of ATP synthase subunit C in the VPM / VPL and the somatosensory cortex of the brain compared with Cln6 nclf control mice injected with PBS (Figure 6; Figure 3C; lower panel).

[0089] Activation of glial cells and astrocytes In addition to abnormal accumulation of storage materials and accumulation of ATP synthase subunit C, other histological markers of disease progression in both human patients and animal models include activation of astrocytes and microglia (Cotman et al., Hum Mol Genet. 2002;11(22):2709-2721, Morgan et al., PLoS One. 2013;8(11):e78694, Pontikis et al., Neurobiol Dis. 2005;20(3):823-836, Palmer et al., Am J Med Genet. 1992;42(4):561-567). In particular, reactive microglia are primed to release inflammatory mediators such as IL1-β26, which can be a major cause of neuronal death in the late stages of CLN6-Batten disease. At 6 and 18 months of age, Cln6 mice treated with scAAV9.CB.CLN6 nclf mice had significantly reduced astrocyte activation (GFAP) and microgliosis (CD68) in the VPM / VPL and somatosensory cortex compared to moribund PBS-treated Cln6 nclf mice (Figure 7 and Figure 8, respectively).

[0090] Figure 7 demonstrates that activated astrocytes were identified in VPM / VPL and somatosensory cortex sections of the thalamus by staining for glial fibrillary acidic protein (GFAP) at 6 and 18 months of age. The graph shows total GFAP+ immunoreactivity.

[0091] Glia activation was also determined in VPM / VPL and somatosensory cortex sections using anti-CD68 staining as a marker of activated microglia. CD68 is a lysosomal protein that is upregulated in cells primed for inflammatory functions such as phagocytosis (Seehafer et al., J Neuroimmunol. 2011;230:169-172). Figure 8 shows that scAAV9.CB.CLN6 injection reduced CD68 immunoreactivity in the somatosensory cortex of 6-month-old Cln6 nclf mice, as well as in the somatosensory cortex of 18-month-old Cln6 nclfDemonstrate a reduction in microgliosis (CD68 reactivity) in both the VPM / VPL of the mouse and the somatosensory cortex. The graph shows total CD68+ immunoreactivity. The inset in Figure 8 shows the morphology of microglia. Untreated Cln6 analyzed in these studies nclf The mice were moribund, and many of the microglia were presumably dying or dead, which is worth noting as contributing to their abnormal morphology. In summary, these results show that a single injection of scAAV9.CB.CLN6 delivered to the CSF on postnatal day 1 nclf can reduce or delay many of the pathological conditions of classical CLN6-Batten disease in the brains of Cln6 mice.

[0092] Improvement in behavior after delivery of scAAV9.CB.CLN6 In an efficacy study of scAAV9.CB.CLN6 starting at 2 months of age and continuing at 2-month intervals, mice underwent a series of behavioral test paradigms including an accelerating rotarod assay and pole climbing to test motor function and coordinated movement, and a Morris water maze to evaluate learning and memory. The animals were followed for 24 months after injection and the study is ongoing.

[0093] Rotarod assay Previous studies have demonstrated that the Cln6 mouse model of CLN6-Batten disease recapitulates many of the movement disorders, cognitive impairments, and survival impairments seen in humans (Morgan et al., PLoS One. 2013;8(11):e78694). In the efficacy study, the rotarod was used as a classical measure of coordinated movement, and PBS-injected Cln6 nclf mice began to show a decline in rotarod performance at 8 months of age compared to wild type. However, Cln6 nclf nclf ​Injection of scAAV9.CB.CLN6 in mice prevented this decline, and the effect persisted throughout the test period (24 months) (Figure 9A). To study the effect of coordinated movement in more detail, animals were subjected to various motor tasks (hindlimb grip, ability to lower themselves from a ledge, and gait assessment) at 12, 18, and 24 months of age, evaluated using a scoring matrix, where the highest score indicated the worst prognosis (Guyenet et al., Journal of visualized experiments: JoVE.201039). PBS-treated Cln6 nclf Compared to PBS-treated mice, mice treated with scAAV9.CB.CLN6 showed significantly lower overall scores at all time points, and these scores increased slightly at 24 months of age (Figure 9B).

