JP2022527015A - Gene therapy for lysosomal disorders - Google Patents

Abstract

The present disclosure relates to compositions and methods for the treatment of central nervous system (CNS) diseases, such as Parkinson's disease (PD) and Gaucher's disease, in several aspects. In some embodiments, the present disclosure provides an expression construct comprising an introductory gene encoding one or more inhibitory nucleic acids targeting one or more CNS disease-related gene products and / or CNS disease-related genes or gene products. .. In some embodiments, the present disclosure provides a method of treating a CNS disease by administering such an expression construct to a subject in need thereof.

Description

Related Applications This application is entitled "AAV VECTORS ENCODING TREM2 AND USES THEREOF" under 35 USC 119 (e) and is filed on April 10, 2019, US Provisional Application No. 62 / 823,223. , "GENE THERAPIES FOR LYSOSOMAL DISORDERS", filed on April 10, 2019, No. 62 / 831,840, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", on April 10, 2019. Filed No. 62 / 831,846, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", filed on April 10, 2019, No. 62 / 831,856, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS" No. 62 / 934,450 filed on November 12, 2019, No. 62 / 954,089 filed on December 27, 2019, entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS". , "GENE THERAPIES FOR LYSOSOMAL DISORDERS", filed on January 13, 2020, No. 62 / 960,471, "GENE THERAPIES FOR LYSOSOMAL DISORDERS", on March 12, 2020. Claiming the benefits of filing date 62/990,246 filed March 16, 2020, entitled 62 / 998,665 filed and "GENE THERAPIES FOR LYSOSOMAL DISORDERS" The entire contents of each of these are incorporated herein by reference.

Background Gaucher's disease is a rare congenital abnormality of glycosphingolipid metabolism due to a deficiency of lysosomal acid β-glucocerebrosidase (Gcase, "GBA"). Patients suffer from non-CNS symptoms as well as findings including hepatosplenomegaly and bone marrow failure leading to pancytopenia, pulmonary disorders / fibrosis and bone defects. In addition, a significant number of patients also suffer from neurological symptoms, including intermittent eye movements and gaze abnormalities, seizures, cognitive impairment, developmental delay, and movement disorders including Parkinson's disease.

There are several therapies that address the main clinical manifestations of peripheral disease and hematopoietic bone marrow and viscera, including the enzyme replacement therapies described below, which bind to defective Gcase and improve stability. Includes molecular drugs and substrate suppression therapies that block the production of substrates that accumulate and cause symptoms and findings in Gaucher's disease. However, other aspects of Gaucher's disease, especially those that affect the skeleton and brain, appear to be refractory to treatment.

Summary This disclosure is in part enumerated in certain central nervous system (CNS) diseases, such as neurodegenerative diseases (eg, neurodegenerative diseases listed in Table 2), synucrane diseases (eg, listed in Table 3). Synuclein disease), tauopathy (tauopathy listed in Table 4), or lysosome storage disease (eg, lysosome storage disease listed in Table 5) with respect to compositions and methods for treating.

Patients with Gaucher's disease (patients with mutations in both chromosomal alleles of the GBA1 gene) as well as those with mutations in only one of the GBA1 alleles are at very high risk of Parkinson's disease (PD). The severity of PD symptoms-difficulty walking, tremor at rest, rigidity, and often depression, insomnia, cognitive decline, etc.-correlates with the degree of decreased enzyme activity. Therefore, patients with Gaucher's disease have the most severe course, while patients with a single mild mutation in GBA1 typically have a more benign course. Mutant carriers are also at high risk for other PD-related disorders, including Lewy body dementias characterized by executive function dysfunction, psychosis, and PD-like dyskinesia, and characteristic motor and cognitive deficits. Includes with multiple system atrophy. There is no cure to change the relentless course of these diseases.

Enzymes such as Gcase (eg, the gene product of the GBA1 gene) and common variants of many genes involved in lysosomal function or transport of polymers to lysosomes (eg, lysosomal membrane protein 1 (LIMP), which is SCARB2). Deficiencies (also referred to as) have been associated with an increased risk of PD and / or Gaucher's disease (neuropathy Gaucher's disease such as type 2 Gaucher's disease or type 3 Gaucher's disease). The present disclosure is in part in one or more genes associated with central nervous system (CNS) diseases such as Gaucher's disease, PD, etc., such as Gcase, GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB, SMPD1, It is based on an expression construct (eg, a vector) that encodes GCH1, RAB7, VPS35, IL-34, TREM2, TMEM106B, or any combination (or part thereof) described above. In some embodiments, the gene product combinations described herein act together (eg, synergistically) to alleviate one or more signs and symptoms of CNS disease when expressed in a subject. ..

Therefore, in some aspects, the present disclosure provides an isolated nucleic acid comprising an expression construct encoding Gcase (eg, the gene product of the GBA1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) Gcase coding sequence. In some embodiments, the nucleic acid sequence encoding Gcase encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 14 (eg, that set forth in NCBI reference sequence NP_0014148.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the Gcase protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a prosaposin (eg, the gene product of the PSAP gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) prosaposin coding sequence. In some embodiments, the nucleic acid sequence encoding prosaposin encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 16 (eg, that set forth in NCBI reference sequence NP_002769.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking a nucleic acid sequence encoding a prosaposin protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding LIMP2 / SCARB2 (eg, the gene product of the SCARB2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) SCARB2 coding sequences. In some embodiments, the nucleic acid sequence encoding LIMP2 / SCARB2 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 18 (eg, that set forth in NCBI reference sequence NP_005497.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the SCARB2 protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a GBA2 protein (eg, the gene product of the GBA2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) GBA2 coding sequences. In some embodiments, the nucleic acid sequence encoding GBA2 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 30, such as that set forth in NCBI reference sequence NP_065995.1. In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the GBA2 protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a GALC protein (eg, the gene product of the GALC gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) GALC coding sequences. In some embodiments, the nucleic acid sequence encoding GALC encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 33 (eg, that set forth in NCBI reference sequence NP_0014144.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the GALC protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a CTSB protein (eg, the gene product of the CTSB gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) CTSB coding sequences. In some embodiments, the nucleic acid sequence encoding CTSB encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 35 (eg, as set forth in NCBI reference sequence NP_001899.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the CTSB protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding an SMPD1 protein (eg, a gene product of the SMPD1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) SMPD1 coding sequences. In some embodiments, the nucleic acid sequence encoding SMPD1 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 37 (eg, that set forth in NCBI reference sequence NP_000534.3). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the CTSB protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a GCH1 protein (eg, the gene product of the GCH1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) GCH1 coding sequence. In some embodiments, the nucleic acid sequence encoding GCH1 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 45 (eg, that set forth in NCBI reference sequence NP_000534.3). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the GCH1 protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a RAB7L protein (eg, the gene product of the RAB7L gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) RAB7L coding sequence. In some embodiments, the nucleic acid sequence encoding RAB7L encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 47 (eg, that set forth in NCBI reference sequence NP_003920.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the RAB7L protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a VPS35 protein (eg, the gene product of the VPS35 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) VPS35 coding sequence. In some embodiments, the nucleic acid sequence encoding VPS35 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 49 (eg, that set forth in NCBI reference sequence NP_060676.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the VPS35 protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding an IL-34 protein (eg, the gene product of the IL34 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) IL-34 coding sequence. In some embodiments, the nucleic acid sequence encoding IL-34 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 55 (eg, that set forth in NCBI reference sequence NP_689669.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the IL-34 protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a TREM2 protein (eg, the gene product of the TREM gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) TREM2 coding sequence. In some embodiments, the nucleic acid sequence encoding TREM2 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 57 (eg, that set forth in NCBI reference sequence NP_061838.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the TREM2 protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a TMEM106B protein (eg, a gene product of the TMEM106B gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) TMEM106B coding sequence. In some embodiments, the nucleic acid sequence encoding TMEM106B encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 63 (eg, that set forth in NCBI reference sequence NP_060844.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 64. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking the nucleic acid sequence encoding the TMEM106B protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a progranulin, eg, a gene product of the PGRN gene (also referred to as the GRN gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells) prosaposin coding sequence. In some embodiments, the nucleic acid sequence encoding progranulin (PRGN (also referred to as GRN)) is an amino acid sequence as set forth in SEQ ID NO: 67 (eg, that set forth in NCBI reference sequence NP_002078.1). Encodes a protein containing. In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 68. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR), eg, an AAV ITR flanking a nucleic acid sequence encoding a prosaposin protein.

Aspects of the present disclosure relate to isolated nucleic acids encoding one or more inhibitory nucleic acids and expression constructs (eg, rAAV vectors). In some embodiments, the one or more inhibitory nucleic acids target a gene associated with a central nervous system (CNS) disease (eg, SNCA, TMEM106B, RPS2 or MAPT). In some embodiments, the inhibitory nucleic acid is expressed alone or in combination with one or more of the gene products described herein (eg, GBA1, PSAP, PRGN, etc.). In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets SNCA, and 2) a GBA1 protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets SNCA, and 2) a PSAP protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets SNCA, and 2) a PGRN protein (eg, GRN protein).

In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets MAPT, and 2) a GBA1 protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets MAPT, and 2) a PSAP protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets MAPT, and 2) a PGRN protein (eg, GRN protein). In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets TMEM106B, and 2) a GBA1 protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets TMEM106B, and 2) a PSAP protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets TMEM106B, and 2) a PGRN protein (eg, GRN protein). In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets RPS25, and 2) a GBA1 protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets RPS25, and 2) a PSAP protein. In some embodiments, the isolated nucleic acid encodes 1) an inhibitory nucleic acid that targets RPS25, and 2) a PGRN protein (eg, a GRN protein).

In some aspects, the present disclosure comprises isolated nucleic acids comprising an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of α-Syn placed beside an AAV reverse end repeat (ITR). offer. In some embodiments, the inhibitory nucleic acid is complementary to at least 6 contiguous nucleotides of the sequence set forth in SEQ ID NO: 90. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, the inhibitory nucleic acid comprises the sequence set forth in any one of SEQ ID NOs: 94-99.
In some aspects, the present disclosure provides an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of TMEM106B, which is flanked by AAV reverse end repeats (ITRs). .. In some embodiments, the inhibitory nucleic acid is complementary to at least 6 contiguous nucleotides of the sequence set forth in SEQ ID NO: 91. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA comprising the nucleic acid sequence set forth in SEQ ID NO: 92 or 93. In some embodiments, the inhibitory nucleic acid comprises the sequence set forth in SEQ ID NO: 65 or 66.

In some aspects, the present disclosure provides an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of MAPT placed beside an AAV reverse end repeat (ITR). .. In some embodiments, the inhibitory nucleic acid is complementary to at least 6 contiguous nucleotides of the sequence set forth in SEQ ID NO: 114. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA comprising the nucleic acid sequence set forth in SEQ ID NO: 123, 124, 127, 128, 131, 132, 135 or 136. In some embodiments, the inhibitory nucleic acid comprises the sequence set forth in SEQ ID NO: 125, 126, 129, 130, 133, 134, 137 or 138.
In some aspects, the present disclosure provides isolated nucleic acids comprising an expression construct encoding a first gene product and a second gene product, where each gene product is independently Table 1. It is selected from the gene products shown in 1 or a part thereof, or the inhibitory nucleic acids targeting the genes or gene products shown in Table 1. In some embodiments, the first gene product is a protein, and the second gene product is a protein. In some embodiments, the first gene product is an inhibitory nucleic acid, and the second gene product is a protein. In some embodiments, the first gene product is an inhibitory nucleic acid, and the second gene product is an inhibitory nucleic acid.

In some embodiments, the first gene product is, or is part of, a Gcase protein. In some embodiments, the second gene product is an inhibitory nucleic acid that targets SNCA. In some embodiments, the interfering nucleic acid is siRNA, shRNA, miRNA, or dsRNA, optionally where the interfering nucleic acid inhibits the expression of the α-Syn protein. In some embodiments, the isolated nucleic acid further comprises one or more promoters, optionally where each of the one or more promoters is independently a chicken-beta actin (CBA) promoter, CAG promoter, CD68. A promoter, or a JeT promoter. In some embodiments, the isolated nucleic acid further comprises an internal ribosome entry site (IRES), optionally where the IRES is positioned between the first gene product and the second gene product. .. In some embodiments, the isolated nucleic acid further comprises a self-cleaving peptide coding sequence, optionally where the self-cleaving peptide is T2A. In some embodiments, the expression construct comprises a first gene product and two adeno-associated virus (AAV) reverse terminal repeat (ITR) sequences flanking the second gene product.

In some aspects, the disclosure provides isolated nucleic acids comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product is independently tabulated. It is selected from the gene product shown in 1 or a part thereof, or an inhibitory nucleic acid targeting the gene or gene product shown in Table 1.
In some embodiments, the first gene product or the second gene product is the Gcase protein or a portion thereof. In some embodiments, the first gene product is the Gcase protein and the second gene product is GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, It is selected from TREM2 and TMEM106B.

In some embodiments, the expression construct encodes an interfering nucleic acid (eg, shRNA, miRNA, dsRNA, etc.) (eg, alone or in addition to another gene product). In some embodiments, the interfering nucleic acid inhibits the expression of α-synuclein (α-synuclein). In some embodiments, the expression construct encodes an inhibitory nucleic acid that targets SNCA, and GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, And one or more gene products selected from TMEM106B. In some embodiments, the interfering nucleic acid targeting α-synuclein comprises the sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, the interfering nucleic acid targeting α-synuclein binds (eg, hybridizes) to the sequence set forth in any one of SEQ ID NOs: 20-25.
In some embodiments, the interfering nucleic acid inhibits TMEM106B expression. In some embodiments, the expression construct encodes an inhibitory nucleic acid that targets TMEM106B, and GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, and. Encodes one or more gene products selected from TREM2. In some embodiments, the interfering nucleic acid targeting TMEM106B comprises the sequence set forth in SEQ ID NO: 65 or 66. In some embodiments, the interfering nucleic acid targeting TMEM106B binds (eg, hybridizes) to the sequence set forth in SEQ ID NO: 65 or 66.

In some embodiments, the interfering nucleic acid inhibits the expression of MAPT. In some embodiments, the expression construct encodes an inhibitory nucleic acid that targets MAPT, and GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2. , And one or more gene products selected from TMEM106B. In some embodiments, the interfering nucleic acid targeting MAPT comprises the sequence set forth in any one of SEQ ID NOs: 123-138. In some embodiments, the interfering nucleic acid targeting MAPT binds (eg, hybridizes) to the sequence set forth in any one of SEQ ID NOs: 123-138.

