Adeno-associated virus delivery of cln3 polynucleotide

Abstract

This disclosure relates to the delivery of recombinant adeno-associated virus (rAAV) to neuronal 3 (CLN3) polynucleotides in ceroid lipofuscinosis. This disclosure provides rAAV and a method for CLN3 gene therapy using said rAAV in neuronal ceroid lipofuscinosis or CLN3-type batten disease.

Description

[Technical Field] This disclosure relates to the delivery of recombinant adeno-associated virus (rAAV) of neuronal 3 (CLN3) polynucleotides in neuronal ceroid lipofuscinosis. This disclosure provides rAAV and methods for using rAAV for CLN3 gene therapy in neuronal ceroid lipofuscinosis (NCL) or CLN3-type batten disease. [Previous Technology] Neuronal cerebrolipofuscinosis (NCL) is a group of serious neurodegenerative diseases. Mutations in the CLN3 gene lead to juvenile NCL or CLN3-Batten Disease (Kitzmü et al., *Human Molecular Genetics*, 2008; 17(2):303-312; Munroe et al., *American Journal of Human Genetics*, 1997; 61:310-316), also known as Spielmeyer-Sjogren-Vogt disease. The mutations interfere with lysosomal storage and clearance processes. At this point, 67 mutations causing the disease have been described. However, 85% of patients have a 1.02 kb deletion of the isotype conjugate, resulting in the deletion of exons 7 and 8. The CLN3 mutations found in patients primarily result in reduced abundance or function of the battenin protein. The typical age of onset for CLN3 type Batten disease is between 4 and 7 years of age, with potentially but rapidly progressive vision loss. Children with early-onset NCL go from normal vision to blindness within months, but may retain light and dark perception for several years. Cognitive and motor decline is usually accompanied by behavioral problems such as aggression (8 to 10 years) and then seizures (10 to 12 years). The characteristic progression of Parkinson's disease occurs between 11 and 13 years of age. Cardiac conduction abnormalities have been reported in individuals in later stages of the disease. High phenotypic variability exists among individuals with CLN3 type Batten disease, but all patients share common low vision or progressive blindness. Furthermore, the physical component of the Unified Batten Disease Rating Scale (UBDRS), validated in 82 patients, showed a steady and measurable decline of 2.86 points per year (2.27 to 3.45, p < 0.0001). The median survival from symptom onset to death is typically 15 years. Treatment options for CLN3 type Bardén disease are broad and aimed at alleviating the disease. These include drug therapies such as EGIS-8332 and talampanel, which target the AMPA receptor; drugs that allow for early cessation of mutations; drugs that help break down accumulated storage material (cystagon / cysteamine); and even immunosuppressive therapies (mycophenolate, prednisolone). Enzyme replacement and stem cell therapies have also been evaluated. Although many treatment options have been investigated, few have been evaluated in clinical settings. There is no treatment that can slow the progression or cure the disease. Patients and their families rely on treatment to improve symptoms and for palliative care. The Cln3Δex7 / 8 mouse model was established in the early 21st century to mimic the most common pathogenic mutation in patients with CLN3-type batten disease: a mutation of approximately 1 kb in exons 7 and 8 of the CLN3 gene that eliminates exons 7 and 8 (Cotman et al., Human Molecular Genetics, 2002; 11(22):2709-2721; Mole et al., European Journal of Paediatr Neurol., 2001; 5:7-10). This mutation was found in 85% of patients via isotype conjugation and in another 15% of patients as a combination of a heterozygous mutation and a point mutation on another allele. Exon loss is predicted to produce frameshift mutations, leading to loss or reduction of activity of truncated protein products (Lerner et al., Cell, September 22, 1995; 82(6):949-57; Kitzmüller et al., Human Molecular Genetics, January 15, 2008; 17(2):303-12). In their initial study, Cotman et al. demonstrated that the CLN3Δex7 / 8 mouse model successfully encapsulated several aspects of CLN3 disease. CLN3Δex7 / 8 animals accumulated spontaneously fluorescent storage material and ATP synthase subunit C in the nervous system at different time points and began to exhibit astrocyte responsiveness in the brain at 10 months of age. Subsequent studies detailed alterations in glutamate receptor function in the cerebellum, corresponding to motor deficits in accelerated swirl bar assays (Cotman et al., Human Molecular Genetics, 2002; 11(22):2709-2721). In terms of behavior, Cln3Δex7 / 8 mice have been characterized at both young and mature age points, with neurodevelopmental motor delay observed in newborn and young adult mice, and gait and hind limb clasping defects observed at 10 to 12 months of age (Cotman et al., Human Molecular Genetics 2002; 11(22):2709-2721; Osório et al., Genes Brain Behav. 