[0094] Morris water maze test In the Morris water maze test, animals were placed in a pool filled with water containing a hidden platform. After training, the time it took the animals to find the hidden platform using environmental cues for orientation was measured as an indication of learning and memory ability.

[0095] PBS-treated Cln6 nclf Mice did not function well in tasks starting at 9 months of age, as indicated by a decline in these abilities to find the hidden platform (Figure 9C). PBS-treated Cln6 nclf Since the swimming speed of mice decreased significantly at 11 and 12 months of age, no conclusions could be drawn about their memory and learning abilities at these later time points (Figure 10A). Cln6 by scAAV9.CB.CLN6 nclfTreatment of the mice corrected this memory and learning impairment up to 12 months after injection (Figure 9C). When wild-type mice were compared to scAAV9.CB.CLN6-treated animals only at later time points in the Morris water maze test, even the treated mice required more time to find the platform at 18 and 24 months, while the swimming speed was the same among all test groups (Figure 9C, Figure 10). To evaluate memory and learning at later time points, mice were subjected to a water maze reversal test in which the platform was moved to a new location at 12, 18, and 24 months of age. Cln6 mice treated with scAAV9.CB.CLN6 nclf Similar to this test, Cln6 mice took significantly longer to find the location of the new platform compared to wild-type mice (Figure 10). Collectively, these results indicate that single treatment with scAAV9.CB.CLN6 prevented much of the motor decline seen in these animals, but did not completely avoid memory and learning impairments when the mice were tested at later time points.

[0096] Improvement in survival after delivery of scAAV9.CB.CLN6 Cln6 nclf Cln6 mice are known to have reduced survival compared to their wild-type counterparts (Guyenet et al., Journal of visualized experiments: JoVE. 201039). Survival of scAAV9.CB.CLN6- and PBS-injected Cln6 nclf mice was compared to that of PBS-injected wild-type mice. Cln6 nclf Single ICV injection of scAAV9.CB.CLN6 into the CSF of Cln6 nclf mice significantly increased their survival compared to PBS-injected Cln6 nclf mice (Figure 9D). The median survival period of PBS-treated mice was 14 months, while scAAV9.CB.CLN6-treated Cln6 nclf mice had a median survival period of 21.5 months. This 65% increase in survival rate was highly significant. Furthermore, the survival curve of scAAV9.CB.CLN6-treated Cln6

[0097] Furthermore, as a measure of overall health, body weight was recorded monthly. Although no differences were observed compared to wild-type animals, the improvement in health status and survival was also emphasized by the ability of the scAAV9.CB.CLN6-treated mice to maintain their body weight, while the PBS-treated Cln6 nclf began to lose weight at approximately 10 - 12 months (Figure 9E).

[0098] A safety study was conducted using 172 wild-type mice treated with PBS and 223 wild-type mice treated with 5×10 10 vg / animal. This study demonstrated that scAAV9.CB.CLN6 showed good tolerance up to 24 weeks and had no adverse effects due to the virus (data not shown). In summary, this is the longest survival extension to date in the CLN6 nclf mouse model and shows the utility of single-dose treatment with scAAV9.CB.CLN6 to reverse both the cytotoxicity and dysfunction of CLN6-Batten disease.

[0099] Example 3 Safety study of scAAV9.CB.CLN6 in non-human primates To test the safety of this treatment in a large animal model relevant to human patients, three 4-year-old male cynomolgus monkeys were administered scAAV9.CB.CLN6 formulated in 1×PBS and 0.001% Pluronic F68.

[0100] The animals were euthanized at 1, 3, or 6 months after injection. Each individual received a single intralumbar injection to directly deliver the viral vector to the CSF at a dose of 6×10 13 viral particles per animal. After injection, the animals were held in the Trendelenburg position with their heads angled downward at 45 degrees for 15 minutes to facilitate targeting of the brain and upper spinal cord regions.

[0101] All subjects recovered well from the injection and showed no abnormal behavior. Hematological and serum chemistry tests were performed at up to five time points (baseline, 1, 2, 3, and 6 months) during the study, and no major abnormalities were revealed. In particular, no evidence of elevated aspartate aminotransferase (AST) or alkaline phosphatase levels was seen, while alanine aminotransferase (ALT) increased slightly in one animal at 1 month after injection (less than 200 units per liter) (Figure 11C).