In some embodiments, the interfering nucleic acid inhibits the expression of RPS25. In some embodiments, the expression construct encodes an inhibitory nucleic acid that targets RPS25, and GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2. , And one or more gene products selected from TMEM106B. In some embodiments, the interfering nucleic acid targeting RPS25 comprises the sequence set forth in any one of SEQ ID NOs: 115-122. In some embodiments, the interfering nucleic acid targeting RPS25 binds (eg, hybridizes) to the sequence set forth in any one of SEQ ID NOs: 115-122. In some embodiments, the expression construct further comprises one or more promoters. In some embodiments, the promoter is a chicken beta actin (CBA) promoter, CAG promoter, CD68 promoter, or JeT promoter. In some embodiments, the promoter is an RNA pol II promoter (eg, or an RNA pol III promoter (eg, U6, etc.)).

In some embodiments, the expression construct further comprises an internal ribosome entry site (IRES). In some embodiments, the IRES is located between the first and second gene products.
In some embodiments, the expression construct further comprises a self-cleaving peptide coding sequence. In some embodiments, the self-cleaving peptide is a T2A peptide.

In some embodiments, the expression construct comprises two adeno-associated virus (AAV) reverse terminal repeat (ITR) sequences. In some embodiments, the ITR sequence is flanked by the first and second gene products (eg, arranged from 5'end to 3'end as follows: ITR-1 gene product: -Second gene product-ITR). In some embodiments, one of the ITR sequences of the isolated nucleic acid lacks a functional terminal degradation site (trs). For example, in some embodiments, one of the ITRs is ΔITR.
The present disclosure relates to an rAAV vector comprising an ITR having a modified "D" region (eg, wild-type AAV2 ITR, modified D sequence compared to SEQ ID NO: 29) in some aspects. In some embodiments, the ITR with the modified D region is the 5'ITR of the rAAV vector. In some embodiments, the modified "D" region comprises an "S" sequence as set forth, for example, in SEQ ID NO: 26. In some embodiments, the ITR having the modified "D" region is the 3'ITR of the rAAV vector. In some embodiments, the modified "D" region comprises a 3'ITR, where the "D" region is located at the 3'end of the ITR (eg, outside or at the end of the ITR with respect to the transgene insert of the vector). do. In some embodiments, the modified "D" region comprises a sequence as set forth in SEQ ID NO: 26 or 27.

In some embodiments, the isolated nucleic acid (eg, rAAV vector) comprises a TRY region. In some embodiments, the TRY region comprises the sequence set forth in SEQ ID NO: 28.
In some embodiments, the isolated nucleic acid described in the present disclosure comprises, comprises, or encodes a peptide having the sequence set forth in any one of SEQ ID NOs: 1-149.
In some aspects, the disclosure provides a vector containing the isolated nucleic acids described in the present disclosure. In some embodiments, the vector is a plasmid or viral vector. In some embodiments, the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector. In some embodiments, the rAAV vector is single-stranded (eg, single-stranded DNA).

In some aspects, the present disclosure provides host cells comprising the isolated nucleic acids described in the present disclosure or the vectors described in the present disclosure.
In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid protein and the isolated nucleic acid or vector described in the present disclosure.
In some embodiments, the capsid protein can cross the blood-brain barrier, eg, AAV9 capsid protein or AAVrh. 10 Capsid protein. In some embodiments, rAAV transfects neurons and non-neurons of the central nervous system (CNS).

In some aspects, the disclosure provides a method of treating a subject having or suspected of having a central nervous system disease, the method comprising subjecting the subject to the composition described in the present disclosure (eg, isolation). Contains administration of the nucleic acid or vector or composition comprising rAAV). In some embodiments, the CNS disease is a neurodegenerative disease such as the neurodegenerative disease shown in Table 2. In some embodiments, the CNS disease is a synuclein disease, such as the synuclein disease shown in Table 3. In some embodiments, the CNS disease is a tauopathy, such as the tauopathy shown in Table 4. In some embodiments, the CNS disease is a lysosomal storage disorder, such as the lysosomal storage disease shown in Table 5. In some embodiments, lysosomal storage disease is a neurological Gaucher disease such as type 2 Gaucher disease or type 3 Gaucher disease.

In some embodiments, the present disclosure relates to Parkinson's disease by administering an isolated nucleic acid encoding GBA1 (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. (For example, Parkinson's disease (PD-GBA) with GBA1 mutation, sporadic Parkinson's disease (sPD)), Gauche disease (eg, neuropathic Gauche disease (nGD), type I Gauche disease (T1GD), II. Selected from Type Gauche disease (T2GD) and Type III Gauche disease (T3GD)), Levy body dementia (DLB), Myotrophic lateral sclerosis (ALS), and Niemann-Pick disease type C (NPC) Regarding how to treat the disease.
In some embodiments, the present disclosure administers an isolated nucleic acid encoding a PGRN (eg, GRN) (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. With frontotemporal dementia (eg, frontotemporal dementia with GRN mutation (FTD-GRN), frontotemporal dementia with MAPT mutation (FTD-tau), and C9ORF72 mutation). Frontotemporal dementia (FTD-C9orf72)), Parkinson's disease (PD), Alzheimer's disease (AD), neuroseroid lipofustinosis (NCL), cerebral cortical basal nucleus degeneration (CBD), motor neuron disease (MND) , Or how to treat Gaucher's disease (GD).

In some embodiments, the present disclosure comprises an isolated nucleic acid (eg, an rAAV vector or rAAV containing the isolated nucleic acid) encoding an inhibitory nucleic acid that targets the GBA1 gene product and SNCA. Synuclein disease (eg, multiple system atrophy (MSA), Parkinson's disease (PD), Parkinson's disease with GBA1 mutation (PD-GBA), Lewy body dementias (DLB), by administration to subjects in need, It relates to a method for treating Lewy body dementias having a GBA1 mutation, and Lewy body disease).
In some embodiments, the present disclosure discloses Parkinson's disease (PD), frontotemporal dementia, by administering an isolated nucleic acid encoding PSAP (eg, rAAV vector or rAAV) to a subject in need thereof. It relates to a method of treating a disease selected from type dementia (eg, frontotemporal dementia with GRN mutation (FTD-GRN)), lithosome accumulation (LSD), or Gaucher's disease (GD).

In some embodiments, the present disclosure comprises administering an isolated nucleic acid encoding TREM2 (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof for Alzheimer's disease. (AD), Nasu-Hakora disease (NHD) or Parkinson's disease (PD).
In some embodiments, the present disclosure relates an isolated nucleic acid encoding an inhibitory nucleic acid targeting MAPT (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. By administration, Alzheimer's disease (AD) or frontotemporal dementia (frontotemporal dementia with MAPT mutation (FTD-Tau), progressive nuclear palsy (PSP), neurodegenerative disease, Lewy body dementias) It relates to a method of treating disease (LBD) or Parkinson's disease.

In some aspects, the disclosure provides a method for treating a subject having or suspected to have Parkinson's disease, wherein the method is a composition as described by the present disclosure (eg, isolation). Containing the administration of a nucleic acid or vector or a composition comprising rAAV) to a subject.
In some embodiments, the composition encodes two or more gene products (eg, CNS disease-related gene products), eg, 2, 3, 4, 5, or more gene products described in this application. Nucleic acid (eg, rAAV genome, eg, capsidized by AAV capsid protein). In some embodiments, the composition comprises two or more (eg, 2, 3, 4, 5, or more) different nucleic acids (eg, two or more rAAV genomes), each encoding one or more different gene products. , For example, individually capsidized by the AAV capsid protein). In some embodiments, two or more different compositions are administered to the subject, and each composition comprises one or more nucleic acids encoding different gene products. In some embodiments, the different gene products are operably linked to the same promoter type (eg, the same promoter). In some embodiments, the different gene products are operably linked to different promoters.

In some embodiments, administration comprises direct injection into the subject's CNS. In some embodiments, the direct injection is an intracerebral injection, an intraparenchymal injection, an intrathecal injection, an intratubal injection, or any combination thereof. In some embodiments, direct injection into the subject's CNS comprises convection enhanced delivery (CED).
In some embodiments, administration comprises peripheral injection. In some embodiments, the peripheral injection is an intravenous injection.

In some aspects, the present disclosure provides methods for treating subjects with or suspected of having central nervous system (CNS) disease, the methods comprising isolated nucleic acids including: Including: (i) an expression construct comprising an introductory gene encoding an inhibitory nucleic acid targeting one or more gene products shown in Table 1 or the genes or gene products shown in Table 1; and (ii). ) Two adeno-associated virus (AAV) reverse terminal repeats (ITR) flanking the expression construct. In some aspects, the disclosure provides methods for treating subjects with or suspected of having central nervous system (CNS) disease, the methods of which are two or more types encoding different gene products. Each type of isolated nucleic acid comprises administering to the subject an isolated nucleic acid of: (i) one or more gene products shown in Table 1 or a gene or gene shown in Table 1. An expression construct containing an introductory gene encoding an inhibitory nucleic acid that targets a gene product; and (ii) two adeno-associated virus (AAV) reverse terminal repeats (ITR) flanking the expression construct.

In some embodiments, the transgene encodes one or more proteins selected from: GBA1, GBA2, PGRN (eg, GRN), TREM2, PSAP, SCARB2, GALC, SMPD1, CTSB, RAB7L, VPS35, GCH1 and IL34. In some embodiments, the transgene encoding one or more gene products comprises a codon-optimized protein coding sequence. In some embodiments, the transgene encodes one or more inhibitory nucleic acids that target SNCA, MAPT, RPS25, and / or TMEM106B.
In some embodiments, the AAV ITR is an AAV2 ITR.
In some embodiments, the isolated nucleic acid is packaged within a recombinant adeno-associated virus (rAAV). In some embodiments, rAAV comprises an AAV9 capsid protein.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the CNS disease is a neurodegenerative disease, synuclein disease, tauopathy, and / or lysosome storage disease (LSD). In some embodiments, the CNS disease is listed in Table 2, Table 3, Table 4, or Table 5.
In some embodiments, administration comprises direct injection into the subject's CNS, optionally where direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intratubal injection or any of them. It is a combination. In some embodiments, the intracisterna magna is an occipital injection into the cisterna magna. In some embodiments, direct injection into the subject's CNS comprises convection enhanced delivery (CED). In some embodiments, the administration comprises a peripheral injection, optionally where the peripheral injection is an intravenous injection. In some embodiments, the subject is administered about 1 × 10 ¹⁰ vg to about 1 × 10 ¹⁶ vg of rAAV.

FIG. 1 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof). FIG. 2 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. The coding sequences for Gcase and LIMP2 are separated by an internal ribosome entry site (IRES). FIG. 3 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. Expression of the Gcase and LIMP2 coding sequences is driven by different promoters.

FIG. 4 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof), LIMP2 (SCARB2) or a portion thereof, and α-Syn interfering RNA. FIG. 5 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), prosaposin (eg, PSAP or a portion thereof), and α-Syn interfering RNA. be. FIG. 6 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and prosaposin (eg, PSAP or a portion thereof). The coding sequences for Gcase and prosaposin are separated by an internal ribosome entry site (IRES).

FIG. 7 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof). In this embodiment, the vector comprises a CBA promoter element (CBA) consisting of four moieties: a CMV enhancer (CMVe), a CBA promoter (CBAp), an exon 1 and an intron (int) to provide a codon-optimized coding sequence for human GBA1. It is constitutively expressed. The 3'region also contains a WPRE regulatory element followed by a bGH poly A tail. The 5'end of the promoter region contains three transcriptional regulatory activation sites, TATA, RBS, and YY1. Adjacent ITRs allow for correct packaging of intervening sequences. Two variants (insertion boxes) of the 5'ITR sequence were evaluated; they have several nucleotide differences within the "D" region of 20 nucleotides of wild-type AAV2 ITR. In some embodiments, the rAAV vector comprises the "D" domain nucleotide sequence shown in the top row. In some embodiments, the rAAV vector comprises a mutant "D" domain (eg, the "S" domain, with nucleotide changes shown in the bottom row).

FIG. 8 is a schematic diagram showing one aspect of the vector shown in FIG. FIG. 9 shows representative data for the delivery of rAAV containing a transgene encoding Gcase (eg, GBA1 or a portion thereof) in a CBE mouse model of Parkinson's disease. Daily IP delivery of PBS vehicle, 25 mg / kg CBE, 37.5 mg / kg CBE, or 50 mg / kg CBE (left to right) was initiated at P8. The survival rate (upper left) was checked twice a day, and the body weight (upper right) was checked daily. All groups started at n = 8. As for the behavior, the total distance traveled in the open field (lower left) was evaluated by P23, and the time until the fall on the rotor rod (lower center) was evaluated by P24. GCase substrate levels were analyzed in the cortex of mice with and without CBE discontinuation (day 3) in PBS and 25 mg / kg CBE treatment groups. Total GluSph & GalSph levels (bottom right) are expressed as pmol per 1 mg of wet weight of tissue. Present the average value. The error bar is SEM. ^* P <0.05; ^** p <0.01; ^*** p <0.001, nominal p-value for the treatment group by linear regression.

FIG. 10 is a schematic diagram illustrating an aspect of study design for maximum rAAV doses in a CBE mouse model. Briefly, rAAV was delivered at P3 by ICV injection and daily CBE treatment was initiated at P8. Behavior was assessed at P24-25 by open field and rotarod assays, and substrate levels were measured at P36 and P38. FIG. 11 shows representative data for survival assessment of maximum rAAV doses in a CBE mouse model. At P3, mice were treated via ICV delivery with either excipients or 8.8e9 vg rAAV-GBA1. Daily IP delivery of either PBS or 25 mg / kg CBE was initiated at P8. At the end of the study, half of the mice were sacrificed on P36 (day 1) one day after the last CBE administration, while the other half were sacrificed on P38 (day 3) for 3 days of CBE. Has passed the cancellation. All treatment groups (excipient + PBS: n = 8, rAAV-GBA1 + PBS: n = 7, excipient + CBE: n = 8, and variant + CBE: n = 9) were weighed daily (upper left) at P36. Weight was analyzed (upper right). Behaviors were assessed by P23 (bottom left) for total distance traveled in the open field and P24 (bottom right) for time to fall on the rotor rod, and each animal was assessed as the median of three trials. Behaviors were assessed by P23 (bottom left) for total distance traveled in the open field and P24 (bottom right) for time to fall on the rotor rod, and each animal was assessed as the median of three trials. Due to lethality, n = 7 in the behavioral assay excipient + CBE group and n = 8 in all other groups. The average value across animals is presented. The error bar is SEM. ^* P <0.05; ^*** p <0.001, nominal p-value for treatment group by linear regression in CBE-treated animals.

