Adeno-associated virus delivery of CLN6 polynucleotide

Abstract

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

Description

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 800,915 filed on February 4, 2019, U.S. Provisional Patent Application No. 62 / 880,641 filed on July 30, 2019, U.S. Provisional Patent Application No. 62 / 881,151 filed on July 31, 2019, U.S. Provisional Patent Application No. 62 / 912,977 filed on October 9, 2019, and U.S. Provisional Patent Application No. 62 / 923,125 filed on October 18, 2019, all of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Incorporated by Reference

[0003] This application contains a Sequence Listing in computer readable form as a separate part of the disclosure (file name: 53894_SeqListing.txt; 24,923 bytes - ASCII text file created on January 31, 2020), which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to the delivery of ceroid lipofuscinosis neuronal 6 (CLN6) polynucleotides by recombinant adeno-associated virus (rAAV). The present disclosure provides rAAV and methods of using rAAV to perform CLN6 gene therapy for neuronal ceroid lipofuscinosis (NCL) or CLN6-Batten Disease. Background Art

[0005] Neuronal ceroid lipofuscinosis (NCL) is a group of serious neurodegenerative diseases, which are collectively known as Batten disease. These diseases affect the nervous system and often lead to problems such as deterioration of movement and thinking ability. Different NCLs are characterized by their genetic causes.

[0006] CLN6 Batten disease can present in two different forms: variant late infantile (vLINCL), the more common form, and adult-onset NCL (also known as 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). For vLINCL (referred to here as CLN6 Batten disease), the age of onset is 18 months to six years, and death usually occurs between the ages of 12 and 15 years. CLN6 Batten disease initially presents with impaired language and delayed motor / cognitive development in early childhood, with most patients confined to a wheelchair within four years of disease onset (Canafoglia et al., Neurology 2015;85(4):316-24). Disease progression includes vision loss, severe motor deficits, recurrent seizures, dementia, and other neurodegenerative symptoms.

[0007] CLN6 is a 311 amino acid protein with seven predicted transmembrane domains and is primarily localized to the endoplasmic reticulum. As with other CLN proteins, its exact function remains unclear; however, it is involved in intracellular trafficking and lysosomal function. There are currently more than 70 characterized pathogenic mutations in CLN6 (Warrier et al., Biochimica et Biophysica Acta. 2013; 1832(11): 1827-30). Most of these mutations result in complete loss of CLN6 protein or produce truncated CLN6 protein products that are considered to be highly unstable and / or non-functional. Several naturally occurring animal models of CLN6-type Batten disease have been described; these include sheep, dogs, and mice models. In Cln6 nclf A spontaneous mutation found in a mouse model (referred to herein as “Cln6 nclf mice) recapitulates many of the pathological and behavioral aspects of the disease (Morgan et al., PLoS One 2013;8(11):e78694). Cln6 nclf The mice contain an extra cytosine insertion (c.307insC, a frameshift after P102) resulting in a premature stop codon homologous to a mutation commonly seen in patients with CLN6-type Batten disease (Gao et al., Am. J. Hum. Genet. 2002;70(2):324-35, Wheeler et al., Am. J. Hum. Genet. 2002;70(2):537-42).

[0008] Currently, there is no therapy that can reverse the symptoms of CLN6 Batten disease. Therefore, there is still a need in the art for treating CLN6 Batten disease. Summary of the invention

[0009] Provided herein are methods and products for CLN6 gene therapy using recombinant AAV.

[0010] Provided herein is a recombinant adeno-associated virus 9 (rAAV9) encoding a CLN6 polypeptide, comprising a rAAV9 genome, the rAAV9 genome comprising, in 5' to 3' order: a hybrid chicken β-actin (CB) promoter and a polynucleotide encoding the CLN6 polypeptide. In some cases, the rAAV9 genome comprises a self-complementary genome. Alternatively, the rAAV9 genome comprises a single-stranded genome.

[0011] A self-complementary recombinant adeno-associated virus 9 (scAAV9) is provided, which encodes the CLN6 polypeptide shown in SEQ ID NO: 1, wherein the genome of the scAAV9 includes, in 5' to 3' order: a first AAV terminal inverted repeat; a hybrid chicken β-actin (CB) promoter including the sequence of SEQ ID NO: 3; a polynucleotide encoding the CLN6 polypeptide shown in SEQ ID NO: 2; and a second AAV terminal inverted repeat. The polynucleotide encoding the CLN6 polypeptide may be at least 90% identical to SEQ ID NO: 2.

[0012] Also provided are scAAV9s having a genome comprising, in 5′ to 3′ order: a first AAV terminal inverted 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 terminal inverted repeat; scAAV9s having a genome comprising, in 5′ to 3′ order: a first AAV terminal inverted repeat; a CB promoter including a 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 terminal inverted repeat; and scAAV9s having a genome comprising the gene cassette shown in the nucleic acid sequence of SEQ ID NO: 4.

[0013] Also provided is an ssAAV9 having a genome comprising, in 5′ to 3′ order: a first AAV terminal inverted 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 terminal inverted repeat; an ssAAV9 having a genome comprising, in 5′ to 3′ order: a first AAV terminal inverted repeat; a CB promoter including a 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 terminal inverted repeat; or an ssAAV9 having a genome comprising the gene cassette shown in the nucleic acid sequence of SEQ ID NO: 4.

[0014] The nucleic acid sequence shown in SEQ ID NO: 4 is Figure 1 A gene cassette is provided in A. An rAAV9 comprising a scAAV9 genome or a ssAAV9 genome is provided, 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.

[0015] Further provided is a nucleic acid molecule comprising: a first AAV terminal inverted repeat; a CB promoter comprising the nucleic acid sequence of SEQ ID NO: 3; a nucleic acid sequence encoding the CLN6 polypeptide of SEQ ID NO: 1; and a second AAV terminal inverted repeat. In some embodiments, the polynucleotide encoding the CLN6 polypeptide may be at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0016] Also provided is a nucleic acid molecule comprising: a first AAV terminal inverted 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 terminal inverted repeat. In addition, a nucleic acid molecule is further provided, comprising: a first AAV terminal inverted repeat; a CB promoter comprising the nucleotide sequence of SEQ ID NO: 3; a nucleic acid encoding the CLN6 polypeptide of SEQ ID NO: 1; a BGH poly-A sequence; and a second AAV terminal inverted repeat. In any of the polynucleotides provided, the CLN6 polypeptide may be encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0017] An rAAV having a scAAV genome or a ssAAV genome is provided, 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.

[0018] The rAAV provided may include any polynucleotide disclosed herein. In addition, a viral particle comprising any disclosed nucleic acid is provided. An rAAV having a self-complementary or single-stranded genome is also provided.

[0019] Also provided is a recombinant adeno-associated virus 9 (rAAV9) virus particle encoding a CLN6 polypeptide, comprising a rAAV9 genome, the rAAV9 genome comprising, in 5′ to 3′ order: a CMV enhancer comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 6; a CB promoter comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 3; and a polynucleotide encoding a CLN6 polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the rAAV9 virus particle provided comprises a self-complementary genome. Alternatively, the rAAV9 virus particle provided comprises a single-stranded genome.

[0020] Further provided is an rAAV9 virus particle, wherein the rAAV9 genome comprises, in 5′ to 3′ order: a first AAV terminal inverted repeat; the CMV enhancer comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 6; the CB promoter comprising a nucleic acid sequence at least 90% identical to 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 terminal inverted repeat. The provided rAAV9 particle comprises a polynucleotide encoding a CLN6 polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1. Any rAAV9 virus particle may optionally further comprise an SV40 intron and / or a BGH poly-A sequence.

[0021] In additional embodiments, the rAAV9 viral particle comprises an AAV9 genome comprising a nucleic acid sequence that is at least 90% identical to the 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.

[0022] In any of the rAAV, ssAAV or scAAV provided, the AAV terminal inverted repeats may be AAV2 terminal inverted repeats.

[0023] Also provided is a nucleic acid molecule comprising a rAAV9 genome, the rAAV9 genome comprising, in 5′ to 3′ order: a first AAV terminal inverted repeat; a CMV enhancer comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 6; a CB promoter comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 3; and a polynucleotide encoding a CLN6 polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 1. The provided nucleic acid molecules include a self-complementary genome and / or a single-stranded genome.

[0024] Further provided are nucleic acid molecules comprising a rAAV9 genome, the rAAV9 genome comprising, in 5′ to 3′ order: a first AAV terminal inverted repeat; the CMV enhancer comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 6; the CB promoter comprising a nucleic acid sequence at least 90% identical to 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 terminal inverted repeat. The provided nucleic acid molecules may include a polynucleotide encoding a CLN6 polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1. Additionally, the nucleic acid molecules may include an AAV9 genome comprising a nucleic acid sequence at least 90% identical to the 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. Any of the provided nucleic acid molecules optionally further include an SV40 intron and / or a BGH poly-A sequence.

[0025] Further provided is a composition comprising: scAAV9 described herein, a nucleic acid molecule described herein, or an rAAV virus particle described herein, and at least one pharmaceutically acceptable excipient. In some cases, the pharmaceutically acceptable excipient comprises a nonionic low permeability 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 include at least a pharmaceutically acceptable excipient comprising a nonionic low permeability compound. For example, the pharmaceutically acceptable excipient may include about 20% to 40% of a nonionic, low permeability compound or about 25% to about 35% of a nonionic low permeability compound. An exemplary composition comprises a mixture of 20 mM Tris (pH 8.0), 1 mM MgCl 2, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% of a non-ionic low-osmotic compound. Another exemplary composition includes: scAAV formulated in 1× PBS including 0.001% Pluronic F68.