[0102] No changes were seen in total protein levels, creatinine, triglycerides, glucose, or ions such as phosphorus, calcium, magnesium, or sodium levels. At the time of euthanasia, extensive histopathology and transgene expression analysis were performed on each animal. At necropsy, no abnormalities were seen in the tissues analyzed, including various brain and spinal cord regions, heart, lungs, liver, spleen, kidneys, small intestine, skeletal muscle (diaphragm, triceps brachii, TA, gastrocnemius), and gonads in animals without bladder infections.

[0103] A single lumbar intrathecal injection delivering scAAV9.CB.CLN6 to the cerebrospinal fluid induced high transgene expression throughout the brain and spinal cord of non-human primates, as shown by fluorescent Western blot. The blot in Figure 11A shows CLN6 expression in the cortex, corpus callosum, periventricular white matter, hippocampus, cerebellum, thalamus, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord, and high transgene expression was seen throughout the brain and spinal cord in all three animals (Figures 11A - B). Collectively, these data indicate that treatment with scAAV9.CB.CLN6 was well tolerated and safe in all three individuals tested.

[0104] Example 4 Clinical Trial of scAAV9.CB.CLN6 Gene Therapy scAAV9.CB.CLN6 is delivered intrathecally to human patients with CLN6-Batten disease.

[0105] The scAAV for clinical trials was manufactured by the Nationwide Children’s Hospital clinical manufacturing facility using the triple transfection method of HEK293 cells under GMP conditions as described in Example 1.

[0106] Patients selected for participation were 1 year of age or older and diagnosed with CLN6 disease as determined by genotype. The first cohort (n = 12) received a single gene transfer dose of 1.5×10 13 vg total scAAV. scAAV9.CB.CLN6 was formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and approximately 20% to approximately 40% nonionic hypotonic compound and delivered once via an intrathecal catheter inserted by lumbar puncture into the subarachnoid space of the lumbar thecal sac between the spinous processes. Safety was evaluated based on clinical grounds and by considering the safety label. There was a minimum of 4 weeks between enrollment of each subject to allow review of safety data at 30 days post-gene transfer.

[0107] The preliminary data provided herein reports on 10 treated patients with a mean follow-up period of 12 months (range 1 to 24 months post-treatment). The preliminary data demonstrated that administration of scAAV9.CB.CLN6 was generally well tolerated. Most adverse events were mild and unrelated to treatment. Any T cell responses and antibody elevations observed were not associated with clinical symptoms and no changes in treatment were required.

[0108] Figure 12 provides preliminary data reporting disease progression measured by the Hamburg motor and language scales after injection in two pairs of siblings under study. These sibling pairs have the same gCLN6 mutant genotype.

[0109] 24-month phase efficacy study Provided herein is data from 8 patients treated for an ongoing clinical study. The 8 patients described herein were administered scAAV9.CB.CLN6 and exposed for at least 17 months. The baseline information of these 8 patients is shown below.

Table 1

[0110] Data from the ongoing 24-month clinical study showed that single intrathecal administration of scAAV9.CB.CLN6 was generally well tolerated. 137 adverse events were reported. Most of the adverse events (AEs) were mild and unrelated to the treatment. There were 9 grade 3 (severe) adverse events (shown as SAEs) reported in 4 patients. 3 of the 9 SAEs were considered possibly related to the treatment. The related events included vomiting (2), upper abdominal pain (1), and fever (1), and all 4 patients recovered. No grade 4 (life-threatening) or grade 5 (death) adverse events were reported. There was no pattern of adverse events related to anti-AAV9 capsid or anti-CLN6 immunogenicity.

[0111] Figure 15 shows efficacy data indicating a positive effect on motor and language functions. In 7 out of 8 patients treated with scAAV9.CB.CLN6, the Hamburg score was maintained or stabilized following the initial change (+1 to -1 point). The oldest patient in this study (treated at 79 months of age) had a 2-point decrease. Natural history data suggest that the Hamburg motor and language decline by 2 to 3 points within 24 months after symptom onset.