FIG. 12 shows representative data for biochemical evaluation of maximum rAAV doses in a CBE mouse model. Before discontinuation of CBE (1) using the cortex of all treatment groups (excipient + PBS: n = 8, variant + PBS: n = 7, excipient + CBE: n = 7, variant + CBE: n = 9). GCase activity (upper left), GluSph level (upper right), GluCer level (lower left), and vector genome (lower right) were measured in the day) or later (day 3) groups. Before discontinuation of CBE (1) using the cortex of all treatment groups (excipient + PBS: n = 8, variant + PBS: n = 7, excipient + CBE: n = 7, variant + CBE: n = 9). GCase activity (upper left), GluSph level (upper right), GluCer level (lower left), and vector genome (lower right) were measured in the day) or later (day 3) groups. The biodistribution is displayed as a vector genome per 1 μg of genomic DNA. Present the average value. The error bar is SEM. ( ^* ) P <0.1; ^** p <0.01; ^*** p <0.001, nominal p-value of treatment group by linear regression in CBE-treated animals, collection date and gender difference as covariates With correction. FIG. 13 shows representative post-dose data of behavioral and biochemical correlations in the CBE mouse model of the excipient + PBS, excipient + CBE, and variant + CBE treatment groups. Throughout the treatment group, rotor rod performance was negatively correlated with GluCer accumulation (A, p = 0.0012 by linear regression) and GluSph accumulation was negatively correlated with increased GCase activity (B, P = 0.0086 by linear regression).

FIG. 14 shows representative data of the biodistribution of variants in the CBE mouse model. The presence of the vector genome in all treatment groups (excipient + PBS: n = 8, variant + PBS: n = 7, excipient + CBE: n = 7, and variant + CBE: n = 9) liver, spleen, kidney , And gonads. The presence of the vector genome in all treatment groups (excipient + PBS: n = 8, variant + PBS: n = 7, excipient + CBE: n = 7, and variant + CBE: n = 9) liver, spleen, kidney , And gonads. The biodistribution is displayed as a vector genome per 1 μg of genomic DNA. The presence of the vector genome was quantified using the vector reference standard curve by quantitative PCR; genomic DNA concentration was assessed by A260 optical density measurement. Present the average value. The error bar is SEM. ^* P <0.05; ^** p <0.01; ^*** p <0.001, nominal p-value of treatment group by linear regression in CBE-treated animals, with correction with collection date and gender difference as covariates ..

FIG. 15 shows representative data for survival assessment of the rAAV dose range in a CBE mouse model. Mice were administered one of three different doses of excipient or rAAV-GBA1: 3.2e9vg, 1.0e10vg, or 3.2e10vg at P3 by ICV delivery. At P8, daily IP treatment of 25 mg / kg CBE was started. Mice treated with excipients and CBE or excipients and PBS were used as controls. All treatment groups started at n = 10 (5M / 5F) per group. All mice were sacrificed 1 day after the final dose of CBE (P38-P40). All treatment groups were weighed daily and the weights were analyzed on P36. The kinetic performance was evaluated by the time to fall with the rotor rod at P24 and the crossing time of the tapered beam at P30. Due to early lethality, the number of mice that participated in the behavioral assay was: excipient + PBS: n = 10, excipient + CBE: n = 9, and 3.2e9vg rAAV-GBA1 + CBE: n = 6. , 1.0e10vg rAAV-GBA1 + CBE: n = 10, 3.2e10vg rAAV-GBA1 + CBE: n = 7. Present the average value. Error bars are SEM; ^* p <0.05; ^** p <0.01, nominal p-value by linear regression in CBE-treated animals, corrected for gender differences as covariates.

FIG. 16 shows representative data for biochemical evaluation of the rAAV dose range in a CBE mouse model. All treatment groups (excipient + PBS: n = 10, excipient + CBE: n = 9, and 3.2e9vg rAAV-GBA1 + CBE: n = 6, 1.0e10vg rAAV-GBA1 + CBE: n = 10, 3. A cortex of 2e10 vg rAAV-GBA1 + CBE: n = 7) was used to measure GCase activity, GluSph levels, GluCer levels, and vector genomes. GCase activity is shown as ng of GCase per 1 mg of total protein. GluSph and GluCer levels are shown as pmol per 1 mg of wet weight of tissue. The biodistribution is shown as a vector genome per 1 μg of genomic DNA. The presence of the vector genome was quantified using the vector reference standard curve by quantitative PCR; genomic DNA concentration was assessed by A260 optical density measurement. The presence of the vector genome was also measured in the liver (E). Present the average value. The error bar is SEM. ^** p <0.01; ^*** p <0.001, nominal p-value by linear regression in CBE-treated animals, corrected for gender differences as covariates.

FIG. 17 shows representative data for tapered beam analysis at maximum dose rAAV-GBA1 in a gene mouse model. Exercise performance of the treatment group (WT + excipient, (n = 5), 4L / PS-NA + excipient (n = 6), and 4L / PS-NA + rAAV-GBA1 (n = 5)), rAAV-GBA1 Analysis was performed by beam walking 4 weeks after administration. Total slip and activation time are shown as the sum of 5 trials on different beams. Velocity and slip per velocity are shown as the average of 5 trials on different beams. Present the average value. The error bar is SEM. FIG. 18 shows representative data for in vitro expression of rAAV constructs encoding progranulin (PGRN) protein (also referred to as GRN protein). The left panel shows the standard curve for the progranulin (PGRN) ELISA assay. The lower panel shows the dose response of PGRN expression as measured by ELISA assay in cell lysates of HEK293T cells transduced with rAAV. MOI = multiplicity of infection (vector genome per cell).

FIG. 19 shows representative data for in vitro expression of the rAAV construct encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids. FIG. 19 shows representative data for in vitro expression of the rAAV construct encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids. FIG. 19 shows representative data for in vitro expression of the rAAV construct encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids. The data show that transfection of HEK293 cells with each construct resulted in overexpression of the transgene of interest compared to mock-transfected cells. FIG. 20 shows an rAAV vector (top) containing a “D” region located “outside” the ITR (eg, proximal to the end of the ITR compared to a transgene insert or expression construct), and the ITR “inside” the vector. ”(Eg, proximal to the transgene insert of the vector) is a schematic diagram showing a wild-type rAAV vector.

FIG. 21 is a schematic diagram showing an aspect of a vector containing an expression construct encoding GBA2 or a portion thereof and α-Syn interfering RNA. FIG. 22 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and galactosylceramidase (eg, GALC or a portion thereof). Expression of the coding sequences for Gcase and galactosylceramidase is separated by the T2A self-cleaving peptide sequence. FIG. 23 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and galactosylceramidase (eg, GALC or a portion thereof). Expression of the coding sequences for Gcase and galactosylceramidase is separated by the T2A self-cleaving peptide sequence.

FIG. 24 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), cathepsin B (eg, CTSB or a portion thereof), and α-Syn interfering RNA. Is. Expression of the coding sequences for Gcase and cathepsin B is separated by the T2A self-cleaving peptide sequence. FIG. 25 shows one embodiment of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), sphingomyelin phosphodiesterase 1 (eg, SMPD1, a portion thereof), and α-Syn interfering RNA. It is a schematic diagram. FIG. 26 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and galactosylceramidase (eg, GALC or a portion thereof). The coding sequences for Gcase and galactosylceramidase are separated by an internal ribosome entry site (IRES).

FIG. 27 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and cathepsin B (eg, CTSB or a portion thereof). Expression of the Gcase and cathepsin B coding sequences is driven by different promoters. FIG. 28 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), GCH1 (eg, GCH1 or a portion thereof), and α-Syn interfering RNA. be. The coding sequences for Gcase and GCH1 are separated by the T2A self-cleaving peptide sequence. FIG. 29 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or part thereof), RAB7L1 (eg, RAB7L1 or part thereof), and α-Syn interfering RNA. be. The coding sequences for Gcase and RAB7L1 are separated by the T2A self-cleaving peptide sequence.

FIG. 30 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), GCH1 (eg, GCH1 or a portion thereof), and α-Syn interfering RNA. be. Expression of the Gcase and GCH1 coding sequences is an internal ribosome entry site (IRES). FIG. 31 is a schematic diagram illustrating an aspect of a vector comprising VPS35 (eg, VPS35 or a portion thereof) and an expression construct encoding an interfering RNA of α-Syn and TMEM106B. FIG. 32 illustrates an aspect of a vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), IL-34 (eg, IL34 or a portion thereof), and α-Syn interfering RNA. It is a figure. The coding sequences for Gcase and IL-34 are separated by the T2A self-cleaving peptide sequence.

FIG. 33 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and IL-34 (eg, IL34 or a portion thereof). The coding sequences for Gcase and IL-34 are separated by an internal ribosome entry site (IRES). FIG. 34 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and TREM2 (eg, TREM2 or a portion thereof). Expression of the Gcase and TREM2 coding sequences is driven by different promoters.

FIG. 35 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and IL-34 (eg, IL34 or a portion thereof). Expression of the Gcase and IL-34 coding sequences is driven by different promoters. 36A-36B show representative data for overexpression of TREM2 and GBA1 in HEK293 cells as measured by qPCR and ELISA and compared to control transduced cells. FIG. 36A shows data for overexpression of TREM2. FIG. 36B shows data for GBA1 overexpression from the same construct.

FIG. 37 shows representative data showing successful silencing of SCNA in vitro by the GFP reporter assay (top) and α-Syn assay (bottom). FIG. 38 shows representative data showing successful silencing of TMEM106B in vitro by the GFP reporter assay (top) and α-Syn assay (bottom). FIG. 39 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding PGRN (also referred to as GRN). FIG. 40 shows data on transduction of HEK293 cells using rAAV with wild-type (circular) ITRs or alternative (eg, “outer”; square) arrangements of ITRs of the “D” sequence. The "outer" placed rAAV with ITR was able to transduce cells as efficiently as rAAV with wild-type ITR.

FIG. 41 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof). FIG. 42 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof). FIG. 43 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and α-Syn interfering RNA.

FIG. 44 is a schematic diagram showing an aspect of a vector containing an expression construct encoding PGRN (also referred to as GRN). FIG. 45 is a schematic diagram showing an aspect of a vector containing an expression construct encoding PGRN (also referred to as GRN). FIG. 46 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an interfering RNA of PGRN (also referred to as GRN) and microtubule-associated protein tau (MAPT). The nucleic acid sequence of this vector is shown in SEQ ID NO: 142.

FIG. 47 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and α-Syn interfering RNA. FIG. 48 is a schematic diagram showing an aspect of a vector containing an expression construct encoding PSAP. FIG. 49 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof).

FIG. 50 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding Gcase (eg, GBA1 or a portion thereof) and galactosylceramidase (eg, GALC or a portion thereof). FIG. 51 illustrates an aspect of a plasmid comprising an rAAV vector comprising an expression construct encoding an interfering RNA of Gcase (eg, GBA1 or a portion thereof), prosaposin (eg, PSAP or a portion thereof), α-Syn interfering RNA. It is a schematic diagram which shows. FIG. 52 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an inhibitory RNA targeting Gcase (eg, GBA1 or a portion thereof), SNCA.

FIG. 53 is a schematic diagram showing an aspect of a plasmid containing an rAAV vector containing an expression construct encoding SNCA. FIG. 54 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 55 is a schematic representation of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets progranulin (PGRN (also referred to as GRN)) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence.

FIG. 56 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 57 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 58 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The "D" sequence of the 3'ITR is located "outside" the vector.

FIG. 59 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 60 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 61 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA.

FIG. 62 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 63 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting Gcase (GBA1) and SNCA. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence. FIG. 64 illustrates an aspect of a plasmid comprising an rAAV vector comprising Gcase (GBA1) and progranulin (PGRN (also referred to as GRN)), and an expression construct encoding an inhibitory RNA targeting TMEM106B. It is a schematic diagram which shows. The inhibitory RNA is located within the intron between the promoter sequence and the Gcase coding sequence.

FIG. 65 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting RPS25. FIG. 66 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting RPS25. FIG. 67 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets MAPT.

FIG. 68 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets MAPT. FIG. 69 is a schematic representation of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets progranulin (PGRN (also referred to as GRN)) and MAPT. The inhibitory RNA is located within the intron between the promoter sequence and the PGRN (also referred to as GRN) coding sequence. FIG. 70 is a schematic diagram illustrating an aspect of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets MAPT.

FIG. 71 is a schematic representation of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets progranulin (PGRN (also referred to as GRN)) and MAPT. The inhibitory RNA is located within the intron between the promoter sequence and the PGRN (also referred to as GRN) coding sequence. FIG. 72 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an inhibitory RNA targeting Gcase (eg, GBA1 or a portion thereof) and SNCA. The nucleic acid sequence of this vector is shown in SEQ ID NO: 141. FIG. 73 is a schematic diagram illustrating an aspect of a vector comprising an expression construct encoding an inhibitory RNA targeting Gcase (eg, GBA1 or a portion thereof) and SNCA. The nucleic acid sequence of this vector is shown in SEQ ID NO: 143.

FIG. 74 is a schematic representation of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA that targets Gcase (GBA1) and prosaposin (PSAP), and SNCA. The nucleic acid sequence of this vector is shown in SEQ ID NO: 144. Figures 75A-75C are charts showing knockdown of MAPT in SY5Y cells due to RNA interference. FIG. 75A shows the stationing of immunofluorescence of an AAV vector using a BGHpA oriented probe. FIG. 75B shows the results of RT-PCR of MAPT expression 3 and 7 days after transduction. FIG. 75C shows general information on the stock of rAAV virus used for transduction.

Detailed Description The present disclosure is based in part on compositions and methods for the expression of a combination of certain gene products (eg, gene products associated with CNS disease) in a subject. The gene product can be a protein, a fragment of a protein (eg, a portion), an interfering nucleic acid that inhibits a CNS disease-related gene, and the like. In some embodiments, the gene product is a protein or protein fragment encoded by a CNS disease-related gene. In some embodiments, the gene product is an interfering nucleic acid (eg, shRNA, siRNA, miRNA, miRNA, etc.) that inhibits a CNS disease-related gene.

A CNS disease-related gene refers to a gene that genetically, biochemically or functionally encodes a gene product associated with a CNS disease, such as PD. For example, individuals with a mutation in the GBA1 gene, which encodes the protein Gcase, have been observed to be at higher risk of developing PD than individuals without a mutation in GBA1. In another example, synuclein disease (eg, PD) is associated with the accumulation of protein aggregates containing the α-synuclein (α-Syn) protein; therefore, SCNA (which encodes α-Syn). It is a PD-related gene. In some embodiments, the expression cassette described herein encodes a wild-type or non-mutant form of a CNS disease-related gene, such as a PD-related gene (or coding sequence thereof). Examples of CNS disease-related genes (eg, PD-related genes, AD-related genes, FTD-related genes, etc.) are listed in Table 1.

Isolated Nucleic Acids and Vectors The isolated nucleic acids may be DNA or RNA. As used herein, the term "isolated" means artificially produced. As used herein, "isolated nucleic acid" was (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii). ) Purified by cleavage and gel separation, etc .; or (iv) refers to nucleic acids synthesized, for example, by chemical synthesis. The isolated nucleic acid can be easily manipulated by recombinant DNA techniques well known in the art.