[0026] Still further provided is a method of treating CLN6 Batten disease in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of any rAAV9 disclosed herein, any scAAV9 disclosed herein, any ssAAV disclosed herein, any nucleic acid molecule described herein, or any composition described herein.

[0027] The present disclosure also provides the use of a therapeutically effective amount of any rAAV9 disclosed herein, any scAAV9 disclosed herein, any ssAAV disclosed herein, any nucleic acid molecule described herein, or any composition described herein for the preparation of a medicament for treating CLN6-type Batten disease.

[0028] Also provided are compositions for treating CLN6 Batten disease, comprising a therapeutically effective amount of any rAAV9 disclosed herein, any scAAV9 disclosed herein, any ssAAV disclosed herein, any nucleic acid molecule described herein, or any composition described herein.

[0029] 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 by a route selected from the group consisting of intrathecal, intracerebroventricular, intraparenchymal, intravenous, and combinations thereof.

[0030] An exemplary dose of scAAV9, ssAAV, or rAAV9 administered via the intrathecal route is about 1×10 11 The scAAV, ssAAV or rAAV9 virus particles of vg were about 1×10 15 vg of the scAAV or AAV9 virus particles; or about 1×10 12 The scAAV, ssAAV or rAAV9 virus particles of vg were about 1×10 14 vg of the scAAV, ssAAV or AAV9 virus particles. For example, about 1×10 13 vg of the scAAV, ssAAV or rAAV9 viral particles to a subject; or about 1.5×10 13 of the scAAV, ssAAV or rAAV9 viral particles to a subject; or about 6×10 13The scAAV, ssAAV or rAAV9 viral particles of vg are administered to a subject.

[0031] Compared to a subject before treatment or an untreated CLN6 Batten disease patient, the methods, uses or compositions disclosed herein for treating CLN6 Batten disease provide a subject with one or more of the following: (a) reduced or slowed lysosomal accumulation of autofluorescent storage materials, (b) reduced or slowed lysosomal accumulation of ATP synthase subunit C, (c) reduced or slowed activation of glial cells (astrocytes and / or microglia), (d) reduced or slowed astrocytosis, (e) reduced or slowed brain mass loss as measured by MRI, (f) reduced or slowed seizures, and (g) stabilization, reduction in progression, or improvement in one or more of a scale used to assess progression and / or improvement of CLN6 Batten disease, such as the Unified Batten Disease Rating System (UBDRS) assessment scale, the Hamburg Motor and Language Scale, or the Mullen Scales of Early Learning. Following administration of a rAAV9, ssAAV9 viral particle or scAAV or nucleic acid molecule disclosed herein, the subject can remain in the Trendelenburg position.

[0032] Still further provided is a method of treating CLN6 disease in a patient in need thereof, the method comprising delivering to the brain or spinal cord of a patient in need thereof a composition comprising: any rAAV viral particle disclosed herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecule described herein, or any composition described herein.

[0033] In addition, the present disclosure provides the use of any of the disclosed rAAV viral particles provided herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecule described herein, or any composition 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.

[0034] Also provided are compositions for delivering the ssAAV9, nucleic acid molecules or compositions to the brain or spinal cord of a patient in need thereof, the compositions comprising any rAAV viral particles disclosed herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecules described herein, or any compositions described herein.

[0035] In any of the methods, uses or compositions provided, the composition can be delivered by intrathecal, intraventricular, intraparenchymal or intravenous injection or a combination thereof. Any of the methods provided further comprises placing the patient in a Trendelenburg position after intrathecal injection of a composition, rAAV9, ssAAV9 or scAAV or nucleic acid molecule disclosed herein.

[0036] In any of the methods, uses or compositions provided, the composition or medicament can include a nonionic low-osmotic contrast agent. For example, the composition can include a nonionic low-osmotic contrast agent, wherein the nonionic low-osmotic contrast agent is selected from the group consisting of iobitrol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, ioxilan and combinations thereof.

[0037] The composition or drug administered may include a pharmaceutically acceptable excipient. For example, the pharmaceutically acceptable excipient may include about 20% to 40% of a non-ionic low permeability compound or about 25% to about 35% of a non-ionic low permeability compound. An exemplary composition includes a 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% of a non-ionic low permeability compound. Another exemplary composition includes a scAAV formulated in 1X PBS and 0.001% Pluronic F68.

[0038] In any of the methods, uses or compositions provided, the composition or drug can be delivered to the brain or spinal cord, the composition or drug can be delivered to the brainstem, or it can be delivered to the cerebellum, it can be delivered to the visual cortex, or it can be delivered to the motor cortex. Further, in any of the methods provided, the composition or drug can be delivered to the brain or spinal cord, the composition can be delivered to nerve cells, glial cells, or both. For example, wherein the delivery to the brain or spinal cord includes delivery to cells of the nervous system, such as neurons, lower motor neurons, microglia, oligodendrocytes, astrocytes, Schwann cells, or a combination thereof.

[0039] The methods, uses, or compositions disclosed herein provide a subject with one or more of the following: (a) reduced or slowed lysosomal accumulation of autofluorescent storage material, (b) reduced or slowed lysosomal accumulation of ATP synthase subunit C, (c) reduced or slowed activation of glial cells (astrocytes and / or microglia), (d) reduced or slowed astrocytosis, (e) reduced or slowed brain mass loss as measured by MRI, (f) reduced or slowed seizures, and (g) stabilization, reduction in progression, or improvement in one or more of a scale used to assess progression and / or improvement of CLN6 Batten disease, such as the Unified Batten Disease Rating System (UBDRS) assessment scale, the Hamburg Motor and Language Scale, or the Mullen Scale of Early Learning (MSEL), compared to a subject before treatment or compared to an untreated CLN6 Batten disease subject.

[0040] In any of the methods, compositions and uses described herein, the treatment, composition or drug stabilizes or slows the disease progression of CLN-6 Batten disease. Specifically, disease progression is assessed using the UBDRS scale, the Hamburg Motor and Language Scale, the Effect of Treatment on Quality of Life Using the Pediatric Quality of Life (PEDSQOL) scale, the Mullen Scale of Early Learning (MSEL), prolonged survival potential or a combination thereof.

[0041] In any method, use or composition described herein, the treatment, composition or drug reduces or slows down one or more symptoms of CLN-6 Batten disease compared to untreated CLN-6 Batten disease patients, the symptoms selected from: (a) brain mass loss; (b) cognitive function loss; and (c) delayed language development. Specifically, the treatment stabilizes or slows the disease progression of CLN-6 Batten disease. For example, disease progression is assessed using the UBDRS scale, the Hamburger Motor and Language Scale, the effect of treatment on quality of life using the Pediatric Quality of Life (PEDSQOL) scale, the Mullen Scale of Early Learning (MSEL), extended survival potential, or a combination thereof.

[0042] In any of the methods, uses, or compositions described herein, 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.

[0043] Since there is no effective cure for CLN6 Batten disease, nclfMouse models were used to test the efficacy of introducing functional human CLN6 via adeno-associated virus (AAV)-mediated gene therapy. Preclinical results presented herein demonstrate that the use of AAV-serotype 9 allows for efficient expression of human CLN6 protein throughout the CNS, where the most affected cells are located. To assess the safety of treatment in a larger animal model, three four-year-old cynomolgus macaques were administered scAAV9.CB.CLN6 via intrathecal lumbar CSF injection and monitored for up to six months post-injection. No adverse effects or pathology were observed, while high levels of transgene expression were found throughout the brain and spinal cord of all animals. A single postnatal intracerebroventricular (ICV) injection of scAAV9.CB.CLN6 into the CSF of mice resulted in the survival of the CLN6 mouse model. nclf Sustained expression of the transgene was induced in mice. Administration of scAAV9.CB.CLN6 reduced typical hallmarks of the disease, including accumulation of autofluorescent storage material and ATP synthase subunit C, reactive gliosis, and loss of dendritic spines. Importantly, this gene therapy treatment could result in broad functional benefits, as it prevents the Cln6 nclf These results strongly emphasize the therapeutic potential of CSF-delivered scAAV9.CB.CLN6 for the treatment of CLN6-type Batten disease.

[0044] The headings herein are for the convenience of the reader and are not limiting.

[0045] The use of "may" and "can" herein is intended to describe various embodiments included in the claims, rather than to indicate uncertainty in the scope of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A to 1C demonstrate in vivo neuronal targeting and expression of human CLN6 protein. Figure 1 A provides a schematic diagram of the scAAV genome of scAAV.CB.CLN6. Figure 1 The graph in B provides CNL6 mRNA and human CLN6 (hCLN6) protein expression levels after transient transfection of HEK293 cells with scAAV.CB.CLN6 plasmid. Figure 1 Images in C provide immunohistochemical staining of GFP and hCLN6 proteins after in utero electroporation of scAAV.CB.CLN6 plasmid.