[0112] Figures 16A - C all provide comparative data of siblings suffering from CLN6 disease. The treated patients showed stabilization compared to untreated siblings who experienced a significant decline in motor and language abilities or died during the same period. These data are provided as the Hamburg score over time: motor + language. Figure 16A provides the aggregated score, Figure 16B provides the Hamburg motor sub - score, and Figure 16C provides the Hamburg language sub - score.

[0113] Figures 12A - C provide in - study sibling comparison data of an in - study pair both treated with scAAV9.CB.CLN6. These data showed that the younger sibling demonstrated an increase or stabilization in Hamburg motor and language scores compared to the older sibling who stabilized following the initial changes. Figure 12A provides the aggregated score, Figure 12B provides the Hamburg motor sub - score, and Figure 12C provides the Hamburg language sub - score.

[0114] Figure 17 compares the data of the first 8 patients treated with scAAV9.CB.CLN6 with the data of an ongoing natural history study of CLN6 patients conducted by Nationwide Children’s Hospital (n = 14). Shown is the Kaplan - Meier curve of the time until the combined score of Hamburg motor and language functions decreases without reversing by more than 2 points from the baseline. The confidence bands are calculated using the estimated survival probability and its standard error. This figure compares the patients in the treatment group and the natural history group whose combined Hamburg motor and language scores decreased by ≥2 points over a 2 - year period, and conveys results supporting the efficacy of the treatment of the present invention, including (i) only 1 treated patient achieved a decline of more than 2 points during the period compared to all 14 untreated patients in the natural history, and (ii) a substantial separation of the treated patients from the untreated patients.

[0115] In summary, 24-month efficacy data showed the following: i) disease stabilization, as opposed to untreated siblings who experienced rapid decline in motor and language abilities; ii) younger patients showed an increase or stabilization in scores; and iii) the majority of older patients showed an initial change followed by stabilization. In addition, the treatment was generally well tolerated.

[0116] Dose escalation study If there are no safety concerns, additional subjects will be enrolled after the first cohort is evaluated at 1 month post-injection. Each subject in cohort 2 (n = 4) will receive escalating doses of the viral vector. There will be at least a 6-week window between the completion of cohort 1 and the start of cohort 2 to allow for safety analysis review from five time points (days 1, 2, 7, 14, and 21) and DSMB review prior to dosing the next subject.

[0117] Disease progression is measured using the UBDRS scale or the Hamburg motor and language scale (mentioned in the forms for carrying out the above invention) and the impact of treatment on quality of life using the Pediatric Quality of Life (PEDSQOL) scale, as well as the likelihood of long-term survival.

[0118] When all patients have completed the 3-year study, a primary analysis of efficacy will be evaluated. The basis for determining efficacy is based on stabilization of the disease or reduction in progression according to the established Unified Batten Disease Rating Scale (UBDRS) or the Hamburg motor and language scale, specifically developed for CLN6-Batten disease. At the end of the 3-year study period, patients will be monitored annually for 5 years in accordance with FDA guidance.

[0119] Example 5 The natural history study demonstrates that scAAV9.CB.CLN6 gene therapy improves motor and language scores Regarding the clinical course over time, to facilitate comparison between the study subjects (the first patients in the CLN6 gene transfer study (n = 8)) and the natural history subjects, the combined Hamburg Scale motor (M) and language (L) scores of the gene transfer patients were matched with the combined Hamburg Scale motor and language score data collected for the patients in a retrospective CLN6 natural history study (PI: Emily de los Reyes, MD; ClinicalTrials.gov identifier: NCT03285425). The gene transfer patients were matched to the natural history patients based on their baseline Hamburg motor and language scores and age at the time of comparison (within 12 months).

[0120] Data for the combined and individual Hamburg motor and language scores (n = 8) indicate that CLN6 gene therapy halts or significantly delays disease progression and has a positive impact on the motor and language function of 7 out of 8 patients (Figure 18). A positive impact refers to patients who maintained their total Hamburg score or those who stabilized following an initial change (+1 to -1 point). The individual motor and language scores were consistent with their respective total scores.