The present disclosure presents in several aspects one or more CNS disease-related genes (eg, PD-related genes) such as Gcase, prosaposin, LIMP2 / SCARB2, GBA2, GALC protein, CTSB protein, IL-34 protein, TREM2. Provided is an isolated nucleic acid (eg, rAAV vector) comprising an expression construct encoding a protein, or TMEM106B protein. The disclosure also discloses isolated nucleic acids encoding one or more inhibitory nucleic acids that target one or more CNS disease-related genes, such as SNCA, TMEM106B, RPS25, and MAPT, in several aspects. (Eg, rAAV vector) is provided. In some embodiments, the isolated nucleic acid encoding the CNS disease-related gene may further comprise a coding sequence for an inhibitory nucleic acid that targets one or more CNS disease-related genes. In some embodiments, the CNS disease-related gene and the inhibitory nucleic acid targeting the CNS disease-related gene are encoded on different nucleic acids.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding Gcase (eg, the gene product of the GBA1 gene). Gcase, also known as β-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the β-glucoside bond of the chemical glucocerebrosid, an intermediate in glycolipid metabolism. In humans, Gcase is encoded by the GBA1 gene located on chromosome 1. In some embodiments, GBA1 encodes the peptide represented by NCBI reference sequence NP_000148.2 (SEQ ID NO: 14). In some embodiments, the isolated nucleic acid is codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells), such as the sequence set forth in SEQ ID NO: 15. Contains code sequences.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a prosaposin (eg, the gene product of the PSAP gene). Prosaposin is a precursor glycoprotein of sphingolipid-activating proteins (saposins) A, B, C, and D and promotes catabolism of glycosphingolipids with short oligosaccharide groups. In humans, the PSAP gene is located on chromosome 10. In some embodiments, PSAP encodes a peptide represented by NCBI reference sequence NP_002769.1 (eg, SEQ ID NO: 16). In some embodiments, the isolated nucleic acid is a codon-optimized (eg, codon-optimized for expression in mammalian cells such as human cells), such as the sequence set forth in SEQ ID NO: 17. Contains a saposin coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding LIMP2 / SCARB2 (eg, the gene product of the SCARB2 gene). SCARB2 refers to a membrane protein that regulates intracellular lysosomal and endosome transport. In humans, the SCARB2 gene is located on chromosome 4. In some embodiments, the SCARB2 gene encodes a peptide represented by NCBI reference sequence NP_005497.1 (SEQ ID NO: 18). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the isolated nucleic acid comprises a codon-optimized SCARB2 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a GBA2 protein (eg, the gene product of the GBA2 gene). GBA2 protein refers to non-lysosomal glucosylceramidase. In humans, the GBA2 gene is located on chromosome 9. In some embodiments, the GBA2 gene encodes a peptide represented by NCBI reference sequence NP_065995.1 (SEQ ID NO: 30). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the isolated nucleic acid comprises a codon-optimized GBA2 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a GALC protein (eg, the gene product of the GALC gene). GALC protein refers to galactosylceramidase (or galactosylceramidase), an enzyme that hydrolyzes the galactose ester bonds of galactosylceramide, galactosylsphingosine, lactosylceramide, and monogalactosyldiglycerides. In humans, the GALC gene is located on chromosome 14. In some embodiments, the GALC gene encodes a peptide represented by NCBI reference sequence NP_000144.2 (SEQ ID NO: 33). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the isolated nucleic acid comprises a codon-optimized GALC coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a CTSB protein (eg, the gene product of the CTSB gene). CTSB protein refers to cathepsin B, a lysosomal cysteine protease that plays an important role in intracellular proteolysis. In humans, the CTSB gene is located on chromosome 8. In some embodiments, the CTSB gene encodes a peptide represented by NCBI reference sequence NP_001899.1 (SEQ ID NO: 35). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the isolated nucleic acid comprises a codon-optimized CTSB coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding the SMPD1 protein (eg, the gene product of the SMPD1 gene). SMPD1 protein refers to sphingomyelin phosphodiesterase 1, a hydrolase involved in sphingolipid metabolism. In humans, the SMPD1 gene is located on chromosome 11. In some embodiments, the SMPD1 gene encodes a peptide represented by NCBI reference sequence NP_000534.3 (SEQ ID NO: 37). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the isolated nucleic acid comprises a codon-optimized SMPD1 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a GCH1 protein (eg, a gene product of the GCH1 gene). The GCH1 protein refers to GTP cyclohydrolase I, a hydrolase that is part of the biosynthetic pathway of folic acid and biopterin. In humans, the GCH1 gene is located on chromosome 14. In some embodiments, the GCH1 gene encodes a peptide represented by NCBI reference sequence NP_000152.1 (SEQ ID NO: 45). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the isolated nucleic acid comprises a codon-optimized GCH1 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a RAB7L protein (eg, the gene product of the RAB7L gene). The RAB7L protein refers to RAB7, which is a GTP-binding protein and is a member of the RAS carcinogenic gene family 1. In humans, the RAB7L gene is located on chromosome 1. In some embodiments, the RAB7L gene encodes a peptide represented by NCBI reference sequence NP_003920.1 (SEQ ID NO: 47). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the isolated nucleic acid comprises a codon-optimized RAB7L coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a VPS35 protein (eg, the gene product of the VPS35 gene). VPS35 protein refers to the vacuolar protein classification-related protein 35, which is part of a protein complex involved in the retrograde transport of proteins from endosomes to the trans-Golgi network. In humans, the VPS35 gene is located on chromosome 16. In some embodiments, the VPS35 gene encodes a peptide represented by NCBI reference sequence NP_060676.2 (SEQ ID NO: 49). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the isolated nucleic acid comprises a codon-optimized VPS35 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding an IL-34 protein (eg, a gene product of the IL34 gene). The IL-34 protein refers to interleukin 34, a cytokine that increases monocyte growth and survival. In humans, the IL34 gene is located on chromosome 16. In some embodiments, the IL34 gene encodes a peptide represented by NCBI reference sequence NP_689669.2 (SEQ ID NO: 55). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the isolated nucleic acid comprises a codon-optimized IL-34 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a TREM2 protein (eg, the gene product of the TREM2 gene). The TREM2 protein refers to a trigger receptor expressed on bone marrow cell 2, which is an immunoglobulin superfamily receptor found on bone marrow cells. In humans, the TREM2 gene is located on chromosome 6. In some embodiments, the TREM2 gene encodes a peptide represented by NCBI reference sequence NP_061838.1 (SEQ ID NO: 57). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the isolated nucleic acid comprises a codon-optimized TREM2 coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding the TMEM106B protein (eg, the gene product of the TMEM106B gene). The TMEM106B protein refers to the transmembrane protein 106B, a protein involved in dendrite morphogenesis and regulation of lysosomal transport. In humans, the TMEM106B gene is located on chromosome 7. In some embodiments, the TMEM106B gene encodes a peptide represented by NCBI reference sequence NP_060844.2 (SEQ ID NO: 63). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 64. In some embodiments, the isolated nucleic acid comprises a codon-optimized TMEM106B coding sequence.

Aspects of the present disclosure relate to isolated nucleic acids comprising an expression construct encoding a progranulin protein (eg, the gene product of the GRN gene). PGRN protein refers to progranulin, a protein involved in development, inflammation, cell proliferation, and protein homeostasis. In humans, the PGRN (also called GRN) gene is located on chromosome 17. In some embodiments, the GRN gene encodes a peptide represented by NCBI reference sequence NP_002078.1 (SEQ ID NO: 67). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 68. In some embodiments, the isolated nucleic acid comprises a codon-optimized PGRN coding sequence (GRN coding sequence).

In some aspects, the present disclosure provides isolated nucleic acids comprising an expression construct encoding a first gene product and a second gene product, where each gene product is independently Table 1. It is selected from the gene product shown in 1 or a part thereof, or an inhibitory nucleic acid targeting the gene or gene product shown in Table 1.

In some embodiments, the gene product is encoded by the coding portion of a naturally occurring gene (eg, cDNA). In some embodiments, the first gene product is a protein (or fragment thereof) encoded by the GBA1 gene. In some embodiments, the gene product is a protein (or fragment thereof) encoded by another gene listed in Table 1, eg, the SCARB2 / LIMP2 gene or the PSAP gene. However, one of ordinary skill in the art recognizes that the order of expression of the first gene product (eg, Gcase) and the second gene product (eg, LIMP2) can generally be reversed (eg, LIMP2 is the first). Gene product of, and Gcase is the second gene product). In some embodiments, the gene product is a fragment (eg, a portion) of a gene listed in Table 1. The protein fragment may contain about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the proteins encoded by the genes listed in Table 1. In some embodiments, the protein fragment comprises 50% to 99.9% (eg, any value between 50% and 99.9%) of the proteins encoded by the genes listed in Table 1.

Pathologically, disorders such as PD and Gaucher's disease are associated with the accumulation of protein aggregates predominantly containing the α-synuclein (α-Syn) protein. As a result, in some embodiments, the isolated nucleic acid described herein comprises an inhibitory nucleic acid that reduces or prevents the expression of the α-Syn protein.
In some aspects, the present disclosure discloses one or more interfering nucleic acids (eg, dsRNA) targeting the microtube-related protein tau, MAPT (eg, the gene product of the MAPT gene) associated with Alzheimer's disease and FTD-tau. , SiRNA, miRNA, amiRNA, etc.) to provide an isolated nucleic acid containing an expression construct.

In general, isolated nucleic acids as described herein are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more inhibitory nucleic acids (eg, dsRNA, siRNA). , ShRNA, miRNA, amiRNA, etc.) may be encoded. In some embodiments, the isolated nucleic acid encodes more than 10 inhibitory nucleic acids. In some embodiments, each of the one or more inhibitory nucleic acids targets a different gene or portion of the gene (eg, the first miRNA targets the first target sequence of the gene and the second. The miRNA targets a second target sequence of a gene that is different from the first target sequence). In some embodiments, each of the one or more inhibitory nucleic acids targets the same target sequence of the same gene (eg, the isolated nucleic acid encodes multiple copies of the same miRNA).

In some aspects, the present disclosure comprises an expression construct encoding one or more interfering nucleic acids (eg, dsRNA, siRNA, miRNA, miRNA, etc.) that target an α-synuclein protein (eg, the gene product of the SNCA gene). Provided with respect to isolated nucleic acids, including. The α-synuclein protein is a protein found in brain tissue and refers to a protein responsible for maintaining the supply of synaptic vesicles at presynaptic terminals and regulating the release of dopamine by clustering synaptic vesicles. In humans, the SNCA gene is located on chromosome 4. In some embodiments, the SNCA gene encodes the peptide represented by NCBI reference sequence NP_001139527.1. In some embodiments, the SNCA gene comprises the sequence set forth in SEQ ID NO: 90.

The inhibitory nucleic acid targeting SNCA may include a region of complementarity (eg, a region of the inhibitory nucleic acid that hybridizes to a target gene such as SNCA), which is 6 to 50 nucleotides in length. In some embodiments, the inhibitory nucleic acid comprises a region of complementarity with SNCA that is about 6-30, about 8-20, or about 10-19 nucleotides in length. In some embodiments, the inhibitory nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 of the SNCA sequence. , 21, 22, 23, 24, or 25 consecutive nucleotides.

In some aspects, the disclosure comprises an expression construct encoding one or more interfering nucleic acids (eg, dsRNA, siRNA, miRNA, amiRNA, etc.) that target the TMEM106B protein (eg, the gene product of the TMEM106B gene). An isolated nucleic acid is provided. The TMEM106B protein refers to the transmembrane protein 106B, a protein involved in dendrite morphogenesis and regulation of lysosomal transport. In humans, the TMEM106B gene is located on chromosome 7. In some embodiments, the TMEM106B gene encodes a peptide represented by NCBI reference sequence NP_060844.2. In some embodiments, the TMEM106B gene comprises the sequence set forth in SEQ ID NO: 91.

The inhibitory nucleic acid targeting TMEM106B may include a complementary region (eg, a region of the inhibitory nucleic acid that hybridizes to a target gene such as TMEM106B), which is 6 to 50 nucleotides in length. In some embodiments, the inhibitory nucleic acid comprises a region of complementarity with TMEM106B, which is about 6-30, about 8-20, or about 10-19 nucleotides in length. In some embodiments, the inhibitory nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 of the TMEM106B sequence. , 21, 22, 23, 24, or 25 consecutive nucleotides.

In some aspects, the present disclosure encodes an expression encoding one or more interfering nucleic acids (eg, dsRNA, siRNA, miRNA, miRNA, etc.) that target the ribosomal protein s25 (RPS25) (eg, the gene product of RPS25). Provided is an isolated nucleic acid containing a construct. RPS25 protein refers to a ribosomal protein, a subunit of the s40 ribosome, a protein complex involved in protein synthesis. In humans, the RPS25 gene is located on chromosome 11. In some embodiments, the RPS25 gene encodes a peptide represented by NCBI reference sequence NP_001019.1. In some embodiments, the RPS25 gene comprises the sequence set forth in SEQ ID NO: 113.

The inhibitory nucleic acid targeting RPS25 may include a complementary region (eg, a region of the inhibitory nucleic acid that hybridizes to a target gene such as RPS25), which is 6 to 50 nucleotides in length. In some embodiments, the inhibitory nucleic acid comprises a region of complementarity with RPS25 that is about 6-30, about 8-20, or about 10-19 nucleotides in length. In some embodiments, the inhibitory nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 of the RPS25 sequence. , 21, 22, 23, 24, or 25 consecutive nucleotides.

In some aspects, the disclosure encodes a microtube-related protein tau, one or more interfering nucleic acids targeting MAPT (eg, the gene product of the MAPT gene) (eg, dsRNA, siRNA, miRNA, amiRNA, etc.). Provided is an isolated nucleic acid containing an expression construct. MAPT protein refers to microtubule-related protein tau, which is a protein involved in microtubule stabilization. In humans, the MAPT gene is located on chromosome 17. In some embodiments, the MAPT gene encodes a peptide represented by the NCBI reference sequence NP_005901.2. In some embodiments, the MAPT gene comprises the sequence set forth in SEQ ID NO: 114.

The inhibitory nucleic acid targeting MAPT may include a region of complementarity (eg, a region of the inhibitory nucleic acid that hybridizes to a target gene such as MAPT), which is 6-50 nucleotides in length. In some embodiments, the inhibitory nucleic acid comprises a region of complementarity with MAPT that is about 6-30, about 8-20, or about 10-19 nucleotides in length. In some embodiments, the inhibitory nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 of the MAPT sequence. , 21, 22, 23, 24, or 25 consecutive nucleotides.

Aspects of the present disclosure relate to isolated nucleic acids encoding one or more gene products (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gene products). .. In some embodiments, one or more gene products are two or more proteins. In some embodiments, one or more gene products are two or more inhibitory nucleic acids. In some embodiments, the one or more gene products are one or more proteins and one or more inhibitory nucleic acids. In some aspects, the disclosure provides isolated nucleic acids comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product is independently tabulated. It is selected from the gene products shown in 1 or a portion thereof, or an inhibitory nucleic acid that targets the genes or gene products shown in Table 1. The sequence encoding the inhibitory nucleic acid may be located in the untranslated region of the expression vector (eg, intron, 5'UTR, 3'UTR, etc.).