[0047] Figure 2 A and Figure 2 B provides a graph showing Cln6 injected with scAAV9.CB.CLN6 nclfFigure 3. Ubiquitous expression of human CLN6 transcripts in the CNS of mice. Figure 2 The images and graphs in A provide representative RT-PCR gels and quantification by densitometry (normalized to GAPDH) at 6 and 18 months post-injection. This analysis demonstrated that Cln6 nclf Mouse (Cln6 nclf +PBS) compared to scAAV9.CB.CLN6 delivery (Cln6 nclf +scAAV9) after the gene expression was increased. Figure 2 The left panel of B provides evidence that at 6 and 18 months post-injection, mice injected with scAAV9.CB.CLN6 (Cln6 nclf +scAAV9)Cln6 nclf Figure 3. Ubiquitous expression of human CLN6 transcripts in the CNS of mice. Figure 2 The right panel of B provides images showing immunohistochemical staining demonstrating that Cln6 was upregulated in mice injected with scAAV9.CB.CLN6 compared with wild-type mice (WT+PBS) at 6 and 18 months after injection. nclf Protein expression in various brain regions of mice. Scale bar 50 μm. Mean + / - SEM. N = 3-9 mice / group. One-Way ANOVA, Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0048] Figure 3 A to Figure 3 C demonstrates the effect of a single scAAV9.CB.CLN6 injection in 2-month-old animals. Figure 3 The images and graphs in A provide representative RT-PCR gels and quantification by densitometry (normalized to GAPDH) at 2 months post-injection. This analysis demonstrated that Cln6 nclf Mouse (Cln6 nclf +PBS) compared to scAAV9.CB.CLN6 delivery (Cln6 nclf +scAAV9) after the gene expression was increased. Figure 3 The upper panel in B provides evidence that 2 months after injection, mice injected with scAAV9.CB.CLN6 (Cln6 nclf +scAAV9)Cln6 nclfFigure 3. Ubiquitous expression of human CLN6 transcripts in the CNS of mice. Figure 3 The lower panel of B provides images showing immunohistochemical staining demonstrating that Cln6 in mice injected with scAAV9.CB.CLN6 was upregulated compared with wild-type mice (WT+PBS) at 2 months after injection. nclf Protein expression in various brain regions of mice. Scale bar 200 μm. Mean + / - SEM. N = 39. One-Way ANOVA, Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 3 The images and graphs in C demonstrate that Cln6 nclf Mouse (Cln6 nclf A single ICV injection of scAAV9.CB.CLN6 at P1 reduced Cln6 compared to PBS nclf Accumulation of autofluorescent storage material (ASM; top) and ATP synthase subunit C (SubC; bottom) in the VPM / VPL and somatosensory cortex of mice. Mean + / - SEM, N = 3-10. (top); Mean + / - SEM, N = 21-72, biological N = 3-10 (bottom) One-way ANOVA, Bonferroni correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar 200 μm (top panel). Scale bar 50 μm (bottom panel).

[0049] Figure 4 A and Figure 4 B demonstrates widespread expression of CLN6 mRNA and hCLN6 protein throughout the brain in the following regions: A: motor cortex, B: somatosensory cortex, C: visual cortex, D: thalamus, E: pons, F: cerebellum, G: Figure 4 The brainstem images provided in A demonstrate Cln6 treated with scAAV9.CB.CLN6 at 2, 6, and 18 months after injection. nclf hCLN6 transcript expression throughout the brain in mice. Figure 4 The images provided in B demonstrate the expression of scAAV9.CB.CLN6 treated with CLN6 at 2, 6, and 18 months after injection. nclf hCLN6 protein expression in whole brain of mice. Scale bar 50 μm.

[0050] Figure 5 Provided is the evidence that Cln6 in wild-type mice (WT) and PBS-treated mice were significantly different at 6 and 18 months after injection. nclfMouse (Cln6 nclf Compared with PBS, scAAV9.CB.CLN6 treated nclf Mouse (Cln6 nclf Images and graphs showing the reduction in accumulation of autofluorescent storage material (ASM) in the VPM / VPL and somatosensory cortex of scAAV. The graphs show the ASM + Cells / 2500μm 2 Number of. Mean + / - SEM, based on time point N = 3-10. One-Way ANOVA, Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar 50 μm.

[0051] Figure 6 Provided is the evidence that Cln6 in wild-type mice (WT) and PBS-treated mice were significantly different at 6 and 18 months after injection. nclf Mouse (Cln6 nclf Compared with PBS, scAAV9.CB.CLN6 treated nclf Mouse (Cln6 nclf Images and graphs showing reduced accumulation of mitochondrial ATP synthase subunit C (SubUnitC) in the VPM / VPL and somatosensory cortex of scAAV (Fig. 2A). Brown staining represents SubUnit C, while blue staining represents methyl green (nuclear). Graphs show total SubUnit C per image field. + Area. Mean + / - SEM, N = 21-72, biological N = 3-10. One-Way ANOVA, Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar 50 μm.

[0052] Figure 7 Provided is the demonstration that wild-type mice (WT) and PBS-injected Cln6 nclf Mouse (Cln6 nclf ) compared to Cln6 injected with scAAV9.CB.CLN6 nclf Mouse (Cln6 nclfFigure 2. Images and graphs of scAAV) in the VPM / VPL and somatosensory cortex at 6 and 18 months of age. Graphs show total GFAP+ immunoreactivity. Mean + / - SEM, N = 16-49 sections, biological N = 3-10 mice / group. One-Way ANOVA, Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar 50 μm. Inset scale bar 10 μm.

[0053] Figure 8 Provided is the demonstration that wild-type mice (WT) and PBS-injected Cln6 nclf Mouse (Cln6 nclf Compared with PBS, Cln6 injected with scAAV9.CB.CLN6 nclf Mouse (Cln6 nclf scAAV9) in the somatosensory cortex of mice 6 months after injection and compared with wild-type mice (WT) and Cln6 mice injected with PBS nclf Mouse (Cln6 nclf Figure 1. Images and graphs showing less microgliosis (CD68 reactivity) in the VPM / VPL and somatosensory cortex 18 months after injection compared to PBS. Graphs show total CD68+ immunoreactivity. Mean + / - SEM, N = 16-49 sections, biological N = 3-10 mice / group. One-Way ANOVA, Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar 50 μm. Inset scale bar 10 μm.

[0054] Fig. 9 A to Fig. 9 E provides evidence that sustained expression of CLN6 rescues Cln6 nclf Diagram of motor, memory, learning, and survival defects in mice. Fig. 9 A demonstrates that Cln6 nclf Mouse (Cln6 nclf Compared with PBS, Cln6 injected with scAAV9.CB.CLN6 nclf Mouse (Cln6 nclf scAAV) had reduced rotarod deficits from 8 to 24 months of age. Fig. 9 B shows that Cln6nclf Mouse (Cln6 nclf Compared with PBS, scAAV9.CB.CLN6 injected nclf Mouse (Cln6 nclf scAAV) corrected hindlimb clasping, gait, and ledge lowering defects in 12- and 18-month-old mice. Fig. 9 C showed that Cln6 nclf Mouse (Cln6 nclf Compared with PBS, scAAV9.CB.CLN6 prevented Cln6 nclf mice (Cln6 nclf scAAV) in 9- to 12-month-old mice with memory and learning deficits in the Morris water maze. Fig. 9 D demonstrated that scAAV9.CB.CLN6 injection prevents Cln6 nclf Animals died early, while Cln6 injected with PBS nclf The animal died at 15 months of age. Fig. 9 E shows the Cln6 expression in wild-type animals (WT) and PBS injected nclf Mouse (Cln6 nclf PBS) compared to mice treated with scAAV9.CB.CLN6 (Cln6 nclf Body weight development of males (left) and females (right) during the study in 144 rats (scAAV) mice. Mean + / - SEM, N = 6-24 for rotarod; N = 7-13 for buckling scores; N = 5-15 for water maze; N = 10-15 for survival curves. Weight N = 3-13. One-way ANOVA with Bonferroni correction or unpaired t-test where appropriate. Log-rank (Mantel-Cox) test for survival curve analysis *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

[0055] Fig.10 Additional behavioral data are provided for animals aged 12 to 24 months. Fig.10 The graph provided in A demonstrates that in the Morris water maze test, untreated Cln6 nclf Animals had significantly slower swimming speeds at 11 and 12 months of age. Fig.10 The graph in B demonstrates that scAAV9.CB.CLN6 did not significantly improve Cln6 in the Morris water maze reversal task at 12, 18, and 24 months of age. nclfMemory and learning deficits in mice. Swimming speed is shown as control. For water maze, N = 5-15; unpaired t-test, mean + / - SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

[0056] Fig.11 A to Fig.11 C provides data demonstrating that scAAV9.CB.CLN6 is highly expressed and well tolerated in non-human primates. Fig.11 A provides a Western blot demonstrating high expression of the transgene in various brain and spinal cord regions of non-human primates treated with scAAV9.CB.CLN6. The blots are representative of 3 animals, and "+" indicates animals treated with scAAV9.CB.CLN6. The following brain regions were tested: cortex (Ctx), corpus callosum (C.Call), periventricular white matter (PVWM:), hippocampus (Hipp), cerebellum (Cere), thalamus (Thal), cervical spinal cord (Cervical), thoracic spinal cord (Thoracic), lumbar spinal cord (Lumbar). Fig.11 The diagram in B provides Figure 1 Quantification of fluorescent western blots in A. Mean + / - SEM, N = 3. Unpaired Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Fig.11 The graph in C demonstrates that delivery of scAAV9.CB.CLN6 does not alter platelet concentrations or elevate liver enzymes in most scAAV9.CB.CLN6 treated non-human primates. Red data points indicate scAAV9.CB.CLN6 treated animals; blue data points indicate PBS treated animals. The enzymes tested were as follows: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (Alk Phos), gamma-glutamyl transferase (GGT).

[0057] Fig. 12A -C provides an analysis of disease progression after injection of scAAV9.CB.CLN6 in the sibling pairs included in the study as measured by the Hamburger Motor and Language Scale.