[0121] Data from the natural history matched comparisons also show improvement in the Hamburg motor and language scores (Figure 19). Using a many-to-one matching methodology, the average Hamburg motor and language scores of the natural history patients matched at the end of the comparison period (plotted in red) are plotted against the respective Hamburg motor and language values (plotted in green) of the gene therapy patients at the last time point. The number of natural history patients in each comparison is shown in each figure, along with the difference in the Hamburg motor and language scores (between the average values of the gene therapy patients and NH patients) at the last time point. Natural history data collected on CLN6-Batten disease patients (n = 11) by Nationwide Children’s Hospital and Dr. Emily de los Reyes shows that the Hamburg motor + language scores decline fairly linearly and almost continuously by 1 point per year from age 2 to age 7 (Figure 20).

[0122] Overall, data from these studies indicate that the majority of CLN6 gene therapy patients show improvement in motor and language scores compared to matched natural history patients.

[0123] Example 6 Analysis of the Mullen Scales of Early Learning The Mullen Scales of Early Learning (MSEL) were used to evaluate whether scAAV.CB.CLN6 gene therapy improved patient learning ability over 12 - 24 months. The MSEL is an individually administered, standardized measure of cognitive function designed for use with children from birth to 68 months. The subscales of the MSEL are gross motor, fine motor, receptive language, expressive language, and early learning composite. (See Mullen EM. (1995). Mullen Scales of Early Learning (AGS ed.). Circle Pines, MN: American Guidance Service Inc).

[0124] The following four domains were analyzed in eight patients: visual reception, fine motor, receptive language, and expressive language. Figures 21A and 21B present the raw scores for the four domains. Higher scores indicate higher function.

[0125] Summary Interim safety and efficacy data suggest that AAV9-CLN6 gene therapy may have the potential to stabilize the progression of atypical late-infantile-onset CLN6 Batten disease. The efficacy results showed a significant treatment effect on motor and language functions. Patients treated with AAV9-CLN6 showed improvement in their Hamburg motor and language scores compared to untreated siblings and the mean values of natural history patients matched for age and baseline scores of Hamburg motor and language. Comparison of treated younger siblings with older siblings further supports the potential benefits of early intervention with gene therapy by AAV9-CLN6. Younger treated patients showed improvement or stabilization of cognitive skills as shown by the MSEL scale.

[0126] Preferred embodiments of the invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the invention. It should be understood that various alternative forms to the embodiments described herein may be employed. The following claims define the scope of the invention, and it is intended that methods and structures within these claims and their equivalents be encompassed thereby.

[0127] All documents referenced in this application are hereby incorporated by reference in their entirety.

Claims

1. 1. A self-complementary recombinant adeno-associated virus 9 (scAAV9) encoding a CLN6 polypeptide, comprising a scAAV9 genome comprising, in 5' to 3' order, a first AAV inverted terminal repeat, a CB promoter comprising the nucleotide sequence of SEQ ID NO:3, a polynucleotide encoding said CLN6 polypeptide of SEQ ID NO:1, and a second AAV inverted terminal repeat.

2. The scAAV9 of claim 1, wherein the scAAV9 genome comprises, in 5' to 3' order, a first AAV inverted terminal repeat, a CMV enhancer, a CB promoter comprising the nucleotide sequence of SEQ ID NO:3, an SV40 intron, a polynucleotide encoding the CLN6 polypeptide of SEQ ID NO:1, and a second AAV inverted terminal repeat.

3. The scAAV9 of claim 1, wherein the scAAV9 genome comprises, in 5' to 3' order, a first AAV inverted terminal repeat, a CB promoter comprising the sequence of SEQ ID NO:3, a polynucleotide encoding the CLN6 polypeptide of SEQ ID NO:1, a bovine growth hormone polyadenylation polyA sequence, and a second AAV inverted terminal repeat.

4. The scAAV9 of any one of claims 1 to 3, wherein the polynucleotide encoding the CLN6 polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

2.

5. The scAAV9 of any one of claims 1 to 3, wherein the polynucleotide encoding the CLN6 polypeptide comprises the nucleic acid sequence of SEQ ID NO:

2.

6. The scAAV9 of any one of claims 1 to 5, wherein the scAAV9 genome comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

4.

7. The scAAV9 of any one of claims 1 to 5, wherein the scAAV9 genome comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:

4.

8. The scAAV9 of any one of claims 1 to 6, wherein the scAAV9 genome comprises the nucleic acid sequence of SEQ ID NO:

4.