In some embodiments, the gene product is encoded by the coding portion of a naturally occurring gene (eg, cDNA). In some embodiments, the first gene product is a protein (or fragment thereof) encoded by the GBA1 gene. In some embodiments, the gene product is an inhibitory nucleic acid that targets a PD-related gene (eg, SNCA) (eg, contains a region that hybridizes to or complements it). Those skilled in the art recognize that the order of expression of the first gene product (eg, Gcase) and the second gene product (eg, inhibitory RNA targeting SNCA) can generally be reversed (eg,). , Inhibitory RNA is the first gene product and Gcase is the second gene product). In some embodiments, the gene product is a fragment (eg, a portion) of a gene listed in Table 1. The protein fragment may contain about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the proteins encoded by the genes listed in Table 1. In some embodiments, the protein fragment comprises 50% to 99.9% (eg, any value between 50% and 99.9%) of the proteins encoded by the genes listed in Table 1. In some embodiments, the gene product (eg, inhibitory RNA) hybridizes to a portion of the target gene (eg, the target gene, eg, SNCA 5,6,7,8,9,10,11, Complementary to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more contiguous nucleotides). In some embodiments, the expression construct is monocistronic (eg, the expression construct encodes a single fusion protein comprising a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (eg, the expression construct encodes two distinct gene products, eg, two different proteins or protein fragments).

A polycistronic expression vector may contain one or more promoters (eg, 1, 2, 3, 4, 5, or more). Any suitable promoter, such as a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (eg, a CNS-specific promoter), or the like can be used. In some embodiments, the promoter is a chicken beta-actin promoter (CBA promoter), CAG promoter (eg, Alexopoulou et al. (2008) BMC Cell Biol. 9: 2; doi: 10.1186 / 1471-2121-9-2). ), CD68 promoter, or JeT promoter (eg, described in Tornoee et al. (2002) Gene 297 (1-2): 21-32). In some embodiments, the promoter is operably linked to a first gene product, a second gene product, or a nucleic acid sequence encoding a first gene product and a second gene product. In some embodiments, the expression cassette comprises one or more additional regulatory sequences, which include, without limitation, a transcription factor binding sequence, an intron splice site, a poly (A) addition site, an enhancer sequence, a repressor. Includes binding sites, or any combination thereof.

In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). Examples of IRES sites are described, for example, in Mokrejs et al. (2006) Nucleic Acids Res. 34 (Database issue): D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, those described in T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, and Liu et al. (2017) Sci Rep. 7: 2193. Is done. In some embodiments, the self-cleaving peptide is a T2A peptide.

In some embodiments, the inhibitory nucleic acid is placed in an intron of the expression construct, eg, an intron upstream of the sequence encoding the first gene product. The inhibitory nucleic acid can be double-stranded RNA (dsRNA), siRNA, microRNA (miRNA), artificial miRNA (amiRNA), or RNA aptamer. In general, the inhibitory nucleic acid binds (eg, hybridizes) to a contiguous nucleotide of about 6 to about 30 (eg, any integer between 6 and 30 including both ends) of the target RNA (eg, mRNA). In some embodiments, the inhibitory nucleic acid molecule is a miRNA or miRNA that targets a miRNA, such as SNCA (a gene encoding an α-Syn protein) or TMEM106B (eg, a gene encoding a TMEM106B protein). In some embodiments, the miRNA does not contain any mismatch with the region of the mRNA of the SNCA to which it hybridizes (eg, the miRNA is "complete"). In some embodiments, the inhibitory nucleic acid is a shRNA (eg, a shRNA targeting SNCA or TMEM106B). In some embodiments, the inhibitory nucleic acid is an artificial miRNA (amiRNA) comprising a miR-155 scaffold and an SCNA or TMEM106B targeting sequence.

In some embodiments, the inhibitory nucleic acid is artificial microRNA (amiRNA). MicroRNAs (miRNAs) typically refer to small, non-coding RNAs found in plants and animals, and function in transcriptional and post-translational regulation of gene expression. MiRNAs are transcribed by RNA polymerases to form hairpin-loop structures called pri-miRNAs, which are subsequently processed by enzymes (eg, Drosha, Pasha, sprisosomes, etc.) to form pre-miRNA hairpin structures. Formed, then it is processed by Dicer to form double-stranded miRNA / miRNA ^* (where ^* refers to the passenger strand of the double-stranded miRNA), and then one of the strands is RNA-induced. It is incorporated within the silencing complex (RISC). In some embodiments, the inhibitory RNA as described herein is a miRNA that targets SNCA or TMEM106B.

In some embodiments, the inhibitory nucleic acid targeting SNCA comprises double chain miRNA / miRNA ^* . In some embodiments, the miRNA strand of the double-stranded miRNA / miRNA ^* comprises or consists of the sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, the miRNA ^* strand of the double-stranded miRNA / miRNA ^* comprises or consists of the sequence set forth in any one of SEQ ID NOs: 20-25.
In some embodiments, the inhibitory nucleic acid targeting TMEM106B comprises double chain miRNA / miRNA ^* . In some embodiments, the miRNA strand of a double-stranded miRNA / miRNA ^* comprises or consists of the sequence set forth in SEQ ID NO: 92 or 93. In some embodiments, the miRNA ^* strand of a double-stranded miRNA / miRNA ^* comprises or consists of the sequence set forth in SEQ ID NO: 92 or 93.

Artificial microRNAs (amiRNAs) are obtained by modification that replaces the natural targeting region of pre-mRNA with the targeting region of interest. For example, naturally occurring, expressed miRNAs can be used as scaffolds or backbones (eg, tri-miRNA scaffolds) by replacing stem sequences with those of miRNAs that target the gene of interest. Artificial precursor microRNAs (pre-amiRNAs) are usually processed to produce one single stable small RNA. In some embodiments, the scAAV vectors and scAAVs described herein include nucleic acids encoding amiRNA. In some embodiments, the pri-miRNA scaffolds of amiRNA are pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, It is derived from a pri-miRNA selected from the group consisting of pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451. In some embodiments, the amiRNA is a nucleic acid sequence targeting SNCA or TMEM106B and an eSIBR amiRNA scaffold (eg, as described in Fowler et al. Nucleic Acids Res. 2016 Mar 18; 44 (5): e48. )including.

In some embodiments, the amiRNA targeting SNCA comprises or consists of the sequence set forth in any one of SEQ ID NOs: 94-99. In some embodiments, the amiRNA targeting TMEM106B comprises or consists of the sequences set forth in SEQ ID NOs: 65-66. In some embodiments, the amiRNA targeting RPS25 comprises or consists of the sequences set forth in SEQ ID NOs: 115-122. In some embodiments, the amiRNA targeting MAPT comprises or consists of the sequences set forth in SEQ ID NOs: 123-138.

In some embodiments, the isolated nucleic acid or vector described by the present disclosure (eg, rAAV vector) is SEQ ID NO: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38. Includes or consists of the sequences set forth in any one of ~ 44, 46, 48, 50 ~ 54, 56, 58 ~ 62, 64 ~ 66, and 68 ~ 145. In some embodiments, the isolated nucleic acid or vector described by the present disclosure (eg, rAAV vector) is SEQ ID NO: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38. Sequences that are complementary (eg, complements thereof) to the sequences shown in any one of ~ 44, 46, 48, 50 to 54, 56, 58 to 62, 64-66, and 68 to 145. Includes or consists of it. In some embodiments, the isolated nucleic acid or vector described by the present disclosure (eg, rAAV vector) is SEQ ID NO: 1-13, 15, 17, 19-29, 31, 32, 34, 36, It comprises or consists of a sequence that is an inverse complement of the sequence shown in any one of 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-145.

In some embodiments, the isolated nucleic acid or vector described by the present disclosure (eg, rAAV vector) is SEQ ID NO: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38. Contains or consists of a portion of the sequence set forth in any one of ~ 44, 46, 48, 50 ~ 54, 56, 58 ~ 62, 64 ~ 66, and 68 ~ 145. Some are SEQ ID NOs: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68. It may contain at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sequence shown in any one of 145. In some embodiments, the nucleic acid sequences described by the present disclosure are nucleic acid sense strands (eg, 5'→ 3'strands), or plus (+) strands in the context of viral sequences. In some embodiments, the nucleic acid sequences described by the present disclosure are nucleic acid antisense strands (eg, 3'→ 5'strands), or negative (-) strands in the context of viral sequences.

Those skilled in the art refer to nucleic acid sequences that include or encode inhibitory nucleic acids (eg, dsRNA, siRNA, miRNA, amiRNA, etc.), any one or more of the thymidines (T) in the sequences provided herein. ) Nucleotides or thymidine (U) nucleotides may be replaced with any other nucleotide suitable for base pairing with adenosine nucleotides (eg, via Watson-Crick base pair). For example, T may be replaced by U, and U may be replaced by T.

The isolated nucleic acids described herein can be present on their own or as part of a vector. In general, the vector can be a plasmid, cosmid, phagemid, bacterial artificial chromosome (BAC), or viral vector (eg, adenoviral vector, adeno-associated virus (AAV) vector, retroviral vector, baculovirus vector, etc.). In some embodiments, the vector is a plasmid (eg, a plasmid containing the isolated nucleic acid described herein). In some embodiments, the rAAV vector is single-stranded (eg, single-stranded DNA). In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the vector is a baculovirus vector (eg, Autographa californica nuclear polyhedrosis (AcNPV) vector).

Typically, an rAAV vector (eg, rAAV genome) is an expression construct containing two or more transgenes (eg, one or more of each of the following: promoters, introns) flanked by two AAV reverse terminal repeat (ITR) sequences. , Enhancer sequence, protein coding sequence, inhibitory RNA coding sequence, poly A tail sequence, etc.). In some embodiments, the transgene of the rAAV vector comprises the isolated nucleic acid described in the present disclosure. In some embodiments, each of the two ITR sequences of the rAAV vector is a full-length ITR (eg, about 145 bp in length, including a functional Rep binding site (RBS) and a terminal degradation site (trs)). be. In some embodiments, one of the ITRs of the rAAV vector is cleaved (eg, shortened or not full length). In some embodiments, truncated ITRs lack functional terminal degradation sites (trs) and are used for the production of self-complementary AAV vectors (scAAV vectors). In some embodiments, the truncated ITR is, for example, the ΔITR described in McCarty et al. (2003) Gene Ther. 10 (26): 2112-8.

Aspects of the present disclosure have one or more modifications (eg, nucleic acid additions, deletions, substitutions, etc.) compared to wild-type AAV ITRs, eg, wild-type AAV2 ITRs (eg, SEQ ID NO: 29). With respect to isolated nucleic acids including ITRs (eg, rAAV vectors). The structure of the wild-type AAV2 ITR is shown in FIG. Wild-type ITRs are generally self-annealed, palindromic, double-stranded T-shaped hairpin structures (which are formed by two cross-arms (formed by sequences called B / B'and C / C', respectively), and more. It contains a region of 125 nucleotides that forms a long stem region (formed by sequence A / A') and a single-stranded terminal region called the "D" region (FIG. 20). Generally, the "D" region of the ITR is located between the stem region formed by the A / A'sequence and the insert containing the transgene of the rAAV vector (eg, "inside" the ITR relative to the end of the ITR. , Or located proximal to the transgene insert or expression construct of the rAAV vector). In some embodiments, the "D" region comprises the sequence set forth in SEQ ID NO: 27. The "D" region has been observed to play an important role in capsid formation of rAAV vectors by capsid proteins, as disclosed, for example, in Ling et al. (2015) J Mol Genet Med 9 (3). ..

The present disclosure partially unmodified the rAAV vector containing the "D" region located "outside" the ITR (eg, near the end of the ITR with respect to the transgene insert or expression construct). It is based on being more efficiently capsidized by the AAV capsid protein than the rAAV vector with (wild-type) ITR. In some embodiments, the rAAV vector with the modified "D" sequence (eg, the "D" sequence in the "outer" position) is less toxic than the rAAV vector with the wild-type ITR sequence. There is.

In some embodiments, the modified "D" sequence comprises at least one nucleotide substitution as compared to the wild-type "D" sequence (eg, SEQ ID NO: 27). The modified "D" sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more compared to the wild-type "D" sequence (eg, SEQ ID NO: 27). May have nucleotide substitutions. In some embodiments, the modified "D" sequence is at least 10,11,12,13,14,15,16,17,18, as compared to the wild-type "D" sequence (eg, SEQ ID NO: 27). Or contains 19 nucleic acid substitutions. In some embodiments, the modified "D" sequence is about 10% to about 99% (eg, 10%, 15%, 20%, 25%) with the wild-type "D" sequence (eg, SEQ ID NO: 27). 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) are the same. In some embodiments, the modified "D" sequence is the sequence set forth in SEQ ID NO: 26, also referred to as the "S" sequence, as described in Wang et al. (1995) J Mol Biol 250 (5): 573-80. including.

The isolated nucleic acid or rAAV vector described in the present disclosure is further described, for example, as shown in SEQ ID NO: 28, or Francois, et al. 2005. The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal. Adeno-Associated Virus Type 2 p5 Element. May contain "TRY" sequences as described in J Virol. In some embodiments, the TRY sequence is placed between the ITR of the isolated nucleic acid or rAAV vector (eg, 5'ITR) and the expression construct (eg, an insert encoding a transgene).

Aspects of the present disclosure relate to constructs configured to express one or more transgenes in subject myeloid cells (eg, CNS myeloid cells such as microglia). Thus, in some embodiments, the construct (eg, a gene expression vector) comprises a protein coding sequence operably linked to a myeloid cell-specific promoter. Examples of myeloid cell-specific promoters are the CD68 promoter, lysM promoter, csf1r promoter, CD11c promoter, c-fes promoter, and F4 / 80 promoter, such as Lin et al. Adv Exp Med Biol. 2010; 706: 149- Includes those described in 56. In some embodiments, the myelogenous cell-specific promoter is the CD68 promoter or the F4 / 80 promoter.

In some aspects, the present disclosure relates to a baculovirus vector comprising the isolated nucleic acid or rAAV vector described in the present disclosure. In some embodiments, the baculovirus vector is described, for example, in Urabe et al. (2002) Hum Gene Ther 13 (16): 1935-43 and Smith et al. (2009) Mol Ther 17 (11): 1888-1896. The Autographa californica nuclear polyhedrosis (AcNPV) vector, as described above.