[0058] Fig.13The nucleic acid sequence of the scAAV9.CB.CLN6 gene cassette is provided (SEQ ID NO: 4). The AAV2 ITR nucleic acid sequence is shown in italics (5'ITR as shown in SEQ ID NO: 9; 3'ITR as shown in SEQ ID NO: 8); the CMV enhancer nucleic acid sequence (SEQ ID NO: 6) is underlined with a dashed line; the CB promoter nucleic acid sequence (SEQ ID NO: 3) is underlined with a single line; the SV40 intron nucleic acid sequence (SEQ ID NO: 11) is underlined with a double line; the nucleic acid sequence of the human CLN6 cDNA sequence (SEQ ID NO: 2) is shown in bold; the nucleic acid sequence of the BGH polyA terminator (SEQ ID NO: 10) is underlined with a dashed line.

[0059] Fig.14 The complete nucleic acid sequence of AAV.CB.CLN6 (SEQ ID NO: 8) is provided.

[0060] Fig.15 Efficacy data for 8 patients treated with scAAV9.CB.CLN6 as measured by the Hamburger Motor and Language Scales are presented.

[0061] Fig.16A -C provides a comparison between treated and untreated siblings. One sibling was treated with scAAV9.CB.CLN6, and its progress as measured by the Hamburger Motor and Language Scale was compared to the natural history of its untreated sibling. This data is provided as Hamburger Score: Motor + Language over time. Fig.17 Kaplan-Meier curves of the combined scores of Hamburger motor and language function relative to baseline until the time of unreversed decrease of 2 or more points are provided. This figure compares data from the first 8 patients treated with scAAV9.CB.CLN6 with data from an ongoing natural history study of CLN6 patients conducted by Nationwide Children's Hospital (n=14). Confidence bands were calculated using the survival probability estimate and its standard error.

[0062] Fig.18 Combined and individual Hamburger motor and language scores from patients treated with scAAV9.CB.CLN6 (n=8) are presented, showing that CLN6 gene therapy halted or substantially slowed disease progression, with a positive impact on motor and language function in 7 of 8 patients.

[0063] Fig.19A natural history matched comparison between patients treated with scAAV9.CB.CLN6 (n=8) compared to natural history patients matched for age and baseline Hamburger Motor and Language Cumulative Scores is presented.

[0064] Fig. 20 The natural history data of CLN6 Batten disease patients (n=11) are provided. In the legend, the dotted line (----) indicates language decline, and the solid gray line indicates motor decline. The blue line (neck line) is the sum of motor decline and language decline. The average Hamburger motor + language score is plotted on the y-axis, and the age in months is plotted on the x-axis. From two to seven years old, there is a fairly linear and almost continuous decline every year.

[0065] Fig.21A -B provides the raw scores for the 4 domains of the Mullen Early Learning Scales. The dashed horizontal lines indicate the scores at screening. Higher scores indicate higher functioning. DETAILED DESCRIPTION

[0066] The present disclosure provides methods and products for treating CLN6-type Batten disease. The methods involve using rAAV as a gene delivery vector to deliver CLN6 polynucleotides to a subject.

[0067] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, containing two 145-nucleotide terminal inverted repeats (ITRs) and can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes and naturally occurring and recombinant forms, unless otherwise specified. There are multiple serotypes of AAV. Each serotype of AAV is associated with a specific clade, whose members have serological and functional similarities. Therefore, a clade can also refer to AAV. For example, the AAV9 sequence is referred to as a "clade F' sequence (Gao et al., J. Virol., 78:6381-6388 (2004). The present disclosure contemplates the use of any sequence within a particular 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 No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., Journal of Virology, 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); portions of the AAV-12 genome are provided in GenBank Accession No. DQ813647; A Portions of the AV-13 genome are provided in Genbank accession number EU285562. See U.S. Patent 9,434,928 for the sequence of the AAVrh.74 genome, which is incorporated herein by reference. The sequence of the AAV-B1 genome is provided in Choudhury et al., Molecular Therapy, 24(7): 1247-1257 (2016). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosome integration are contained in the ITR. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding rep and cap genes.Two rep promoters (p5 and p19) combined with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The Rep proteins have a variety of enzymatic properties that are ultimately responsible for replicating 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 start sites are responsible for 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).

[0068] AAV has unique characteristics, which makes it attractive as a carrier for delivering exogenous DNA to 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. Moreover, AAV infects many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can basically continue the life span of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome has infectivity as cloned DNA in a plasmid, which makes the construction of a recombinant genome possible. In addition, since the signal for instructing AAV replication, genome encapsidation and integration is included in the ITR of the AAV genome, the genome of about 4.3kb (encoding replication and structural capsid protein, rep-cap) in part or all of the interior can be replaced by exogenous DNA such as a gene cassette containing a promoter, DNA of interest and a polyadenylation signal. In some cases, rep and cap proteins are provided in trans. Another notable feature of AAV is that it is an extremely stable and robust virus. It readily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cold storage of AAV less critical. AAV can even be lyophilized. Finally, AAV-infected cells do not tolerate repeated infection.

[0069] 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.

[0070] 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' terminal inverted repeats (ITRs). In some embodiments, the rAAV genome includes ITRs from an AAV serotype that is different from the AAV serotype from which the AAV genome was 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 terminal inverted repeats (ITRs). In some embodiments, the rAAV genome includes a "gene cassette". Exemplary gene cassettes are in Figure 1 A and shown in the nucleic acid sequence of SEQ ID NO: 4. The rAAV genome may be a self-complementary (sc) genome, which is referred to herein as a "scAAV genome". Alternatively, the rAAV genome may be a single-stranded (ss) genome, which is referred to herein as a "ssAAV genome".

[0071] The term "scAAV" refers to a rAAV virus or rAAV viral particle comprising a self-complementary genome. The term "ssAAV" refers to a rAAV virus or rAAV viral particle comprising a single-stranded genome.

[0072] The rAAV genome provided herein may include a polynucleotide encoding a CLN6 polypeptide. The CLN6 polypeptide includes the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide having 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 which encodes a polypeptide having CLN6 activity (e.g., when treated, the clearance rate of lysosomal autofluorescent storage materials is increased, the lysosomal accumulation of ATP synthase subunit C is reduced, and the activation of astrocytes and microglia in the patient is reduced, compared to, for example, the patient before treatment).

[0073] In some cases, the rAAV genome provided herein includes a polynucleotide encoding a CLN6 polypeptide, wherein the polynucleotide has a nucleotide sequence as 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., at least one of increased clearance of lysosomal autofluorescent storage material, reduced lysosomal accumulation of ATP synthase subunit C, and reduced activation of astrocytes and microglia in a patient during treatment compared to, for example, the patient before treatment).

[0074] In some embodiments, the rAAV genome provided herein includes a polynucleotide sequence encoding a polypeptide having CLN6 activity and hybridizing to the nucleic acid sequence of SEQ ID NO: 2 or its complementary sequence under stringent conditions. The term "stringent" is used to refer to conditions that are generally understood to be stringent in the art. The stringency of hybridization is mainly determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing include, but are not limited to, 0.015M sodium chloride, 0.0015M sodium citrate at 65°C to 68°C, or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at 42°C. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, New York, 1989).

[0075] In some embodiments, the rAAV genome provided herein includes one or more AAV ITRs flanking 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 functions in a target cell to form a gene cassette. Examples of promoters are chicken beta actin promoters and P546 promoters. Other promoters contemplated herein include but are not limited to simian virus 40 (SV40) early promoters, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukosis virus promoters, Epstein-Barr virus (Epstein-Barrvirus) immediate early promoters, Rous sarcoma virus promoters, and human gene promoters, such as but not limited to actin promoters, myosin promoters, elongation factor-1a promoters, hemoglobin promoters, and creatine kinase promoters. Further provided herein is the CB promoter sequence shown in SEQ ID NO: 3, 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, which is a promoter having CB transcription promoting activity. Other examples of transcription control elements are tissue-specific control elements, for example, promoters that allow specific expression in neurons or specific expression in astrocytes. Examples include neuron-specific enolase and glial fibrillary acid protein promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters. The gene cassette may also contain an intron sequence to facilitate processing of the CLN6 RNA transcript when expressed in mammalian cells. An example of such an intron is the SV40 intron.

[0076] "Packaging" refers to the series of intracellular events that lead to the assembly and encapsidation of AAV particles. The term "production" refers to the process of producing rAAV (infectious, encapsulated rAAV particles) by packaging cells.

[0077] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus, respectively. AAV rep and cap are referred to herein as AAV "packaging genes".

[0078] AAV "helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses, and poxviruses such as vaccinia virus. Adenoviruses can encompass many different subgroups, although the most commonly used is adenovirus type 5 of subgroup C. Many adenoviruses of humans, non-human mammals, and birds are known and available from deposits such as ATCC. Viruses of the herpes virus family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); these can also be obtained from deposits such as ATCC.

[0079] "One or more helper virus functions" refers to one or more functions encoded in the helper virus genome that allow AAV replication and packaging (in conjunction with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in a variety of ways, including by providing a helper virus or providing, for example, a polynucleotide sequence encoding one or more essential functions to a trans-producing cell.

[0080] The rAAV genome provided herein lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome (e.g., ITR) contemplated herein 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 are also contemplated herein, and include capsids with various post-translational modifications, such as glycosylation and deamidation. Deamidation of asparagine or glutamine side chains results in conversion of asparagine residues to aspartic acid or isoaspartic acid residues, and conversion of glutamine to glutamate or isoglutamate is contemplated in the rAAV capsids provided herein. See, e.g., Giles et al., Molecular Therapy, 26(12): 2848-2862 (2018). Modified capsids are also contemplated herein that include targeting sequences that direct rAAV to affected tissues and organs in need of treatment.