9. The scAAV9 of any one of claims 1 to 8, wherein the AAV inverted terminal repeat is an AAV2 inverted terminal repeat.

10. The scAAV9 of any one of claims 1 to 10, wherein the rAAV9 genome comprises a single-stranded genome.

11. A nucleic acid molecule comprising a first AAV inverted terminal repeat, a CB promoter comprising the sequence of SEQ ID NO:3, a nucleic acid sequence encoding a CLN6 polypeptide of SEQ ID NO:1, and a second AAV inverted terminal repeat.

12. The nucleic acid molecule of claim 11, comprising a first AAV inverted terminal repeat, a CB promoter comprising the nucleotide sequence of SEQ ID NO:3, an SV40 intron, a nucleic acid sequence encoding the CLN6 polypeptide of SEQ ID NO:1, and a second AAV inverted terminal repeat.

13. The nucleic acid molecule of claim 11, comprising a first AAV inverted terminal repeat, a CB promoter comprising the nucleotide sequence of SEQ ID NO:3, a nucleic acid sequence encoding the CLN6 polypeptide of SEQ ID NO:1, a bovine growth hormone polyadenylation poly A sequence, and a second AAV inverted terminal repeat.

14. The nucleic acid molecule of any one of claims 11 to 13, wherein the nucleic acid encoding the CLN6 polypeptide comprises a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

2.

15. The nucleic acid molecule according to any one of claims 11 to 13, wherein the nucleic acid encoding the CLN6 polypeptide comprises the nucleic acid sequence of SEQ ID NO:

2.

16. The nucleic acid molecule according to any one of claims 11 to 15, comprising a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

4.

17. A nucleic acid molecule according to any one of claims 11 to 15, comprising a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:

4.

18. A nucleic acid molecule according to any one of claims 11 to 15, comprising the nucleic acid sequence of SEQ ID NO:

4.

19. 19. The nucleic acid molecule of any one of claims 11 to 18, wherein the AAV inverted terminal repeat is an AAV2 inverted terminal repeat.

20. A self-complementary recombinant adeno-associated virus 9 (scAAV9) comprising a nucleic acid molecule according to any one of claims 11 to 19.

21. The scAAV9 of claim 20, wherein the scAAV9 comprises a single-stranded genome.

22. 20. An rAAV particle comprising a polynucleotide sequence according to any one of claims 11 to 19.

23. The rAAV particle of claim 22, wherein the rAAV particle comprises a single-stranded genome.

24. 1. A recombinant adeno-associated virus 9 (rAAV9) virion encoding a CLN6 polypeptide, the rAAV9 genome comprising, in 5' to 3' order, a CMV enhancer comprising a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:6, a chicken beta-actin promoter comprising a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:3, and a polynucleotide that encodes a CLN6 polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:

1.

25. The rAAV9 viral particle of claim 24, wherein the rAAV9 genome comprises a self-complementary genome.

26. 25. The rAAV9 viral particle of claim 24, wherein the rAAV9 genome comprises a single-stranded genome.

27. 27. The rAAV9 viral particle of any one of claims 24 to 26, wherein the rAAV9 genome comprises, in 5' to 3' order, a first AAV inverted terminal repeat, the CMV enhancer comprising a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:6, the chicken β-actin promoter comprising a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:3, the polynucleotide encoding a CLN6 polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a second AAV inverted terminal repeat.

28. The rAAV9 viral particle of any one of claims 24 to 27, wherein the polynucleotide encoding the CLN6 polypeptide comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

2.

29. The rAAV9 viral particle of any one of claims 24 to 28, wherein the rAAV9 genome comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

4.

30. The rAAV9 viral particle of any one of claims 24 to 28, wherein the rAAV9 genome comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:

4.

31. The rAAV9 viral particle of any one of claims 24 to 30, wherein the AAV inverted terminal repeat is an AAV2 inverted terminal repeat.

32. The rAAV9 viral particle of any one of claims 24 to 31, wherein the rAAV9 genome further comprises an SV40 intron.

33. The rAAV9 viral particle of any one of claims 24 to 32, wherein the rAAV9 genome further comprises a BGH polyA sequence.