In some aspects, the disclosure provides host cells comprising the isolated nucleic acids or vectors described herein. The host cell can be a prokaryotic cell or a eukaryotic cell. For example, the host cell can be a mammalian cell, a bacterial cell, a yeast cell, an insect cell, or the like. In some embodiments, the host cell is a mammalian cell, eg, a HEK293T cell. In some embodiments, the host cell is a bacterial cell, such as an E. coli cell.

rAAV
In some aspects, the disclosure relates to recombinant AAV (rAAV) (eg, 1, 2, 3, 4) comprising a transgene encoding one or more isolated nucleic acids described herein. 5, 6, 7, 8, 9, 10, or more gene products described herein, and / or rAAV vectors encoding inhibitory nucleic acids targeting the gene products described herein). .. The term "rAAV" generally refers to viral particles comprising an rAAV vector capsidized with one or more AAV capsid proteins. The rAAV described in the present disclosure may comprise a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10, or variants thereof. In some embodiments, the capsid protein is an AAV9 capsid protein or a variant thereof. In some embodiments, the AAV9 capsid protein variant comprises mutations at one or more positions corresponding to T492, Y705, and Y731 of SEQ ID NO: 147 (eg, corresponding to those positions in AAV6). In some embodiments, one or more mutations are selected from T492V, Y705F, Y731F, or a combination thereof. In some embodiments, the AAV9 capsid protein variant comprises the amino acid sequence set forth in SEQ ID NO: 149.

In some embodiments, rAAV is a capsid protein from a non-human host, eg, AAVrh. 10, AAVrh. Contains rhesus monkey AAV capsid proteins such as 39. In some embodiments, the rAAV described herein comprises a capsid protein that is a variant of the wild-type capsid protein, which is, for example, at least 1, 2, 3 with respect to the wild-type AAV capsid protein from which it is derived. A capsid protein variant comprising 4, 5, 6, 7, 8, 9, 10, or more than 10 (eg, 15, 20, 25, 50, 100, etc.) amino acid substitutions (eg, mutations). In some embodiments, the AAV capsid protein variant is an AAV1RX capsid protein, as described, for example, in Albright et al. Mol Ther. 2018 Feb 7; 26 (2): 510-523. In some embodiments, the capsid protein is AAV1RX and comprises the amino acid sequence set forth in SEQ ID NO: 146 (or encoded by the nucleic acid sequence set forth in SEQ ID NO: 145). In some embodiments, the capsid protein variant is an AAV TM6 capsid protein, as described, for example, in Rosario et al. Mol Ther Methods Clin Dev. 2016; 3: 16026. In some embodiments, the AAV6 capsid protein variant is an AAV-TM6 capsid protein and comprises the amino acid sequence set forth in SEQ ID NO: 148.

In some embodiments, the rAAV described herein spreads readily through the CNS, especially when introduced into the CSF space or directly into the brain parenchyma. Thus, in some embodiments, the rAAV described herein comprises a capsid protein that is capable of crossing the blood-brain barrier (BBB). For example, in some embodiments, rAAV may be AAV9 or AAVrh. Includes a capsid protein with 10 serotypes. The generation of rAAV is described, for example, in Samulski et al. (1989) J Virol. 63 (9): 3822-8 and Wright (2009) Hum Gene Ther. 20 (7): 698-706. In some embodiments, rAAV comprises a capsid protein that specifically or preferentially targets myeloid cells, such as microglial cells. In some embodiments, rAAV transduces microglial cells.

In some embodiments, the rAAV described herein, including, for example, a recombinant rAAV genome that is capsidized by an AAV capsid protein to form rAAV capsid particles, is produced by a baculovirus vector expression system (BEVS). Will be done. The generation of rAAV using BEVS is described, for example: Urabe et al. (2002) Hum Gene Ther 13 (16): 1935-43, Smith et al. (2009) Mol Ther 17 (11) :. 1888-1896, US Pat. No. 8,945,918, US Pat. No. 9,879,282, and International PCT Publication WO 2017/184879. However, rAAV can be produced using any suitable method (eg, using recombinant rep and cap genes). In some embodiments, the rAAV disclosed herein is produced in HEK293 (human fetal kidney) cells.

Pharmaceutical Compositions In some aspects, the present disclosure provides pharmaceutical compositions comprising the isolated nucleic acids or rAAVs described herein and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not negate the biological activity or properties of the compound and is relatively non-toxic, such as the material. Can be administered to an individual without causing unwanted biological effects or interacting with any component of the composition in which it is contained in a detrimental manner.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspension. Turbids, diluents, excipients, thickeners, solvents or encapsulants, etc., which are useful compounds in the present invention so that they can perform their intended function in or to the patient. Means anything that is carried or related to being carried. Further ingredients that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA). It is described; it is incorporated herein by reference.

The compositions provided herein (eg, pharmaceutical compositions) can be administered by any route including: transintestinal (eg, oral), parenteral, intravenous, intramuscular, intraarterial. , Intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (by powder, ointment, cream, and / or drops), mucosa, nasal cavity, buccal , Sublingual; intratracheal infusion, bronchial infusion, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specific planned routes are oral administration, intravenous administration (eg, systemic intravenous injection), topical administration by blood and / or lymphatic supply, and / or direct administration to the affected area. In general, the most appropriate route of administration is various factors, such as the nature of the drug (eg, its stability in the gastrointestinal environment) and / or the condition of the subject (eg, whether the subject can tolerate oral administration). ) Etc. will depend on it. In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the subject's eye.

In some embodiments, the composition comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) different rAAVs, where each rAAV is a different gene product. Includes isolated nucleic acids encoding (eg, different proteins or inhibitory nucleic acids). Different rAAVs may contain capsid proteins of the same or different serotypes.

Methods Aspects of the present disclosure relate to compositions for the expression of one or more CNS disease-related gene products in a subject for treating a CNS-related disease. One or more CNS disease-related gene products may be encoded by one or more isolated nucleic acids or rAAV vectors. In some embodiments, the subject receives a single vector (eg, isolated nucleic acid, rAAV, etc.) encoding one or more (1, 2, 3, 4, 5, or more) gene products. Be administered. In some embodiments, the subject is administered with multiple (eg, 2, 3, 4, 5, or more) vectors (eg, isolated nucleic acid, rAAV, etc.), where each vector is different. Encodes a CNS disease-related gene product.
CNS-related diseases can be neurodegenerative diseases, synuclein diseases, tauopathy, or lysosomal storage disorders. Examples of neurodegenerative diseases and their related genes are listed in Table 2.

"Synuclein disease" is a disease characterized by accumulation, overexpression or activity of alpha-synuclein (a gene product of SNCA) in a subject (eg, compared to a healthy subject, eg, a subject without synuclein disease). Or refers to a failure. Examples of synuclein diseases and their related genes are listed in Table 3.
"Tauopathy" refers to a disease or disorder characterized by the accumulation, overexpression or activity of tau protein in a subject (eg, a healthy subject without tauopathy). Examples of tauopathy and their related genes are listed in Table 4.
"Lysosome storage disease" refers to a disease characterized by an abnormal assembling of toxic cell products in a subject's lysosome. Examples of lysosomal storage disorders and their related genes are listed in Table 5.

In some embodiments, the present disclosure relates to Parkinson's by administering an isolated nucleic acid encoding GBA1 (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. Diseases (eg, Parkinson's disease with GBA1 mutation (PD-GBA), sporadic Parkinson's disease (sPD)), Gauche disease (eg, neuropathic Gauche disease (nGD), type I Gauche disease (T1GD), type II) Selected from Gauche disease (T2GD) and type III Gauche disease (T3GD)), Levy body dementia (DLB), muscular atrophic lateral sclerosis (ALS), and Niemann-Pick disease type C (NPC). Regarding how to treat the disease.
In some embodiments, the present disclosure relates an isolated nucleic acid encoding a PGRN (also referred to as GRN) (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. By administration, frontotemporal dementia (eg, frontotemporal dementia with GRN mutation (FTD-GRN), frontotemporal dementia with MAPT mutation (FTD-tau), and C9ORF72 mutation) Frontotemporal dementia (FTD-C9orf72)), Parkinson's disease (PD), Alzheimer's disease (AD), neuroseroid lipofustinosis (NCL), cerebral cortical basal nucleus degeneration (CBD), motor neuron disease (MND) ), Or a method of treating Gaucher's disease (GD).

In some embodiments, the present disclosure comprises an isolated nucleic acid (eg, an rAAV vector or rAAV containing the isolated nucleic acid) encoding an inhibitory nucleic acid that targets the GBA1 gene product and SNCA. Synuclein disease (eg, multiple system atrophy (MSA), Parkinson's disease (PD), Parkinson's disease mutation with GBA1 mutation (PD-GBA), Lewy body dementias (DLB)) by administration to the subject in need , Lewy body dementias with GBA1 mutation, and Lewy body disease).
In some embodiments, the present disclosure relates to Parkinson's disease by administering an isolated nucleic acid encoding PSAP (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. (PD), treating a disease selected from frontotemporal dementia (eg, frontotemporal dementia with GRN mutation (FTD-GRN)), lithosome accumulation (LSD), or Gaucher's disease (GD). Regarding how to do it.

In some embodiments, the present disclosure comprises administering an isolated nucleic acid encoding TREM2 (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof for Alzheimer's disease. (AD), Nasu-Hakora disease (NHD), frontotemporal dementia with MAPT mutation (FTD-Tau), or Parkinson's disease (PD).
In some embodiments, the present disclosure relates an isolated nucleic acid encoding an inhibitory nucleic acid targeting MAPT (eg, an rAAV vector or rAAV containing the isolated nucleic acid) to a subject in need thereof. By administration, Alzheimer's disease (AD) or frontotemporal dementia (frontotemporal dementia with MAPT mutation (FTD-Tau), tauopathy, progressive nuclear palsy (PSP), neurodegenerative disease, Lewy It relates to a method for treating body disease (LBD) or Parkinson's disease).

As used herein, "treating" or "treating" means (a) preventing or delaying the onset of CNS disease; (b) reducing the severity of CNS disease; ( c) To reduce or prevent the onset of symptoms characteristic of CNS disease; (d) and / or to prevent the exacerbation of symptoms characteristic of CNS disease. Symptoms of CNS disease include, for example, motor dysfunction (eg, tremors, stiffness, clumsiness, difficulty walking, paralysis), cognitive dysfunction (eg, dementia, depression, anxiety, psychiatric disorders), memory impairment. , Emotional and behavioral dysfunction will be included.
The present disclosure relates, in part, to compositions for the expression of a combination of CNS disease-related genes (eg, PD-related gene products) in a subject that act together (eg, synergistically) to treat the disease. Based on.
Accordingly, in some aspects, the disclosure provides a method of treating a subject who has or is suspected of having a CNS-related disease (eg, Parkinson's disease, AD, FTD, etc.), wherein the method provides the subject. The present disclosure comprises administering the composition described in this disclosure (eg, a composition comprising an isolated nucleic acid or vector or rAAV).

In some embodiments, the subject has one or more signs or symptoms or has a genetic predisposition to the neurodegenerative diseases listed in Table 2 (eg, mutations in the genes listed in Table 1). In some embodiments, the subject has one or more signs or symptoms or has a genetic predisposition to synuclein disease listed in Table 3 (eg, mutations in the genes listed in Table 1). In some embodiments, the subject has one or more signs or symptoms or has a genetic predisposition to the tauopathy listed in Table 4 (eg, mutations in the genes listed in Table 1). In some embodiments, the subject has one or more signs or symptoms or has a genetic predisposition to lysosomal storage disease listed in Table 5 (eg, mutations in the genes listed in Table 1).
The present disclosure is based, in part, on a composition for the expression of one or more CNS disease-related gene products in a subject for treating Gaucher's disease. In some embodiments, Gaucher's disease is a neurological Gaucher's disease, such as type 2 Gaucher's disease or type 3 Gaucher's disease. In some embodiments, the subject has no PD or PD symptoms.

Accordingly, in some aspects, the present disclosure provides a method for treating a subject having or suspected of having neurological Gaucher's disease, the method of which is described by the present disclosure (eg, the composition). , A composition comprising an isolated nucleic acid or vector or rAAV), comprising administering to the subject.
The present disclosure is based, in part, on a composition for the expression of one or more CNS disease-related gene products in a subject for the treatment of Alzheimer's disease or frontotemporal dementia (FTD). In some embodiments, the subject does not have Alzheimer's disease.
Accordingly, in some aspects, the disclosure provides a method for treating a subject having or suspected of having FTD, wherein the method is the composition described by the present disclosure (eg, isolated). A composition comprising a nucleic acid or vector or rAAV) that comprises administering to a subject. In some embodiments, subjects with Alzheimer's disease or frontotemporal dementia (FTD) are administered progranulin (PGRN (also referred to as GRN)) or rAAV encoding a portion thereof.

In some aspects, the disclosure provides a method for delivering a transgene to microglial cells, the method comprising administering to the subject rAAV as described herein.
In some embodiments, rAAV, which encodes a Gcase protein for treating Type 2 or 3 Gaucher's disease or Parkinson's disease with a GBA1 mutation, is administered to the subject as a single dose, and rAAV is subsequently administered to the subject. Not administered.
In some embodiments, the rAAV encoding the Gcase protein is administered via a single intracisterna magna intraoccipital injection. In some embodiments, the injection into the cisterna magna is performed fluoroscopically.

The subject is typically a mammal, preferably a human. In some embodiments, the subject is 1 month to 10 years old (eg, 1 month old, 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old). , 12 months old, 13 months old, 14 months old, 15 months old, 16 months old, 17 months old, 18 months old, 19 months old, 20 months old, 21 months old, 22 months old, 23 months old, 24 months old, 3 years old, 4 years old, 5 years old, 6 years old Years, 7 years, 8 years, 9 years, 10 years, or any age between them). In some embodiments, the subject is 2 to 20 years old. In some embodiments, the subject is 30 to 100 years old. In some embodiments, the subject is older than 55 years.

In some embodiments, the composition is administered directly to the subject's CNS, eg, by direct injection into the subject's brain and / or spinal cord. Examples of CNS direct dosing methods include, but are not limited to, intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination described above. In some embodiments, the composition is administered to the subject by intramagna (ICM) injection. In some embodiments, direct injection into the subject's CNS results in introduction gene expression (eg, first gene product, second gene product, and the like) in the subject's midbrain, striatum and / or cerebral cortex. If so, the expression of the third gene product) is brought about. In some embodiments, direct injection into the CNS results in transgene expression (eg, a first gene product, a second gene product, and a third gene product, if applicable) in the spinal cord and / or CSF of the subject. (Expression of).

In some embodiments, direct injection into the subject's CNS comprises convection enhanced delivery (CED). Convection-enhanced delivery involves placing a surgical exposure of the brain and a small-diameter catheter directly into the target area of the brain, followed by injecting a therapeutic agent (eg, the composition described herein or rAAV) directly into the subject's brain. It is a treatment strategy including. CEDs are described, for example, in Debinski et al. (2009) Expert Rev Neurother. 9 (10): 1519-27.

In some embodiments, the composition is administered to the periphery of the subject, for example by peripheral injection. Examples of peripheral injections include subcutaneous injections, intravenous injections, intraarterial injections, intraperitoneal injections, or any combination thereof. In some embodiments, the peripheral injection is an intraarterial injection, eg, an injection into the carotid artery of interest.