2009 April; 8(3): 337-345). CLN3Δex7 / 8 mice did not appear to exhibit functional visual impairment, but had a slight survival defect compared to 12-month-old control mice (Cotman et al., Human Molecular Genetics 2002; 11(22):2709-2721; Seigel et al., Molecular Cell Neurosci. 2002 April; 19(4):515-27). In summary, the Cln3Δex7 / 8 mouse model carrying the most common human mutation exhibits many cellular and behavioral changes consistent with CLN3-type batten disease, making it a suitable model for testing therapies. Therefore, there remains a need in this field to treat CLN3 type batten disease. [Summary of the Invention] This article provides methods and products for CLN3 gene therapy using recombinant AAV. This document provides a recombinant adeno-associated virus 9 (rAAV9) encoding the CLN3 polypeptide, comprising an rAAV9 genome including, in a 5' to 3' sequence, a p546 promoter and a polynucleotide encoding the CLN3 polypeptide. In some embodiments, the rAAV9 genome comprises a self-complementary genome. In some embodiments, the rAAV9 genome comprises a single-stranded genome. A self-complementary recombinant adeno-associated virus 9 (scAAV9) is provided, encoding the CLN3 polypeptide shown in SEQ ID NO: 1, wherein the genome of said scAAV9 comprises, in a 5' to 3' sequence: a first AAV terminal inverted repeat; a p546 promoter comprising the sequence of SEQ ID NO: 3; a polynucleotide encoding the CLN3 polypeptide shown in SEQ ID NO: 1; and a second AAV terminal inverted repeat. The polynucleotide encoding said CLN3 polypeptide may be at least 90% identical to that of SEQ ID NO: 2. Also provided is a scAAV9 having a genome comprising, in a 5' to 3' order, the following: a first AAV terminal inverted repeat; a P546 promoter comprising the sequence of SEQ ID NO: 3; an SV40 intron; a polynucleotide encoding the CLN3 polypeptide of SEQ ID NO: 1; and a second AAV terminal inverted repeat; a scAAV9 having a genome comprising, in a 5' to 3' order, the following: a first AAV terminal inverted repeat; a P546 promoter comprising the sequence of SEQ ID NO: 3; a polynucleotide encoding the CLN3 polypeptide of SEQ ID NO: 1; a bovine growth hormone polyadenylated poly A sequence; and a second AAV terminal inverted repeat. In an exemplary embodiment, the scAAV9 has a genome comprising the gene cassette shown in SEQ ID NO: 4. A single-stranded recombinant adeno-associated virus 9 (ssAAV9) is provided, encoding the CLN3 polypeptide shown in SEQ ID NO: 1, wherein the genome of said ssAAV9 comprises, in a 5' to 3' sequence: a first AAV terminal inverted repeat; a p546 promoter comprising the sequence of SEQ ID NO: 3; a polynucleotide encoding the CLN3 polypeptide shown in SEQ ID NO: 1; and a second AAV terminal inverted repeat. The polynucleotide encoding said CLN3 polypeptide may be at least 90% identical to that of SEQ ID NO: 2. Also provided is an ssAAV9 having a genome comprising, in a 5' to 3' order, the following: a first AAV terminal inverted repeat; a P546 promoter comprising the sequence of SEQ ID NO: 3; an SV40 intron; a polynucleotide encoding the CLN3 polypeptide of SEQ ID NO: 1; and a second AAV terminal inverted repeat; ssAAV9 having a genome comprising, in a 5' to 3' order, the following: a first AAV terminal inverted repeat; a P546 promoter comprising the sequence of SEQ ID NO: 3; a polynucleotide encoding the CLN3 polypeptide of SEQ ID NO: 1; a bovine growth hormone polyadenylated poly A sequence; and a second AAV terminal inverted repeat. The nucleic acid sequence shown in SEQ ID NO: 4 is the gene cassette provided in Figure 1A. rAAV9 with an scAAV9 genome or an ssAAV9 genome is provided, comprising a nucleic acid sequence that is at least 90% identical to, or at least 95% identical to, or at least 98% identical to, the nucleic acid sequence of SEQ ID NO: 4. Also provided is a nucleic acid molecule comprising: a first AAV terminal inverted repeat; a P546 promoter comprising the sequence of SEQ ID NO: 3; a nucleic acid sequence encoding the CLN3 polypeptide of SEQ ID NO: 1; and a second AAV terminal inverted repeat. In some embodiments, the polynucleotide encoding the CLN3 polypeptide is at least 90% identical to SEQ ID NO: 2. Also provided are nucleic acid molecules comprising: a first AAV terminal inverted repeat; a P546 promoter comprising the nucleotide sequence of SEQ ID NO: 3; an SV40 intron; a nucleic acid sequence encoding the CLN3 polypeptide of SEQ ID NO: 1; and a second AAV terminal inverted repeat. Additionally, polynucleotides comprising: a first AAV terminal inverted repeat; a P546 promoter comprising the nucleotide sequence of SEQ ID NO: 3; a nucleic acid sequence encoding the CLN3 polypeptide of SEQ ID NO: 1; a bovine growth hormone polyadenylated poly A sequence; and a second AAV terminal inverted repeat. In any of the provided