34. 1. A nucleic acid molecule comprising a rAAV9 genome comprising, in 5' to 3' order, a first AAV inverted terminal repeat, a CMV enhancer having a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:6, a chicken β-actin promoter having a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:3, a polynucleotide encoding a CLN6 polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a second AAV inverted terminal repeat.

35. 35. The nucleic acid molecule of claim 34, wherein the rAAV9 genome comprises a self-complementary genome.

36. 35. The nucleic acid molecule of claim 34, wherein the rAAV9 genome comprises a single-stranded genome.

37. 37. The nucleic acid molecule of any one of claims 34 to 36, wherein the rAAV9 genome comprises, in 5' to 3' order, a first AAV inverted terminal repeat, the CMV enhancer having a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:6, the chicken β-actin promoter having a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:3, the polynucleotide encoding a CLN6 polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a second AAV inverted terminal repeat.

38. The nucleic acid molecule of any one of claims 34 to 37, wherein the polynucleotide encoding the CLN6 polypeptide comprises an amino acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:

2.

39. The nucleic acid molecule of any one of claims 34 to 38, wherein the rAAV9 genome comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

4.

40. The nucleic acid molecule of any one of claims 34 to 38, wherein the rAAV9 genome comprises a sequence that is at least 95% identical to SEQ ID NO:

4.

41. 41. The nucleic acid molecule of any one of claims 34 to 40, wherein the AAV inverted terminal repeat is an AAV2 inverted terminal repeat.

42. The nucleic acid molecule of any one of claims 34 to 41, wherein the rAAV9 genome further comprises an SV40 intron.

43. The nucleic acid molecule of any one of claims 34 to 42, wherein the rAAV9 genome further comprises a BGH polyA sequence.

44. 10. A composition comprising an scAAV9 according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 19, 31 or 34 to 43, an rAAV9 viral particle according to any one of claims 22 to 33, and a pharma- ceutically acceptable excipient, carrier, or diluent.

45. 45. The composition of claim 44, wherein the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

46. 13. A method of treating CLN6-Batten disease in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising the scAAV9 of any one of claims 1-10, a rAAV9 viral particle of any one of claims 22-33, a nucleic acid of any one of claims 11-19, 31 or 34-43, or a composition of claim 44 or claim 45.

47. 47. The method of claim 46, wherein the composition is administered via a route selected from the group consisting of intrathecal, intraventricular, intraparenchymal, intravenous, and combinations thereof.

48. 48. The method of claim 47, wherein the composition is administered intrathecally.

49. 48. The method of claim 47, wherein the composition is administered intracerebroventricularly.

50. The method of claim 4746, wherein the composition is administered intravenously.

51. Approximately 1×10 １１ ~Approx. 1×10 １５ 51. The method of any one of claims 46-50, wherein the rAAV9 viral particles are administered in a concentration of 0.01 to 0.1% by weight.

52. Approximately 1×10 １２ ~Approx. 1×10 １４ 52. The method of any one of claims 46-51, wherein the rAAV9 viral particles are administered in an amount of 1000 μg / kg.

53. The treatment, compared to untreated CLN-6 Batten disease patients, (a) reduced brain volume, (b) loss of cognitive function, and (c) selected for language delay; 53. The method of any one of claims 46 to 52, which stabilizes or delays one or more symptoms of CLN6-Batten disease.

54. 53. The method of any one of claims 46-52, wherein said treatment stabilizes or slows disease progression of CLN-6 Batten disease.

55. 55. The method of claim 54, wherein disease progression is assessed using the UBDRS scale, the Hamburg Motor and Language Scale, the impact of treatment on quality of life using the Pediatric Quality of Life (PEDSQOL) scale, Mullen's Scale of Early Learning (MSEL), likelihood of long-term survival, or a combination thereof.

56. 56. The method of any one of claims 46-55, wherein the subject is aged 80 months or less, 75 months or less, 70 months or less, 65 months or less, 62 months or less, 60 months or less, 55 months or less, 50 months or less, or 40 months or less.

57. 57. The method of any one of claims 46-56, further comprising placing the subject in the Trendelenberg position after administration of the rAAV9 viral particles.