[0081] The DNA plasmid provided herein includes the rAAV genome described herein. The DNA plasmid can be transferred into cells that allow infection with the helper virus of AAV (e.g., adenovirus, adenovirus or herpes virus deleted by E1) to assemble the rAAV genome into infectious virus particles with AAV9 capsid protein. The technology for producing rAAV is standard in the art, wherein the rAAV genome to be packaged, rep and cap genes, and helper virus functions are provided to cells. The production of rAAV particles requires the following components to be present in a single cell (represented herein as packaging cells): rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not therein), and helper virus functions. AAV rep and cap genes can be from any AAV serotype that can derive recombinant viruses, and can be from AAV serotypes different from rAAV genome ITRs. The production of pseudotype rAAV is disclosed in, for example, WO 01 / 83692, which is incorporated herein by reference in its entirety. In various embodiments, the AAV capsid protein can be modified to enhance the delivery of recombinant rAAV. Modifications to capsid proteins are generally known in the art. See, for example, US 2005 / 0053922 and US 2009 / 0202490, the disclosures of which are incorporated herein by reference in their entirety.

[0082] The method of generating packaging cells is to create a cell line that stably expresses all the necessary components for rAAV production. For example, a plasmid (or plasmids) comprising a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separate from the rAAV genome, and a selectable marker such as a neomycin resistance gene can be integrated into the genome of the cell. The rAAV genome can be introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79: 2077-2081), adding a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23: 65-73) or by direct blunt end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 259: 4661-4666). The packaging cell line can then 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.Other non-limiting examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0083] The general principles of rAAV particle production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermona et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. 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, Journal of Virology, 63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 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. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing references are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the references relating to the production of rAAV particles.

[0084] Further 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 (homologous 293 lines). In another embodiment, the packaging cells can be cells of untransformed cancer cells, such as low-passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus embryonic lung cells).

[0085] Also provided herein are rAAVs (e.g., infectious encapsidated rAAV particles) comprising the rAAV genome of the present disclosure. The genome of rAAV lacks AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome of rAAV. The rAAV genome can be a self-complementary (sc) genome. rAAVs with sc genomes are referred to herein as scAAVs. The rAAV genome can be a single-stranded (ss) genome. rAAVs with single-stranded genomes are referred to herein as ssAAVs.

[0086] An exemplary rAAV provided herein is a scAAV named "scAAV9.CB.CLN6". The scAAV9.CB.CLN6scAAV contains a scAAV genome including a human CLN6 cDNA controlled by a 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 Poly A) terminator sequence (downstream of the human CLN6 cDNA). The sequence of this scAAV9.CB.CLN6 gene cassette is shown in SEQ ID NO: 4. The scAAV genome is packaged in an AAV9 capsid and contains AAV2 ITRs (one ITR upstream of the CB promoter and another ITR downstream of the BGH Poly A terminator sequence).

[0087] rAAV can be purified by standard methods in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV from helper virus are known in the art and can be included in, for example, Clark et al., Human Gene Therapy, 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0088] Compositions comprising rAAV are also provided. The composition comprises an rAAV encoding a CLN6 polypeptide. The composition may comprise two or more rAAVs encoding different polypeptides of interest. In some embodiments, the rAAV is a scAAV or a ssAAV.

[0089] The compositions provided herein include rAAV and one or more pharmaceutically acceptable excipients. Acceptable excipients are nontoxic to the recipient and are preferably inert at the doses and concentrations employed, and include, but are not limited to, buffers such as phosphate [e.g., phosphate buffered saline (PBS)], citrate 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). The compositions provided herein may include a pharmaceutically acceptable aqueous excipient containing a nonionic low permeability compound, such as iobitrol, iohexol, iomeprol, iopamidol, iopentol, iopromide, iofosmol, ioxilan, wherein the aqueous excipient containing the nonionic low permeability compound may have one or more of the following properties: about 180 mgI / mL, an osmotic pressure of about 322 mOsm / kg water as measured by vapor pressure osmotic pressure method, an osmotic pressure 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% to 40% of a nonionic low permeability compound or about 25% to about 35% of a nonionic low permeability compound. An exemplary composition includes a 20 mM Tris (pH 8.0), 1 mM MgCl 2 , 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% of a non-ionic low-osmotic compound. Another exemplary composition includes scAAV or rAAV virus particles formulated in 1X PBS and 0.001% Pluronic F68.

[0090] The dose of rAAV to be administered in the methods of the present disclosure will vary depending on, for example, the specific rAAV, the mode of administration, the time of administration, the therapeutic goal, the individual, and the one or more cell types targeted, and can be determined by methods standard in the art. The dose can be expressed in units of viral genomes (vg). The doses contemplated herein include about 1×1011 , about 1×10 12 , about 1×10 13 , about 1.1×10 13 , about 1.2×10 13 , about 1.3×10 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 To about 1×10 16 or more total viral genomes. 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 about 6×10 13 To about 1.0×10 14 Doses of vg are also contemplated. One dose exemplified herein is 6×1013 vg. Another dose exemplified herein is 1.5×10 13 .

[0091] Methods of transducing target cells (including but not limited to cells of the nervous system, neural or glial cells) with rAAV are provided. Cells of the nervous system include lower motor neurons, microglia, oligodendrocytes, astrocytes, Schwann cells, or a combination thereof.

[0092] The term "transduction" is used to refer to the administration / delivery of CLN6 polynucleotides to target cells in vivo or in vitro by the replication-deficient rAAV disclosed herein, resulting in the expression of functional polypeptides by the recipient cells. Transduction of cells with the rAAV disclosed herein results in the sustained expression of the polypeptide or RNA encoded by the rAAV. Therefore, the present disclosure provides a method for administering / delivering rAAV encoding CLN6 polypeptides to a subject by an intrathecal, intraventricular, intraparenchymal or intravenous route or any combination thereof. Intrathecal delivery refers to delivery to the subarachnoid space of the brain or spinal cord. In some embodiments, intrathecal administration is by intracisternal administration.

[0093] Intrathecal administration is illustrated in this article. These methods include transducing target cells (including but not limited to nerves and / or glial cells) with one or more rAAVs described herein. In some embodiments, the rAAV viral particles including polynucleotides encoding CLN6 polypeptides are administered or delivered to the brain and / or spinal cord of the patient. In some embodiments, the polynucleotides are delivered to the brain. The brain regions envisioned for delivery include but are not limited to motor cortex, visual cortex, cerebellum and brainstem. In some embodiments, the polynucleotides are delivered to the spinal cord. In some embodiments, the polynucleotides are delivered to lower motor neurons. The polynucleotides can be delivered to nerves and glial cells. Glial cells are microglia, oligodendrocytes or astrocytes. In some embodiments, the polynucleotides are delivered to Schwann cells.

[0094] In some embodiments of the methods provided herein, the patient is maintained in a Trendelenburg position (head down position) after administration of the rAAV (e.g., about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes). For example, the patient can be tilted in the head down position from about 1 degree to about 30 degrees, from about 15 degrees to about 30 degrees, from about 30 degrees to about 60 degrees, from about 60 degrees to about 90 degrees, or from about 90 degrees to about 180 degrees.

[0095] The methods provided herein include administering an effective dose or effective multiple doses of a composition including rAAV provided herein to a subject in need (e.g., an animal including but not limited to a human patient). If the dose is administered before the development of CLN6 Batten disease, the administration is preventive. If the dose is administered after the development 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, which slows or prevents the progression of the disease, reduces the extent of the disease, causes remission (partial or complete) of the disease and / or prolongs survival. Compared to the subject before treatment or compared to an untreated subject, the methods provided herein result in stabilization, reduction in progression or improvement of one or more scales for assessing the progression and / or improvement of CLN6 Batten disease, such as the Unified Batten Disease Rating System (UBDRS), the Hamburger Motor and Language Scale or the Mullen Scale of Early Learning (MSEL). UBDRS assessment scale (as described in Marshall et al., Neurology, 2005 65(2): 275-279) [comprising the UBDRS physical assessment scale, the UBDRS seizure assessment scale, the UBDRS behavioral assessment scale, the UBDRS ability assessment scale, the UBDRS symptom onset sequence, and the UBDRS clinical global impression (CGI)]; the Pediatric Quality of Life Scale (PEDSQOL) scale, motor function, language function, cognitive function, and survival. The methods provided herein can result in one or more of the following: a reduction or slowing of lysosomal accumulation of autofluorescent storage material, a reduction or slowing of lysosomal accumulation of ATP synthase subunit C, a reduction or slowing of glial activation (astrocytic and / or microglial) activation, a reduction or slowing of astrocytic proliferation, and a reduction or delay in brain mass loss as measured by MRI compared to subjects before treatment or compared to untreated subjects.

[0096] Combination therapies are also provided. Combination as used herein encompasses simultaneous treatment or sequential treatment. Combinations of the methods described herein with standard medical treatments are specifically contemplated.

[0097] Further, combinations of compositions used according to the present invention (eg, a combination of scAAV9.P546.CLN6 and a contrast agent disclosed herein) (simultaneous or sequential treatment) are specifically contemplated.

[0098] While delivery to a subject in need after birth is contemplated, intrauterine delivery of the fetus is also contemplated.

[0099] Examples

[0100] Although the following examples describe specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, only those limitations that appear in the claims shall apply to the present invention.