polynucleotides, the CLN3 polypeptide may be encoded by the nucleic acid sequence shown in SEQ ID NO: 2 or a sequence that is at least 90% identical to SEQ ID NO: 2. rAAV9, scAAV9, or ssAAV9, including any polynucleotide, are available. rAAV with a single-stranded genome is also available. Further provided are rAAV9 viral particles encoding the CLN3 polypeptide, wherein the rAAV9 genome comprises, in a 5' to 3' sequence: a first AAV terminal inverted repeat; a P546 promoter comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 3; a polynucleotide encoding the CLN3 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 particles may include a polynucleotide encoding the CLN3 polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1. Additionally, the rAAV9 viral particles 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 rAAV9 viral particle may further include an SV40 intron and / or a BGH poly-A sequence. In any of the provided rAAV, ssAAV, and scAAV, the AAV terminal reverse repeat can be the AAV2 terminal reverse repeat. Also provided is a nucleic acid molecule comprising the rAAV9 genome, comprising, in a 5' to 3' sequence: a first AAV terminal inverted repeat; a P546 promoter comprising a nucleic acid sequence at least 90% identical to that of SEQ ID NO: 3; and a polynucleotide encoding a CLN3 polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 1. The provided nucleic acid molecule may comprise a self-complementary genome or a single-stranded genome. Further provided is a nucleic acid molecule comprising the rAAV9 genome, comprising, in a 5' to 3' order: a first AAV terminal inverted repeat; a P546 promoter comprising a nucleic acid sequence at least 90% identical to that of SEQ ID NO: 3; a polynucleotide encoding a CLN3 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 molecule may include a polynucleotide encoding a CLN3 polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1. Additionally, the nucleic acid molecule may include the 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, and at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4. Any provided nucleic acid molecule may further include an SV40 intron and / or a BGH poly-A sequence. Further, compositions comprising: scAAV9, ssAAV9, nucleic acid molecules, or rAAV viral particles as described herein, and at least one pharmaceutically acceptable excipient are provided. In some cases, the pharmaceutically acceptable excipient includes a nonionic hypotonic compound, a buffer, a polymer, a salt, or a combination thereof. In some embodiments, the polymer is a copolymer. In some embodiments, the copolymer is poloxamer. For example, the composition may include at least a pharmaceutically acceptable excipient comprising a nonionic hypotonic compound. For example, a pharmaceutically acceptable excipient may comprise about 20% to 40% of a nonionic, hypotonic compound or about 25% to about 35% of a nonionic hypotonic compound. An exemplary composition comprises 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 nonionic hypotonic compound. Another exemplary composition comprises scAAV formulated in 1X PBS and 0.001% Prönnicke F68. Further, a method for treating a subject with CLN3 type Barten disease is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of any of the following: any rAAV9 viral particles disclosed herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecule described herein, or any composition described herein. In any of the methods provided, the composition rAAV9, ssAAV9, scAAV9 and / or nucleic acid molecules are administered via a route selected from the group consisting of: intrathecal, intravenous, intracranial, intravenous, and combinations thereof. The use of therapeutically effective amounts of any rAAV9 viral particles disclosed herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecules described herein, or any composition described herein for the preparation of a medicament for the treatment of CLN3 type Barten disease in subjects of need. Compositions comprising therapeutically effective amounts of the following are also provided: any rAAV9 viral particles disclosed herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecule described herein, or any composition described herein for the treatment of CLN3 type batten disease in subjects of need. An exemplary dose of scAAV9, ssAAV9, or rAAV9 administered via the intrathecal route is approximately 1 × 10¹¹ vg of scAAV9, ssAAV9, or rAAV9 viral particles per subject to approximately 2 × 10¹⁵ vg; or approximately 1 × 10¹¹ vg of scAAV9, ssAAV9, or rAAV9 viral particles per subject to approximately 1 × 10¹⁵ vg of scAAV9, ssAAV9, or AAV9 viral particles per subject to approximately 2 × 10¹⁵ vg of scAAV9, ssAAV9, or rA ... Vg of scAAV9, ssAAV9, or AAV9 viral particles. For example, administering approximately 1 × 10¹³ Vg of scAAV9, ssAAV9, or AAV9 viral particles to the subject; or