In some embodiments, the compositions described in the present disclosure (eg, a composition comprising an isolated nucleic acid or vector or rAAV) are administered to the CNS of interest both peripherally and directly. For example, in some embodiments, the subject is administered the composition by intraarterial injection (eg, injection into the carotid artery) and intraparenchymal injection (eg, intraparenchymal injection by CED). In some embodiments, direct and peripheral injections into the CNS are simultaneous (eg, simultaneous). In some embodiments, the direct injection is given prior to the peripheral injection (eg, between 1 minute and 1 week, or earlier). In some embodiments, the direct injection is given after the peripheral injection (eg, for 1 minute to 1 week, or thereafter).
In some embodiments, the subject is administered an immunosuppressive agent prior to (eg, between 1 month and 1 minute prior) or simultaneously with the composition as described herein. In some embodiments, the immunosuppressive drug is a corticosteroid (eg, prednisone, budesonide, etc.), an mTOR inhibitor (eg, cilolimus, everolimus, etc.), an antibody (eg, adalimumab, etanercept, natalizumab, etc.), or methotrexate. ..

The amount of the composition described in the present disclosure to be administered to a subject (eg, a composition comprising an isolated nucleic acid or vector or rAAV) will vary depending on the method of administration. For example, in some embodiments, the rAAV described herein is, for example, between about 109 genomic copies (GC) / kg and about ^{10 14} GC / kg (eg, about ¹⁰⁹ GC / kg, about). It is administered at a titer of 10 ¹⁰ GC / kg, about 10 ¹¹ GC / kg, about 10 ¹² GC / kg, about 10 ¹² GC / kg, or about 10 ¹⁴ GC / kg). In some embodiments, the subject is administered with a high titer (eg,> 10 ¹² genomic copy GC / kg rAAV) by injection into the CSF space or by intraparenchymal injection. In some embodiments, rAAV as described herein is administered to a subject by intravenous injection at a dose ranging from about 1 × 10 ¹⁰ vector genome (vg) to about 1 × 10 ¹⁷ vg. To. In some embodiments, rAAV as described herein is administered to a subject by injection into a cisterna magna at a dose ranging from about 1 × 10 ¹⁰ vg to about 1 × 10 ¹⁶ vg. ..

The compositions described in the present disclosure (eg, a composition comprising an isolated nucleic acid or vector or rAAV) may be applied once or multiple times (eg, 2, 3, 4, 5, 6, 7, 8) to a subject. 9, 10, 20, or more) can be administered. In some embodiments, the composition is administered to the subject continuously (eg, chronically), eg, via an infusion pump.

Example 1: rAAV vector AAV vectors are generated using cells such as HEK293 cells for triple plasmid transfection. The ITR sequence is flanked by an expression construct containing a promoter / enhancer element for each transgene of interest, a 3'poly A signal, and a post-translational signal such as the WPRE element. Multiple gene products such as GBA1, LIMP2, prosaposin can be simultaneously expressed by: fusion of protein sequences; or use of 2A peptide linkers such as T2A and P2A (because it interferes with the formation of peptide bonds). , 2 peptide fragments with amino acids added); or by the use of IRES elements; or by expression in two separate expression cassettes. The presence of short intron sequences that are efficiently spliced upstream of the expressed gene can improve expression levels. shRNA and other regulatory RNAs may be included within these sequences. Examples of the expression constructs described in this disclosure are shown in FIGS. 1-8, 21-35, 39 and 41-45 and Table 6 below.

Example 2: Cell-based assay for viral transduction into GBA-deficient cells GBA1-deficient cells can be, for example, fibroblasts, monospheres, or hES cells from GD patients, or induced pluripotent stem cells derived from the patient. Acquired as iPSC). These cells accumulate substrates such as glucosylceramide and glucosylsphingosine (GluCer and GluSph). Treatment of wild-type or mutant cultured cell lines with Gcase inhibitors, such as CBE, is also used to obtain GBA-deficient cells.

Using such a cell model, lysosomal defects are quantified with an antibody against this protein or phospho-αSyn in terms of the accumulation of protein aggregates such as α-synuclein, followed by fluorescence microscopy. And image it. Imaging of lysosomal abnormalities is also performed by ICC of protein markers such as LAMP1, LAMP2, LIMP1, LIMP2, or by uptake with dyes such as Lysotracker, or by uptake of fluorescent dextran or other markers via the endocytosis compartment. .. Imaging of the accumulation of autophagy markers due to defects in fusion with lysosomes such as LC3 is also feasible. Western blotting and / or ELISA is used to quantify the abnormal accumulation of these markers. Also, the accumulation of glycolipid substrates and GBA1 products is measured using standard approaches.
Treatment endpoints (such as alleviation of PD-related pathologies) are measured in the context of expression of transduction of AAV vectors to confirm and quantify activity and function. Gcase can be quantified using a protein ELISA measure or also by a standard Gcase activity assay.

Example 3: In vivo Assay Using Mutant Mice This example describes an in vivo assay for AAV vectors using mutant mice. In vivo studies of AAV vectors as described above in mutant mice include, for example, Liou et al. (2006) J. Biol. Chem. 281 (7): 4242-4253, Sun et al. (2005) J. Lipid Res. 46: 2102-2113, and Farfel-Becker et al. (2011) Dis. Model Mech. 4 (6): 746-752.
Intrathecal or intraventricular delivery of vehicle controls and AAV vectors (eg, 2 × 10 ¹¹ vg / mouse dose) is performed using concentrated AAV stock, eg, at an infusion volume of 5-10 μL. Intrasubstantial delivery by convection enhanced delivery.
Treatment begins before or after the onset of symptoms. The endpoints to be measured are substrate accumulation in CNS and CSF, Gcase enzyme accumulation by ELISA, enzyme activity accumulation, motor and cognitive endpoints, lysosomal dysfunction, and α-synuclein monomer, protofibril or fibril accumulation. be.

Example 4: Chemical Model of Disease This example describes an in vivo assay for AAV vectors using a chemically induced mouse model of Gaucher's disease (such as the CBE mouse model). In vivo studies of these AAV vectors are performed in a chemically induced mouse model of Gaucher's disease, as described, for example, in Vardi et al. (2016) J Pathol. 239 (4): 496-509.
Intrathecal or intraventricular delivery of vehicle controls and AAV vectors (eg, 2 × 10 ¹¹ vg / mouse dose) is performed using concentrated AAV stock, eg, at an infusion volume of 5-10 μL. Intrasubstantial delivery by convection enhanced delivery. Peripheral delivery is achieved by tail vein injection.
Treatment begins before or after the onset of symptoms. The endpoints to be measured are substrate accumulation in CNS and CSF, Gcase enzyme accumulation by ELISA, enzyme activity accumulation, motor and cognitive endpoints, lysosomal dysfunction, and α-synuclein monomer, protofibril or fibril accumulation. be.

Example 5: Clinical study of patients with PD, LBD, Gaucher's disease In some embodiments, patients with a particular form of Gaucher's disease (eg, GD1) are Parkinson's disease (PD) or Lewy body dementias (LBD). There is a high risk of developing. This example describes a clinical trial to assess the safety and efficacy of rAAV described herein in patients with Gaucher's disease, PD and / or LBD.
Clinical trials of such vectors for the treatment of Gaucher's disease, PD and / or LBD are similar to those described in Grabowski et al. (1995) Ann. Intern. Med. 122 (1): 33-39. Perform using study design.

Example 6: Treatment of Peripheral Diseases In some embodiments, a patient with a particular form of Gaucher's disease is a peripheral nerve, as described, for example, in Biegstraaten et al. (2010) Brain 133 (10): 2909-2919. Shows symptoms of disability.
This example describes an in vivo assay of the AAV vector described herein for the treatment of peripheral neuropathy associated with Gaucher's disease (eg, type 1 Gaucher's disease). Briefly, patients with type 1 Gaucher disease identified as having signs or symptoms of peripheral neuropathy receive rAAV as described herein. In some embodiments, signs and symptoms of a subject's peripheral neuropathy are monitored after administration of rAAV, eg, using the methods described in Biegstraaten et al.
Levels of transduced gene products described in the present disclosure present in a patient (eg, patient's serum, peripheral tissue (eg, liver tissue, spleen tissue, etc.)), eg, Western blot analysis, enzyme function assay, or imaging. Assay by test.

Example 7: CNS-type treatment This example describes an in vivo assay for rAAV described herein for the treatment of CNS-type Gaucher's disease. Briefly, patients with Gaucher's disease identified as having CNS-type Gaucher's disease (eg, type 2 or type 3 Gaucher's disease) are administered rAAV as described herein. Levels of the transfected gene product described in the present disclosure present in the patient's CNS (eg, in the patient's CNS serum, in the patient's cerebrospinal fluid (CSF), or in the patient's CNS tissue), eg, Western. Assay by blot analysis, enzyme function assay, or imaging test.

Example 8: Gene therapy for Parkinson's disease in subjects with a mutation in GBA1 This example describes administration of a recombinant adeno-associated virus (rAAV) encoding GBA1 to a patient with Parkinson's disease characterized by a mutation in the GBA1 gene. do.
The rAAV-GBA1 vector insert is composed of four parts: CMV enhancer (CMVe), CBA promoter (CBAp), exon 1, and intron (int), and is a codon-optimized coding sequence (CDS) for human GBA1 (maroon). ) Constituently expresses a CBA promoter element (CBA). The 3'region also includes a post-transcriptional regulatory element of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), followed by a bovine growth hormone poly A signal (bGH poly A) tail. Adjacent ITRs allow for correct packaging of intervening sequences. Two variants of the 5'ITR sequence (FIG. 7, insertion box, sequence below) were evaluated; these variants have several nucleotide differences within the 20 nucleotide "D" region of the ITR, which is the package. It is believed to affect the efficiency of Nucleotides and Expression. The rAAV-GBA1 vector product comprises the "D" domain nucleotide sequence shown in FIG. 7 (insertion box, top sequence). Variant vectors include a mutant "D" domain that acts similarly in preclinical studies (referred to herein as the "S" domain, nucleotide changes are shaded). The skeleton contains genes that confer kanamycin resistance and stuffer sequences to prevent reverse packaging. A schematic diagram showing the rAAV-GBA1 vector is shown in FIG. The rAAV-GBA1 vector is packaged in rAAV using the AAV9 serotype capsid protein.

rAAV-GBA1 is administered to the subject as a single dose via intraoccipital injection guided by X-ray fluoroscopy into the cisterna magna (intra-cisterna; ICM). One aspect of the rAAV-GBA1 dosing regimen study is as follows:
Two dose levels determined based on the results of non-clinical pharmacology and toxicology studies in patients (N = 12) with a single dose of rAAV-GBA1 (3e13vg (low dose); 1e14vg (high dose), etc.) Administer with one of.
Early studies included daily delivery of conduritol-b-epoxide (CBE), a GCase inhibitor to evaluate the efficacy and safety of the rAAV-GBA1 vector and the S variant construct of rAAV-GBA1. It was performed with a mouse model (as described further below). Early studies were also performed on a gene mouse model carrying a homozygous GBA1 mutation and partially deficient in saposin (4L / PS-NA). Additional dose range studies in mouse and non-human primates (NHP) will be performed to further evaluate the safety and efficacy of the vector.

Two slightly different versions of the 5'reverse end repeat (ITR) of the AAV backbone were tested to assess manufacturability and transgene expression (FIG. 7). The 20 bp "D" domain within the 145 bp 5'ITR is believed to be required for the generation of optimal viral vectors, but mutations within the "D" domain are also in some cases transgenes. It has been reported to increase the expression of. Therefore, in addition to the viral vector rAAV-GBA1 carrying the intact "D" domain, a second vector morphology carrying the mutant D domain (referred to herein as the "S" domain) was also evaluated. Both rAAV-GBA1 and the variant express the same transgene. Although both vectors produced effective viruses in vivo as detailed below, rAAV-GBA1 containing the wild-type "D" domain was selected for further development.

To establish a CBE model of GCase deficiency, young mice were administered CBE, a specific inhibitor of GCase. Mice were given CBE daily by IP injection starting 8 days after birth (P8). Three different CBE doses (25 mg / kg, 37.5 mg / kg, 50 mg / kg) and PBS were tested to establish a model exhibiting behavioral phenotypes (FIG. 9). High doses of CBE resulted in dose-dependent lethality. All mice treated with 50 mg / kg CBE died by P23, and 5 of the 8 mice treated with 37.5 mg / kg CBE died by P27. Mice treated with 25 mg / kg CBE were not lethal. CBE-injected mice did not show general dyskinesia in the open-field assay (move at the same distance and rate as mice treated with PBS), but mice treated with CBE were measured by the rotarod assay. Showed impaired motor coordination and balance.

Mice that survived to the end of the study were sacrificed the day after the last CBE administration (P27, "Day 1") or 3 days after CBE discontinuation (P29, "Day 3"). Lipid analysis was performed in the cortex of mice treated with 25 mg / kg CBE to assess accumulation of GCase substrate in both the 1st and 3rd day cohorts. GluSph and GalSph levels (measured in total in this example) were significantly accumulated in CBE-treated mice compared to PBS-treated controls and were consistent with GCase deficiency.
Based on the above studies, a CBE dose of 25 mg / kg was selected because it caused behavioral disorders without affecting survival. To achieve GBA1 distribution throughout the brain and transgene expression during CBE treatment, rAAV-GBA1 or excipients are delivered 3 days after birth (P3) by intraventricular (ICV) injection, followed daily. CBE or PBS treatment with IP was started at P8 (FIG. 10).

CBE-treated mice treated with rAAV-GBA1 performed statistically significantly better in rotarods than mice treated with excipients (FIG. 11). Mice in the variant-treated group were not different from mice treated with excipients in terms of other behavioral indicators such as total distance traveled during the study (FIG. 11).
At the completion of the survival study, half of the mice were sacrificed for biochemical analysis the day after the last dose of CBE (P36, "Day 1") or 3 days after discontinuation of CBE (P38, "Day 3"). (Fig. 12). GCase activity was assessed in the cortex using a fluorescent enzyme assay performed in a biological triplicate. GCase activity was increased in mice treated with rAAV-GBA1, whereas CBE treatment decreased GCase activity. In addition, mice treated with both CBE and rAAV-GBA1 exhibited similar GCase activity levels as in the PBS-treated group, which means that delivery of rAAV-GBA1 can overcome the inhibition of GCase activity induced by CBE treatment. Is shown. Lipid analysis was performed in the motor cortex of mice to determine the levels of the substrates GluCer and GluSph. Both lipids accumulated in the brains of CBE-treated mice, and rAAV-GBA1 treatment significantly reduced substrate accumulation.