[0101] In this example, a self-complementary AAV carrying the CLN6 cDNA under the control of the hybrid chicken β-actin (CB) promoter (designated scAAV9.CB.CLN6) was generated. IVC injection (6×10 13 vg / animal) to postnatal day 1 CSF of mice is sufficient to induce stable, robust expression of CLN6 protein throughout the CNS for up to 18 months. Progression of CLN6-type Batten disease is associated with accumulation of ASM, aggregation of ATP synthase subunit C, decreased synaptic spine density, increased GFAP reactivity in astrocytes, and increased CD68 staining in microglia. nclf Mice showed increased ASM and ATP synthase subunit C and decreased dendritic spines at two months of age, and increased GFAP and CD68 reactivity at six months of age. nclf Injection of scAAV9.CB.CLN6 into mice reduced the accumulation of ASM and ATP synthase subunit C and increased dendritic spine density, and decreased the levels of CD68+ microglia and GFAP+ astrocyte reactivity.

[0102] Example 1

[0103] Production of scAAV9.CB.CLN6

[0104] The human CLN6 cDNA clone was obtained from Origene, Rockville, Maryland. 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 was generated, which includes the human CLN6 (hCLN6) gene under the control of the chicken-β-actin (CB) hybrid promoter. Figure 1 A schematic diagram of the plasmid construct showing the CLN6 cDNA inserted between the AAV2 ITRs is provided in A. The plasmid construct also contains the CP promoter, the Simian Virus 40 (SV40) chimeric intron, and the bovine growth hormone (BGH) polyadenylation signal (BGH PolyA).

[0105] scAAV9.CB.CLN6 was generated under cGMP conditions by a transient triple plasmid transfection procedure using a double-stranded AAV2-ITR-based CB-CLN6 vector encoding the Rep2Cap9 sequence as previously described (Gao et al., Journal of Virology, 78:6381-6388 (2004)), and the adenoviral helper plasmid pHelper (Stratagene, Santa Clara, CA) in HEK293 cells (36). The purity and titer of the vector were assessed by 4% to 12% sodium dodecyl sulfate-acrylamide gel electrophoresis and silver staining and qPCR analysis. After cloning, transgene expression was verified in HEK293 cells by in utero ICV electroporation at embryonic day 15.5 and in vivo (see Figure 1 B and Figure 1 C) This analysis confirms neuronal targeting and expression of human CLN6 protein in vivo.

[0106] Example 2

[0107] Analysis in CLN6 nclf Expression of CSF-delivered scAAV9.CB.CLN6 in mice

[0108] Cell targeting and expression

[0109] To confirm the expression and biodistribution of virally introduced human CLN6 in mice, scAAV9.CB.CLN6 was administered to CLN6 cells via a single intracerebroventricular (ICV) injection within 24 h after birth. nclf Mice were injected with equal volumes of PBS of wild-type and CLN6 nclf Mice were used as controls. The NCH viral vector core titer was used, and the effective administration dose was 5×10 10 vg / mouse. 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.

[0110] RT-PCR analysis of hCLN6 expression at 2, 6, and 18 months after injection demonstrated that compared with the PBS-injected control, the Cln6 nclf Sustained and robust hCLN6 expression in the cortex of mice ( Figure 2 A, Figure 3A). These results are similar to previously reported scAAV9-CB-GFP expression levels (see, e.g., 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). Figure 2 In A, the top gel and graph are representative RT-PCR gels and densitometry (normalized to GAPDH). These data demonstrate that, compared with PBS-injected Cln6 nclf The bottom gel and graph show CLN6 protein expression measured by Western blot. ICV delivery of scAAV9.CB.CLN6 vectors shows that Cln6 nclf The expression of hCLN6 protein in the cerebral cortex of mice was significantly increased.

[0111] To examine the regional distribution of transgene expression, a Modified in situ hybridization method to visualize hCLN6 transcripts. nclf Mice maintained widespread hCLN6 transduction in all regions of the brain at 2, 6, and 18 months of age, including the somatosensory cortex and VPM / VPL nuclei of the thalamus, two regions that have been shown to be overexpressed in Cln6 nclf The earliest affected mice in the disease progression ( Figure 2 B, left; Figure 3 B; top picture, Figure 4 A).

[0112] To examine the expression of hCLN6 protein in the CNS, anti-hCLN6 antibody was used to perform a PCR reaction from the Cln6 cells injected with scAAV9.CB.CLN6. nclf Immunoblots of cortical lysates collected from controls injected with PBS. Consistent with RNA expression, robust hCLN6 protein expression was seen throughout the CNS at 2, 6, and 18 months ( Figure 2 B, right picture, Figure 3 B, lower panel). In addition, immunolabeling of brain tissue using anti-hCLN6 antibody confirmed the expression of hCLN6 in scAAV9.CB.CLN6-treated nclf Expression in the whole brain of mice ( Figure 4 B). Taken together, these findings demonstrate that CSF delivery of scAAV9.CB.CLN6 by ICV injection enables stable production of hCLN6 transcripts and protein in disease-relevant regions of the CNS.

[0113] Improved pathology after scAAV9.CB.CLN6 delivery

[0114] Accumulation of Autofluorescent Storage Material (ASM)

[0115] Accumulation of autofluorescent storage material (ASM) is a hallmark histological marker of Batten disease progression (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). Accumulation of ASM is a strong indicator of disease progression in 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 envisaged herein that a reduction in ASM is used as an indicator of successful treatment.

[0116] At 2, 6, and 18 months after treatment, Cln6 in mice injected with scAAV9.CB.CLN6 increased compared with that in mice injected with PBS. nclf Mice showed reduced ASM accumulation in the VPM / VPL nuclei of the thalamus and somatosensory cortex ( Figure 5 , Figure 3 C). Because PBS-treated Cln6nclf mice died at 15 months of age ( Fig. 9 D), Cln6 treated with PBS from 12 to 14 months of age nclf Mice were treated with scAAV9.CB.CLN6 at 18 months of age. nclf Comparison of mice. It is noteworthy that these 18-month-old Cln6 nclf The amount of ASM accumulated in the mice was comparable to that in age-matched untreated wild-type mice. Fig. 9 E demonstrates that male (left) and female (right) scAAV9.CB.CLN6-treated mice have similar body weights as wild-type mice as they age, whereas untreated Cln6 nclf The mice lost weight during the study. nclf Mouse (Cln6 nclfPBS) begin to lose weight at 10 to 11 months of age (males) and 13 to 14 months of age (females).

[0117] Accumulation of mitochondrial protein ATP synthase subunit C

[0118] In CLN6 from wild type, PBS injected nlcf mice or Cln6 injected with scAAV9.CB.CLN6 nclf The accumulation of ATP synthase subunit C was analyzed in the brain tissue of mice. In healthy individuals, this protein is part of the respiratory chain in the mitochondrial membrane, but in patients with Batten disease, the protein abnormally accumulates in lysosomes (Palmer et al., Am J Med Genet. 1992; 42(4): 561-567). nclf In mice, subunit C accumulation was evident at 2 months compared with wild-type animals in the ventral posteromedial and ventral posterolateral nuclei of the thalamus (VPM / VPL regions), brain regions frequently affected in NCL mouse models (Morgan et al., PLoS One 2013;8(11):e78694; Pontikis et al., Neurobiol Dis. 2005;20(3):823-836). nclf Compared with mice treated with scAAV9.CB.CLN6 at 2, 6, and 18 months, nclf The accumulation level of ATP synthase subunit C in the VPM / VPL and somatosensory cortex of mice was significantly reduced ( Figure 6 ; Figure 3 C; lower figure).

[0119] Glial and astrocyte activation

[0120] In addition to abnormal accumulation of storage material and accumulation of ATP synthase subunit C, other histological markers of disease progression in human patients and animal models include activation of astrocytes and microglia (Cotman et al., Human 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). Specifically, reactive microglia are primed to release proinflammatory mediators such as IL1-β26, which may be a key cause of neuronal cell death in the late stages of CLN6 Batten disease. At 6 and 18 months, compared with moribund PBS-treated Cln6 nclf Compared with mice injected with scAAV9.CB.CLN6 nclf The mice showed significantly reduced astrocyte activation (GFAP) and microglial proliferation (CD68) in the VPM / VPL and somatosensory cortex ( Figure 7 and Figure 8 ;)

[0121] Figure 7 Demonstration of identification of activated astrocytes in VPM / VPL thalamus and somatosensory cortex sections by staining for glial fibrillary acidic protein (GFAP) at 6 and 18 month time points. Graphs show total GFAP+ immunoreactivity.

[0122] Glial activation was also determined in VPM / VPL and somatosensory cortex sections using anti-CD68 staining as a marker for activated microglia. CD68 is a lysosomal protein that is upregulated in cells that initiate proinflammatory functions such as phagocytosis (Seehafer et al., J Neuroimmunol. 2011; 230: 169-172). Figure 8 Demonstration that scAAV9.CB.CLN6 injection reduced 6M Cln6 nclf In the somatosensory cortex of mice, and 18M Cln6 nclf Microgliosis (CD68 reactivity) in the VPM / VPL and somatosensory cortex of mice. Graphs show total CD68+ immunoreactivity. Figure 8 The inlet in the figure shows the morphology of microglia. Of note, the untreated Cln6 nclfThe mice were moribund, and many microglia were likely dying or dead, leading to their unusual morphology. Taken together, these results indicate that a single injection of scAAV9.CB.CLN6 delivered into the CSF on postnatal day 1 can reduce or delay the onset of Cln6 nclf Many typical CLN6-type Batten disease pathologies in the mouse brain.