administering approximately 1.5 × 10¹³ Vg of scAAV9, ssAAV9, or AAV9 viral particles to the subject; or administering approximately 3.4 × 10¹³ Vg of scAAV9, ssAAV9, or AAV9 viral particles to the subject; or administering approximately 6 × 10¹³ Vg of scAAV9, ssAAV9, or AAV9 viral particles to the subject; or administering approximately 1.2 × 10¹⁴ Vg of scAAV9, ssAAV9, or AAV9 viral particles to the subject; or administering approximately 2 × 10¹⁴ Vg of scAAV9, ssAAV9, or AAV9 viral particles to the subject. Compared to pre-treatment subjects or untreated CLN3-barten disease patients, the methods, drugs, or compositions used for treatment resulted in subjects exhibiting one or more of the following: (a) reduced or slowed lysosomal aggregation of spontaneously fluorescent storage material; (b) reduced or slowed lysosomal aggregation of ATP synthase subunit C; (c) reduced or slowed glial cell activation (astrocytes and / or microglia); (d) reduced or slowed astrocytic proliferation; (e) reduced or slowed brain volume loss as measured by MRI; (f) reduced or slowed seizures; and (g) stabilization, reduced or slowed progression, or improvement in one or more of the scales used to assess the progression and / or improvement of CLN3-barten disease (e.g., the Unified Barten Disease Rating System (UBDRS) assessment scale or the Hamburg Motor and Language Scales). Subjects were able to maintain the Trendlenberg position after administration of rAAV9, ssAAV9, or the scAAV or nucleic acid molecules disclosed herein. Furthermore, a method for treating CLN3 disease in patients in need is provided, the method comprising delivering a composition comprising any of the following to the brain or spinal cord of the patient in need: any rAAV virus provided herein, any scAAV9 disclosed herein, any ssAAV9 disclosed herein, any nucleic acid molecule described herein, any composition described herein, or any drug described herein. In any of the methods or uses provided, the composition or drug may be delivered by intrathecal, intraventricular, intraparenchymal, or intravenous injection, or a combination thereof. Any of the methods provided may further include placing the patient in the Trendlenberg position after intrathecal injection of the composition, rAAV9 viral particles, or scAAV or the nucleic acid molecules disclosed herein. In any of the methods or uses provided, the composition or drug may include a nonionic low-osmotic contrast agent. For example, the composition includes a nonionic low-osmotic contrast agent selected from the group consisting of: iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, ioxilan, and combinations thereof. The administered composition or drug may include pharmaceutically acceptable excipients. For example, pharmaceutically acceptable excipients include about 20% to 40% of a nonionic, hypotonic compound or about 25% to about 35% of a nonionic hypotonic compound. One exemplary composition includes scAAV formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% of a nonionic hypotonic compound. Another exemplary composition includes scAAV formulated in 1X PBS and 0.001% Pronek F68. In any of the methods or uses provided, when delivering the composition or drug to the brain or spinal cord, it may be delivered to the brainstem, or to the cerebellum, or to the visual cortex, or to the motor cortex. Additionally, in any of the methods or uses provided, when delivering the composition or drug to the brain or spinal cord, it may be delivered to nerve cells, glial cells, or both. For example, delivery to the brain or spinal cord includes delivery to cells of the nervous system, such as neurons, lower motor neurons, microglia, oligodendrocytes, astrocytes, Schwann cells, or combinations thereof. Compared with pre-treatment or untreated subjects, the method, use, or administration of the composition or drug results in one or more of the following in the subject: (a) reduced or slowed lysosomal aggregation of spontaneously fluorescent storage material; (b) reduced or slowed lysosomal aggregation of ATP synthase subunit C; (c) reduced or slowed glial cell activation (astrocytes and / or microglia); (d) reduced or slowed astrocytic proliferation; (e) reduced or slowed brain volume loss as measured by MRI; (f) reduced or slowed seizures; and (g) one or more of the scales used to assess the progression and / or improvement of CLN3 Barten disease (e.g., the Unified Barten Disease Assessment System (UBDRS) Assessment Scale or the Hamburg Motor and Language Scale) remain stable, have reduced or slowed progression, or have improved. The title of this article is for the reader's convenience and is not restrictive. The use of "may" and "can" in this document describes the various embodiments included in the scope of the patent application, rather than indicating any uncertainty regarding the scope of the patent application.