58. 11. A method of treating CLN6 disease in a patient in need thereof, comprising delivering a composition comprising the scAAV of any one of claims 1-10, the rAAV9 viral particle of any one of claims 22-33, the nucleic acid of any one of claims 11-19, 31 or 34-4334, or the composition of claim 44 or claim 45 to the brain or spinal cord of a patient in need thereof.

59. 56. The method of claim 55, wherein the composition is delivered by intrathecal, intraventricular, intraparenchymal, or intravenous injection, or a combination thereof.

60. 57. The method of claim 56, further comprising placing the patient in the Trendelenberg position after intrathecal injection of the composition.

61. The method of any one of claims 55 to 57, wherein the composition comprises a non-ionic low osmolarity contrast agent.

62. The method of claim 5861, wherein the non-ionic low osmolarity contrast agent is selected from the group consisting of iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, ioxilan, and combinations thereof.

63. 60. The method of any one of claims 55 to 59, wherein said delivering to the brain or spinal cord comprises delivery to the brain stem.

64. 60. The method of any one of claims 55 to 59, wherein said delivering to the brain or spinal cord comprises delivery to the cerebellum.

65. 60. The method of any one of claims 55 to 59, wherein said delivering to the brain or spinal cord comprises delivery to the visual cortex.

66. 60. The method of any one of claims 55 to 59, wherein said delivering to the brain or spinal cord comprises delivery to the motor cortex.

67. 64. The method of any one of claims 55 to 63, wherein said delivering to the brain or spinal cord comprises delivery to neuronal cells, glial cells, or both.

68. 64. The method of any one of claims 55-63, wherein said delivering to the brain or spinal cord comprises delivery to a cell of the nervous system, said cell of the nervous system being a neuron, a lower motor neuron, a microglial cell, an oligodendrocyte, an astrocyte, a Schwann cell, or a combination thereof.

69. The treatment, compared to untreated CLN-6 Batten disease patients, (a) reduced brain volume, (b) loss of cognitive function, and (c) selected for language delay; 69. The method of any one of claims 58 to 68, which stabilizes or delays one or more symptoms of CLN6-Batten disease.

70. 69. The method of any one of claims 58-68, wherein said treatment stabilizes or slows disease progression of CLN-6 Batten disease.

71. 71. The method of claim 70, wherein disease progression is assessed using the UBDRS scale, the Hamburg Motor and Language Scale, the impact of treatment on quality of life using the Pediatric Quality of Life (PEDSQOL) scale, Mullen's Scale of Early Learning (MSEL), likelihood of long-term survival, or a combination thereof.

72. 72. The method of any one of claims 58-71, wherein the patient is aged 80 months or less, 75 months or less, 70 months or less, 65 months or less, 62 months or less, 60 months or less, 55 months or less, 50 months or less, or 40 months or less.

73. 23. Use of a therapeutically effective amount of a scAAV9 according to any one of claims 1 to 10, a rAAV9 viral particle according to any one of claims 22 to 33, a nucleic acid according to any one of claims 11 to 19, 31 or 34 to 43, or a composition according to claim 44 or claim 45, for the preparation of a medicament for treating CLN6-Batten disease in a subject.

74. 13. A composition comprising a therapeutically effective amount of the scAAV9 of any one of claims 1 to 10, the rAAV9 viral particle of any one of claims 22 to 33, the nucleic acid of any one of claims 11 to 19, 31 or 34 to 43, or the composition of claim 44 or claim 45, for treating CLN6-Batten disease in a subject.

75. 13. Use of a composition comprising the scAAV of any one of claims 1 to 10, the rAAV9 viral particle of any one of claims 22 to 33, the nucleic acid of any one of claims 11 to 19, 31 or 34 to 4334, or the composition of claim 44 or claim 45, for the preparation of a medicament for delivering the rAAV viral particle, nucleic acid or composition to the brain or spinal cord of a patient in need thereof.

76. 11. A composition for treating CLN6 disease in a patient in need of such treatment, said composition comprising a scAAV of any one of claims 1-10, a rAAV9 viral particle of any one of claims 22-33, a nucleic acid of any one of claims 11-19, 31 or 34-4334, or a composition of claim 44 or claim 45, to the brain or spinal cord of a patient in need of the composition.

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