[0123] Behavioral improvements after delivery of scAAV9.CB.CLN6

[0124] In the efficacy study of scAAV9.CB.CLN6, mice were subjected to a battery of behavioral testing paradigms starting at 2 months of age and continuing at 2-month intervals, including: accelerating rotarod assay, and pole climbing to test motor function and coordination, and Morris water maze to assess learning and memory. Animals were followed for 24 months after injection and the study is ongoing.

[0125] Rotarod assay

[0126] Previous work has shown that Cln6 in CLN6-type Batten disease nclf The mouse model recapitulates many of the motor, cognitive, and survival deficits found in humans (Morgan et al., PLoS One 2013;8(11):e78694). In efficacy studies, using the rotarod as a classic measure of motor coordination, PBS-injected Cln6 mice showed significant differences in motor function compared to wild-type controls. nclf Mice began to show decreased rotarod performance at 8 months of age. However, injection of Cln6 with scAAV9.CB.CLN6 nclf The mice prevented this decline, and the effect lasted throughout the study period (24 months) ( Fig. 9 A). To further investigate the effects of motor coordination in detail, animals were subjected to various motor tasks (hindlimb clasping, ability to lower oneself from a ledge, and gait assessment) at 12, 18, and 24 months of age and were evaluated using a scoring matrix, with the highest scores associated with the worst prognosis (Guyenet et al., Journal of visualized experiments: JoVE. 201039). nclf Compared with mice treated with scAAV9.CB.CLN6, mice treated with scAAV9.CB.CLN6 showed significantly lower combined scores at all time points, with only a slight increase in scores at 24 months of age ( Fig. 9 B).

[0127] Morris water maze test

[0128] In the Morris water maze test, animals are placed in a water-filled pool containing a hidden platform. After training, the time it takes the animals to use environmental cues to locate the hidden platform is measured as a marker of learning and memory ability.

[0129] PBS-treated Cln6 nclf The mice performed poorly on the task at nine months of age, indicating a reduced ability to find the hidden platform. Fig. 9 C) Cln6 due to PBS treatment nclf The swimming speed of mice decreased significantly at 11 and 12 months of age, so we were unable to draw any conclusions about their memory and learning abilities at these later time points ( Fig.10 A) Treatment of Cln6 with scAAV9.CB.CLN6 nclf The mice had corrected these memory and learning deficits 12 months after the injection ( Fig. 9 C). When wild-type mice were compared to scAAV9.CB.CLN6 treated animals only at later time points in the Morris water maze test, we found that even treated mice required more time to find the platform at 18 and 24 months of age, whereas swimming speed was the same between all tested groups ( Fig. 9 C, Fig.10 B) To assess memory and learning at later time points, mice were subjected to a water maze reversal test at 12, 18, and 24 months of age, in which the platform was moved to a novel location. In this test, Cln6 treated with scAAV9.CB.CLN6 showed significantly higher levels of leukemia compared to wild-type mice. nclf Mice took significantly longer to find the new platform location ( Fig.10 A). Taken together, these results indicate that a single treatment with scAAV9.CB.CLN6 prevented much of the motor decline seen in these animals, but was unable to fully protect against memory and learning deficits when the mice were tested at later time points.

[0130] Improved survival after delivery of scAAV9.CB.CLN6

[0131] Known Cln6 nclf The survival rate of mice was reduced compared with wild-type mice (Guyenet et al., Journal of Visualized Experiments: JoVE. 201039). nclf The survival of the mice was compared with that of wild-type mice injected with PBS. nclf Compared with mice, a single ICV injection of scAAV9.CB.CLN6 into Cln6 nclf The CSF of mice significantly increased their survival rate ( Fig. 9D) Although the median survival of PBS-treated mice was 14 months, the Cln6 nclf The median survival of mice was 21.5 months. This 65% increase in survival is very significant. nclf The survival curves of the mice were not significantly different from those of wild-type animals.

[0132] Furthermore, body weight was recorded monthly as a measure of overall health. The ability of scAAV9.CB.CLN6 treated mice to maintain their body weight also highlights the improvement in health and survival, as no differences were observed compared to wild-type animals, whereas PBS treated Cln6 nclf Starts to lose weight around 10 to 12 months ( Fig. 9 E).

[0133] 172 wild-type mice were treated with PBS and 5 × 10 10 A safety study was conducted in 223 wild-type mice treated with vg / animal. This study demonstrated that scAAV9.CB.CLN6 was well tolerated over 24 weeks with no adverse effects attributable to the virus (data not shown). In summary, this is the best Cln6 study to date. nclf The longest survival extension in the mouse model and indicates the utility of scAAV9.CB.CLN6 alone to restore cellular and functional defects in CLN6-type Batten disease.

[0134] Example 3

[0135] Safety study of scAAV9.CB.CLN6 in non-human primates

[0136] To test the safety of this treatment in a large animal model more relevant to human patients, three four-year-old male cynomolgus monkeys were administered scAAV9.CB.CLN6 formulated in 1× PBS and 0.001% Pluronic F68.

[0137] The animals were sacrificed 1, 3, or 6 months after injection. Each individual received a single lumbar intrathecal injection with 6 × 10 13 The viral vector was delivered directly into the CSF at a dose of 100 viral particles. After injection, the animals were kept in the Trendelenburg position for 15 min with the head facing downward at a 45-degree angle to facilitate targeting of the brain and upper spinal cord regions.

[0138] All subjects recovered well from the injection and did not show any abnormal behavior. During the study period (baseline, 1 month, 2 months, 3 months and 6 months), hematology and serum chemistry were performed at up to 5 time points and did not show major abnormalities. Specifically, no evidence of elevated aspartate aminotransferase (AST) or alkaline phosphatase levels was found, while alanine aminotransferase (ALT) was slightly increased (less than 200 units / liter) in one animal 1 month after injection ( Fig.11 C).

[0139] There were no changes in total protein levels, creatinine, triglycerides, glucose or ions such as phosphorus, calcium, magnesium or sodium. Extensive histopathology and transgene expression analysis was performed on each animal at sacrifice. With the exception of one animal that showed bladder infection at necropsy, no abnormalities were found in any of the tissues analyzed, including various brain and spinal cord regions, heart, lung, liver, spleen, kidney, small intestine, skeletal muscle (diaphragm, triceps, TA, gastrocnemius), gonads.

[0140] A single lumbar intrathecal injection of scAAV9.CB.CLN6 into the cerebrospinal fluid induced high expression of the transgene throughout the brain and spinal cord of non-human primates, as shown by fluorescent western blots. Fig.11 The blot in A shows expression of CLN6 in the cortex, corpus callosum, periventricular white matter, hippocampus, cerebellum, thalamus, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. High expression of the transgene was found throughout the brain and spinal cord of all three animals ( Fig.11 A to Fig.11 B). In summary, these data indicate that treatment with scAAV9.CB.CLN6 was well tolerated and safe in all three subjects tested.

[0141] Example 4

[0142] Clinical trials of scAAV9.CB.CLN6 gene therapy

[0143] scAAV9.CB.CLN6 was delivered intrathecally to human patients with CLN6-type Batten disease.

[0144] The scAAV used for clinical trials was produced by the Nationwide Children's Hospital Clinical Manufacturing Facility using a triple transfection method of HEK293 cells under GMP conditions as described in Example 1.

[0145] Patients were selected for participation if they were one year old or older and had a diagnosis of CLN6 disease as determined by genotype. The first cohort (n = 12) received a one-time gene transfer dose of 1.5 × 10 13 vg total scAAV. scAAV9.CB.CLN6 was prepared in 20 mM Tris (pH 8.0), 1 mM MgCl 2 , 200 mM NaCl, 0.001% poloxamer 188, and about 20% to about 40% nonionic low-osmotic compound, and delivered once into the subarachnoid space of the lumbar intervertebral sac via an intrathecal catheter inserted by lumbar puncture. Safety was assessed based on clinical conditions and by reviewing the safety label. There was at least four weeks between enrollment of each subject to allow review of safety data at day 30 after gene transfer.

[0146] The preliminary data provided herein report ten patients treated, and the average follow-up time is 12 months (1 month to 24 months after treatment). Preliminary data demonstrate that the administration of scAAV9.CB.CLN6 is generally well tolerated. Most adverse events are mild and unrelated to treatment. Any T cell responses and antibody elevations observed are not associated with clinical manifestations and do not require changes in treatment.

[0147] Figure 12 provides preliminary data reporting disease progression in sibling pairs in two studies as measured by the Hamburger Motor and Language Scale post-injection. These sibling pairs had the same gCLN6 mutation genotype.

[0148] Twenty-four-month efficacy study

[0149] Data from eight treated patients in an ongoing clinical study are provided herein. The eight patients described herein were administered and exposed to scAAV9.CB.CLN6 for at least 17 months. Baseline information for these eight patients is provided below.

[0150]

[0151] Data from an ongoing 24-month clinical study indicate that a single intrathecal administration of scAAV9.CB.CLN6 is generally well tolerated. 137 adverse events were reported. Most adverse events (AEs) were mild and unrelated to treatment. Nine grade 3 (serious) adverse events (expressed as SAEs) were reported in four patients. Three of the nine SAEs were considered possibly related to treatment. Related events included vomiting (2), upper abdominal pain (1), and fever (1) and all four patients recovered. No grade 4 (life-threatening) or grade 5 (fatal) adverse events were reported. There was no pattern of adverse events associated with anti-AAV9 capsid or anti-CLN6 immunogenicity.

[0152] Fig.15 Efficacy data are provided showing positive effects on motor and language function. In 7 of 8 patients treated with scAAV9.CB.CLN6, Hamburger scores remained unchanged or had an initial change (+1 to -1 point) followed by stabilization. The oldest patient in this study (treated at 79 months of age) dropped two points. Natural history data suggest that Hamburger motor and language drop by 2-3 points within 24 months of symptom onset.