Implementation Method

Claims

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

4.

2. A self-complementary recombinant adeno-associated virus 9 (scAAV9) comprising the nucleic acid molecule as claimed in claim 1.

3. scAAV9 as in claim 2, wherein the scAAV9 comprises a single-stranded genome.

4. An rAAV particle comprising a nucleic acid molecule as claimed in claim 1.

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

6. A composition comprising: Such as scAAV9 in request item 2 or 3, nucleic acid molecules in request item 1, or rAAV viral particles in request item 4 or 5; And pharmaceutically acceptable excipients, carriers or diluents.

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

8. Use of a scAAV9 as claimed in claim 2 or 3, a nucleic acid molecule as claimed in claim 1, an rAAV virus particle as claimed in claim 4 or 5, or a composition as claimed in claim 6 or 7, for the preparation of a pharmaceutical product for treating an individual with CLN3-Batten Disease.

9. As claimed in claim 8, wherein the pharmaceutical product is formulated for administration via a route selected from the group consisting of: intrathecal, intravenous, intracerebral, intravenous, and combinations thereof.

10. As claimed in claim 8, wherein the pharmaceutical product is formulated for intrathecal administration.

11. As requested in item 8, wherein the group of medicines is formulated for intraventricular administration.

12. As claimed in claim 8, wherein the pharmaceutical product is formulated for intravenous administration.

13. As claimed in any of claims 8 to 12, wherein the pharmaceutical product comprises 1 × 10¹² to 1 × 10¹⁵ vg of the rAAV virus particles.

14. As claimed in any of claims 8 to 12, wherein the pharmaceutical product comprises 6 × 10¹³ to 1.2 × 10¹⁴ vg of the rAAV virus particles.

15. As claimed in any of claims 8 to 12, administration of the pharmaceutical product reduces one or more symptoms of CLN3 type Barten disease, said symptoms being selected from: (a) reduced or slowed lysosomal aggregation of spontaneously fluorescent storage material, (b) reduced or slowed lysosomal aggregation of ATP synthase subunit C, (c) reduced or slowed glial cell activation (astrocytes and / or microglia), (d) reduced or slowed astrocyte proliferation, (e) reduced or slowed brain volume loss as measured by MRI, (f) reduced or slowed seizures, (g) one or more of the UBDRS assessment scales being stable, reduced or delayed in progression, or improved, said reduction, stabilization, or improvement being relative to the subject or untreated CLN3 type Barten disease patients before administration of the composition.

16. As requested in any of items 8 to 12, wherein the individual is placed in the Trendelenburg position after being given the rAAV9 virus particle.

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