[0153] Fig.16A -C provides sibling comparison data, all of whom had CLN6 disease. Treated patients showed stabilization relative to untreated siblings who experienced a substantial decline in motor and language abilities or died over the same time period. These data are provided as Hamburger Scores: Motor + Language over time. Fig.16A A cumulative score is provided, and Fig. 16B A burger sports subscore is provided, and Fig. 16C A Hamburger language subscore is provided.

[0154] Fig. 12A -C presents data comparing siblings in the study, all treated with scAAV9.CB.CLN6. These data show that younger siblings showed increases or stabilization in Hamburger motor and language scores compared to older siblings who had initial changes followed by stabilization. Fig. 12A These cumulative scores are provided, and Fig. 12B A burger sports subscore is provided, and Fig. 12C A Hamburger language subscore is provided.

[0155] Fig.17 Data from the first 8 patients treated with scAAV9.CB.CLN6 were compared to data from an ongoing natural history study of CLN6 patients conducted by Nationwide Children's Hospital (n=14). Shown are Kaplan-Meier curves of the combined Hamburger motor and language function scores relative to baseline until a 2 or more point decrease was not reversed. Confidence bands were calculated using the survival probability estimate and its standard error. The figure compares patients in the treatment group with a ≥2 point decrease in the combined Hamburger motor and language score over a 2-year period relative to the natural history group, and conveys results supporting the efficacy of the treatment of the present invention, including: (i) only 1 treated patient achieved a ≥2 point decrease over the period compared to all 14 natural history untreated patients; and (ii) a clear separation of treated patients from untreated patients.

[0156] In summary, the 24-month efficacy data demonstrated the following: i) stabilization of disease, compared to untreated siblings who experienced a rapid decline in their motor and language abilities; ii) younger patients showed increases in scores or stabilization; and iii) most older patients showed initial changes followed by stabilization. In addition, treatment was generally well tolerated.

[0157] Dose escalation study

[0158] If there are no safety issues, additional subjects will be enrolled after the first cohort is evaluated one month after injection. Each subject in the cohort 2 (n=4) receives an increasing dose of viral vector. There is at least a six-week time window between the completion of the cohort 1 and the start of the cohort 2 to allow for a review of the safety analysis from five time points (day 1, day 2, day 7, day 14 and day 21) and DSMB review before giving the next subject.

[0159] Disease progression was measured using the UBDRS scale or the Hamburger Motor and Language Scale (referenced above in the detailed description) and the effect of treatment on quality of life and potential to prolong survival using the Pediatric Quality of Life (PEDSQOL) scale.

[0160] The primary analysis evaluating efficacy will be when all patients complete the three-year study period. Efficacy is determined based on disease stabilization or reduction in disease progression based on the well-developed Unified Batten Disease Rating Scale (UBDRS) specifically developed for CLN6 Batten disease or the Hamburg Motor and Language Scale. After completing the three-year study period, patients will be monitored annually for five years per FDA guidance.

[0161] Example 5

[0162] Natural history study demonstrates that scAAV9.CB.CLN6 gene therapy improves motor and language scores

[0163] To facilitate comparison of study subjects (first patient in CLN6 gene transfer study (n=8)) with natural history subjects regarding clinical course over time, combined Hamburger Scale motor (M) and language (L) scores from gene transfer patients were matched with combined Hamburger Scale motor and language score data collected on patients in the retrospective CLN6 natural history study (PI: Emily de los Reyes, MD; ClinicalTrials.gov identifier: NCT03285425). Gene transfer patients were matched to natural history patients based on baseline Hamburger motor and language scores and age at the time of comparison (within 12 months).

[0164] Combined and separate data (n=8) of Hamburg motor and language scores showed that CLN6 gene therapy halted or substantially slowed disease progression, with a positive impact on motor and language function in 7 of 8 patients ( Fig.18 A positive effect was defined as a patient maintaining a combined Hamburger score or having an initial change (+1 to -1 point) followed by stabilization. Individual motor and language scores were consistent with the corresponding combined scores.

[0165] Data for natural history matched comparisons also showed improvements in Hamburger motor and language scores ( Fig.19 ). Using a many-to-one matching approach, the mean Hamburger motor and language scores of matched natural history patients at the last time point of the comparison period (for the corresponding gene transfer patients) are plotted in red compared to the corresponding Hamburger motor and language values ​​of the gene transfer patients at the last time point (plotted in green). Fig.19 The number of natural history patients in each comparison is provided in each figure along with the difference between Hamburger motor and language scores at the last time point (between the mean of the gene transfer patients and the NH patients). Natural history data for Batten disease patients (n=11) collected at Nationwide Children's Hospital and Dr. Emily de los Reyes' study showed a fairly linear and almost continuous decline in Hamburger motor + language scores from two to seven years of age ( Fig. 20 ).

[0166] In summary, data from these studies indicate that the majority of CLN6 gene transfer patients exhibit improvements in motor and language scores compared with matched natural history patients.

[0167] Example 6

[0168] Analysis of the Mullen Early Learning Scale

[0169] The Mullen Scale for Early Learning (MSEL) is used to assess whether scAAV.CB.CLN6 gene therapy improves the patient's learning ability within 12 to 24 months. MSEL is a normalized measure of cognitive function administered alone, which is designed for children from birth to 68 months. The subscales of MSEL are overall movement, fine movement, receptive language, expressive language, and early learning composite. (See Mullen EM. (1995). Mullen Scales of Early Learning (AGS Editor) Circle Pines, Minnesota: American Guidance Service Inc.).

[0170] The following four domains were analyzed in eight patients: visual receptive, fine motor, receptive language, and expressive language. Fig.21A and 21B Raw scores for the four domains are provided. Higher scores indicate higher function.

[0171] Summarize

[0172] Interim safety and efficacy data suggest that AAV9-CLN6 gene therapy has the potential to stabilize progression of variant late infantile-onset CLN6-type Batten disease. Efficacy results demonstrated meaningful treatment effects on motor and language function. Patients treated with AAV9-CLN6 demonstrated improvements in Hamburger motor and language scores compared to untreated siblings, and the means of natural history patients were matched for age and Hamburger motor and language baseline scores. Comparison of younger and older treated siblings further supports the potential benefit of early intervention gene therapy with AAV9-CLN6. Younger treated patients demonstrated improvements or stabilization of cognitive skills, as measured on the MSEL scale.

[0173] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, many variations, changes and substitutions will now occur to those skilled in the art. It should be understood that various alternatives to the embodiments described herein may be adopted. It is contemplated that the following claims define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

[0174] All documents mentioned in this application are incorporated herein by reference in their entirety.

Claims

1. A nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO:

4.

2. A self-complementary recombinant adeno-associated virus 9 (scAAV9), comprising the nucleic acid molecule according to claim 1.

3. The scAAV9 of claim 2, wherein the scAAV9 comprises a single-stranded genome.

4. An rAAV particle comprising the nucleic acid molecule according to claim 1.

5. The rAAV particle of claim 4, wherein the rAAV particle comprises a single-stranded genome.

6. A composition comprising: the scAAV9 according to claim 2 or 3, the nucleic acid molecule according to claim 1, or the rAAV9 virus particle according to claim 4 or 5; and a pharmaceutically acceptable excipient, carrier or diluent.

7. The composition of claim 6, wherein the excipient comprises a nonionic low permeability compound, a polymer, or a combination thereof.

8. The composition of claim 6, wherein the excipient comprises a buffer.

9. The composition of claim 6, wherein the excipient comprises a salt.

10. A composition for treating CLN6 Batten disease in a subject, comprising: a therapeutically effective amount of scAAV9 according to claim 2.

11. A composition for treating CLN6 Batten disease in a subject, comprising: a therapeutically effective amount of the rAAV9 virus particles according to claim 4.

12. The composition of claim 10, wherein the composition is formulated for administration by a route selected from the group consisting of intrathecal, intracerebroventricular, intraparenchymal, intravenous, or a combination thereof.

13. The composition of claim 12, wherein the composition is formulated for intrathecal administration.

14. The composition of claim 12, wherein the composition is formulated for intracerebroventricular administration.

15. The composition of claim 12, wherein the composition is formulated for intravenous administration.

16. The composition of claim 11, wherein the composition is formulated for intrathecal administration.

17. The composition according to claim 16, wherein the composition comprises 1×10 11 to 1×10 15 vg of the rAAV9 viral particles.

18. The composition according to claim 16, wherein the composition comprises 1×10 12 to 1×10 14 vg of the rAAV9 viral particles.

19. The composition of claim 10, wherein administration of the composition stabilizes or alleviates one or more symptoms of CLN6-type Batten disease compared to untreated CLN6-type Batten disease patients, the symptoms selected from: (a) Loss of brain mass; (b) loss of cognitive function; and (c) Delayed language development.

20. The composition of claim 10, wherein administration of the composition stabilizes or slows disease progression of CLN-6 Batten disease.

21. The composition of claim 20, wherein disease progression is assessed using the UBDRS scale, the Hamburg Motor and Language Scale, the Effect of Treatment on Quality of Life Using the Pediatric Quality of Life (PEDSQOL) scale, the Mullen Scales of Early Learning (MSEL), prolonged survival potential, or a combination thereof.

22. The composition of claim 10, wherein the subject is 80 months or younger.

23. The composition of claim 16, wherein the subject is placed in a Trendelenburg position following administration of the rAAV9 viral particles.

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