Lipid levels were negatively correlated with both GCase activity and performance on the rotarod throughout the treatment group. Increased GCase activity after administration of rAAV-GBA1 was associated with a decrease in substrate and an increase in motor function (FIG. 13). As shown in FIG. 14, preliminary biodistribution was assessed by the presence of vector genomes using qPCR measurements (more than 100 vector genomes per μg of genomic DNA are defined as positive). Mice treated with rAAV-GBA1 both with and without CBE are positive for the rAAV-GBA1 vector genome in the cortex, indicating that ICV delivery results in delivery of rAAV-GBA1 to the cortex. In addition, the vector genome was detected in the liver, little in the spleen, and none in the heart, kidney or gonads. There was no statistically significant difference between the 1st and 3rd day groups for all measurements.

A large study in the CBE model further investigated the effective dose of rAAV-GBA1 in the CBE model. Using a 25 mg / kg CBE dose model, the excipient or rAAV-GBA1 was delivered on P3 via ICV and PBS or CBE treatment with daily IP was initiated on P8. Considering the similarities between groups with and without CBE discontinuation observed in previous studies, all mice were sacrificed 1 day after the final CBE administration (P38-40). As a result of evaluating the effect of three different rAAV-GBA1 doses, the following five groups were obtained with 10 mice (5M / 5F) per group.
Excipient ICV + PBS IP
Excipient ICV + 25 mg / kg CBE IP
3.2e9vg (2.13e10vg / brain 1g) rAAV-GBA1 ICV + 25mg / kg CBE IP
1.0e10vg (6.67e10vg / brain 1g) rAAV-GBA1 ICV + 25mg / kg CBE IP
3.2e10vg (2.13e11vg / brain 1g) rAAV-GBA1 ICV + 25mg / kg CBE IP.

The highest dose of rAAV-GBA1 relieved the failure of weight gain at P37 associated with CBE treatment. In addition, this dose statistically significantly increased performance on rotor rods and tapered beams compared to the excipient + CBE treatment group (FIG. 15). Fatality was observed in several groups including both excipient-treated and rAAV-GBA1 treated groups (excipient + PBS: 0; excipient + 25 mg / kg CBE: 1; 3.2e9 vg). rAAV-GBA1 + 25 mg / kg CBE: 4; 1.0 e10 vg rAAV-GBA1 + 25 mg / kg CBE: 0; 3.2 e10 vg rAAV-GBA1 + 25 mg / kg CBE: 3).

At the completion of the survival test, mice were sacrificed for biochemical analysis (Fig. 16). Cortical GCase activity was evaluated by fluorescence assay in a biological triplet. CBE-treated mice showed a decrease in GCase activity, whereas mice treated with the high dose rAAV-GBA1 showed a statistically significant increase in GCase activity compared to CBE treatment. CBE-treated mice also had accumulations of GluCer and GluSph, both of which were rescued by administration of high doses of rAAV-GBA1.

In addition to the established chemical CBE model, rAAV-GBA1 is also evaluated in a 4L / PS-NA genetic model; it is homozygous for the V394L GD mutation of Gba1 and affects the localization and activity of GCase. The giving saposin is partially deficient. These mice exhibit impaired exercise intensity, coordination, and balance, as evidenced by their performance in beam walking, rotarod, and wire hang assays. Usually, these mice have a lifespan of less than 22 weeks. In early studies, 3 μl of maximal titer virus was delivered by ICV at P23 with a final dose of 2.4 e10 vg (6.0 e10 vg / brain 1 g). Treatment groups with 6 mice per group are:
WT + Excipient ICV
4L / PS-NA + excipient ICV
4L / PS-NA + 2.4e10vg (6.0e10vg / brain 1g) rAAV-GBA1 ICV.

Exercise performance by the beam gait test was evaluated 4 weeks after delivery of rAAV-GBA1. The group of mutant mice treated with rAAV-GBA1 tended to have less total slip and less slip per velocity compared to mutant mice treated with excipients, and restored motor function to near WT levels (Figure). 17). Motor phenotypes become more severe as these mice age, so their performance in this and other behavioral tests will be assessed at a later point in time. Upon completion of the survival test, lipid levels, GCase activity, and biodistribution will be assessed in these mice.

Additional low doses of rAAV-GBA1 are currently being tested using the CBE model and correspond to 0.03, 0.1, and 1 times the proposed high clinical doses of Phase 1. Each group contains 10 mice (5M / 5F) in each group:
Excipient ICV
Excipient ICV + 25 mg / kg CBE IP
3.2e8vg (2.13e9vg / brain 1g) rAAV-GBA1 ICV + 25mg / kg CBE IP
1.0e9vg (6.67e9vg / brain 1g) rAAV-GBA1 ICV + 25mg / kg CBE IP
1.0e10vg (6.67e10vg / brain 1g) rAAV-GBA1 ICV + 25mg / kg CBE IP.

In addition to motor phenotype, lipid levels and GCase activity are assessed in the cortex. Time course and analysis of treatment will also be performed.
A larger dose range study was started to evaluate efficacy and safety data. Ten 4L / PS-NA mice (5M / 5F per group) were injected with 10 μl of rAAV-GBA1. Using allometric brain weight calculations, doses correlate with 0.15, 1.5, 4.4, and 14.5 times the proposed high clinical doses of Phase 1. The injection group consists of:
WT + Excipient ICV
4L / PS-NA + excipient ICV
4L / PS-NA + 4.3e9vg (1.1e10vg / brain 1g) rAAV-GBA1 ICV
4L / PS-NA + 4.3e10vg (1.1e11vg / g / brain) rAAV-GBA1 ICV
4L / PS-NA + 1.3e11vg (3.2e11vg / brain 1g) rAAV-GBA1 ICV
4L / PS-NA + 4.3e11vg (1.1e12vg / brain 1g) rAAV-GBA1 ICV.

正の生体内分布は、１００ｖｇ／１μｇゲノムＤＮＡより大として定義されることに注意。
略語：ＢＤ＝生体内分布；ＮＳ＝有意性なし；Ｔ＝傾向；Ｓ＝有意；Ｎ／Ａ＝適用されず；＋＝正；－＝負。 The outline of the non-clinical study of the CBE model is shown in Table 7 below.

Note that the positive biodistribution is defined as greater than 100 vg / 1 μg genomic DNA.
Abbreviations: BD = biodistribution; NS = no significance; T = tendency; S = significant; N / A = not applicable; + = positive;-= negative.

Example 9: In vitro analysis of rAAV vector The rAAV construct was tested in vitro and in vivo. FIG. 18 shows representative data for in vitro expression of the rAAV construct encoding the progranulin (PGRN (also referred to as GRN)) protein. The left panel shows the standard curve for the progranulin (PGRN) ELISA assay. The lower panel shows the dose response of PGRN expression as measured by ELISA assay in cell lysates of HEK293T cells transduced with rAAV. MOI = multiplicity of infection (vector genome per cell).
Pilot studies were performed to assess the in vitro activity of rAAV vectors encoding prosaposin (PSAP) and SCARB2 alone or in combination with GBA1 and / or one or more inhibitory RNAs. One construct encoding PSAP and progranulin (PGRN (also referred to as GRN)) was also tested. The vectors tested include those shown in Table 4. "Opt" refers to a nucleic acid sequence codon optimized for expression in mammalian cells (eg, human cells). FIG. 19 shows representative data showing that transfection of HEK293 cells with each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells.

Pilot studies were performed to assess the in vitro activity of the rAAV vector encoding TREM2 alone or in combination with one or more inhibitory RNAs. The vectors tested include those shown in Table 8. "Opt" refers to a nucleic acid sequence codon optimized for expression in mammalian cells (eg, human cells). 36A-36B show representative data showing that transfection of HEK293 cells with each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells.

Example 10: Test of SNCA and TMEM106B shRNA construct HEK293 cells Human fetal kidney 293 cell line (HEK293) was used in this study (# 85120602, Sigma-Aldrich). HEK293 cells were prepared in 10% fetal bovine serum [FBS] [D-MEM [# 11995065, Thermo Fisher Scientific]] containing 100 units / ml penicillin and 100 μg / ml streptomycin (# 15140122, Thermo Fisher Scientific). # 10028147, Thermo Fisher Scientific] was replenished).

Plasmid Transfection plasmid transfection was performed using Lipofectamine 2000 Transfection Reagent (# 11668019, Thermo Fisher Scientific) and as directed by the manufacturer. Briefly, HEK293 cells (# 12022001, Sigma-Aldrich) were plated in antibiotic-free medium at a density of ³ × 105 cells / ml. The next day, the plasmid and Lipofectamine 2000 reagent were mixed with Opti-MEM solution (# 31985062, Thermo Fisher Scientific). After 5 minutes, the mixture was added to the HEK293 culture. After 72 hours, cells were harvested for RNA or protein extraction or image analysis was performed. For image analysis, the plates were precoated with 0.01% poly-L-lysine solution (P8920, Sigma-Aldrich) prior to plating the cells.

Gene expression analysis by quantitative real-time PCR (qRT-PCR) Relative gene expression levels are measured by quantitative real-time PCR (qRT-PCR) using the Power SYBR Green Cells-to-CT kit (# 4402955, Thermo Fisher Scientific). Determined according to the manufacturer's instructions. HEK293 cells plated with candidate plasmids in 48-well plates (7.5 × 10 ⁴ cells / well) with 0.5 μg of plasmid and 1.5 μl in Lipofectamine 2000 transfection reagent (50 μl of Opti-MEM solution). Transfection was performed temporarily using a reagent). After 72 hours, RNA was extracted from the cells and used for reverse transcription according to the manufacturer's instructions to synthesize cDNA. In quantitative PCR analysis, 2-5 μl of cDNA product was duplicated with Power SYBR Green PCR Master Mix (# 4367659, Thermo Fisher Scientific) using a gene-specific primer pair (final concentration 250 nM). The primer sequences for the SNCA, TMEM106B, and GAPDH genes are as follows: For SNCA, 5'-AAG AGG GTG TTC TCT ATG TAG GC-3'(SEQ ID NO: 71), 5'-GCT CCT CCA ACA TTT GTC ACT T- 3'(SEQ ID NO: 72); for TMEM106B, 5'-ACA CAG TAC CTA CCG TTA TAG CA-3'(SEQ ID NO: 73), 5'-TGT TGT CAC AGT AAC TTG CAT CA-3'(SEQ ID NO: 74) And for GAPDH, 5'-CTG GGC TAC ACT GAG CAC C-3'(SEQ ID NO: 75), 5'-AAG TGG TCG TCG TCG AGG GCA ATG-3' (SEQ ID NO: 76). Quantitative PCR was performed with the QuantStudio 3 real-time PCR system (Thermo Fisher Scientific). Expression levels were normalized by the housekeeping gene GAPDH and calculated using the comparative CT method.

Fluorescence Image Analysis The SNCA and TMEM106B knockdown plasmids were validated using an EGFP reporter plasmid containing the 3'-UTR of the human SNCA gene downstream of the EGFP coding region. Lipofectamine 2000 transfection reagent ( ¹⁰ μl Opti -Transfected simultaneously using 0.04 μg reporter plasmid, 0.06 μg knockdown plasmid and 0.3 μl reagent in MEM solution. After 72 hours, the fluorescence intensity of the EGFP signal was measured at excitation 488 nm / emission 512 nm using a Varioskan LUX multimode reader (Thermo Fisher Scientific). Cells were fixed at room temperature with 4% PFA and incubated with D-PBS containing 40 μg / ml 7-aminoactinomycin D (7-AAD) for 30 minutes at room temperature. After washing with D-PBS, the fluorescence intensity of the 7-AAD signal was measured using a Varioskan reader at excitation 546 nm / emission 647 nm to quantify cell numbers. Normalized EGFP signals per 7-AAD signal level were compared to control knockdown samples.

Enzyme-linked immunosorbent assay (ELISA)
Knockdown plasmids at the protein level were validated using an α-synuclein reporter plasmid containing the 3'-UTR of the human SNCA gene or the TMEM106B gene downstream of the SNCA coding region. The level of α-synuclein protein was determined by ELISA (# KHB0061, Thermo Fisher Scientific) using lysate extracted from HEK293 cells. HEK293 cells (7.5 × 10 ⁴ cells / well) plated with the candidate plasmid on a 48-well plate with Lipofectamine 2000 transfection reagent (0.1 μg reporter plasmid, 0.15 μg in 25 μl Opti-MEM solution). Knockdown plasmid, and 0.75 μl of reagent) were used for temporary transfection. After 72 hours, cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer (# 89900, Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (# P8340, Sigma-Aldrich) and sonicated for a few seconds. After incubating on ice for 30 minutes, the lysate was centrifuged at 20,000 xg at 4 ° C. for 15 minutes and the supernatant was collected. Protein levels were quantified. Plates were read at 450 nm with a Varioskan plate reader and concentrations were calculated using SoftMax Pro 5 software. The measured protein concentration was normalized to the total protein concentration determined by the bicinchoninic acid assay (# 23225, Thermo Fisher Scientific).

FIG. 37 and Table 9 show representative data showing successful silencing of SNCA in vitro by the GFP reporter assay (top) and α-Syn assay (bottom). FIG. 38 and Table 10 show representative data showing successful silencing of TMEM106B in vitro by the GFP reporter assay (top) and α-Syn assay (bottom).

Example 11: ITR "D" sequence arrangement and cellular transduction The effect of the ITR "D" sequence arrangement on cell transduction of the rAAV vector was investigated. HEK293 cells are rAAVs encoding Gcase, 1) wild-type ITR (eg, a "D" sequence proximal to the transduction gene insert and distal to the end of the ITR), or 2 ) Transduction using an ITR having an "D" sequence located "outer" of the vector (eg, a "D" sequence located proximal to the end of the ITR and distal to the transgene insert). did. The data show that rAAV with a "D" sequence in the "outer" position retains the ability to be packaged and efficiently transduces cells (FIG. 40).

Example 12: In vitro test of progranulin rAAV FIG. 39 is a schematic diagram showing an aspect of a vector containing an expression construct encoding PGRN (also referred to as GRN). Progranulins include rAAV vectors encoding PGRN (codon-optimized PGRN (also referred to as codon-optimized GRN)) in the CNS of GRN-deficient rodents that are heterozygous or homozygous for GRN deficiency. ) Is overexpressed by injection by either intraparenchymal injection or intrathecal injection into a large tank or the like.
Mice are injected at 2 or 6 months of age, raised to 6 or 12 months and analyzed for one or more of the following: levels of expression of GRN at RNA and protein levels, behavioral assays (eg). : Improvement of exercise), survival assay (eg, improvement of survival), microglial and inflammatory markers, gliosis, nerve cell loss, lipofuscinosis, and / or relief of lysosomal marker accumulation such as LAMP1. Assays for GRN-deficient mice include, for example, Arrant et al. (2017) Brain 140: 1477-1465; Arrant et al. (2018) J. Neuroscience 38 (9): 2341-2358; and Amado et al. (2018). Doi: https://doi.org/10.1101/30869; the entire contents are incorporated herein by reference.

Example 13: In vitro test of MAPT rAAV SY5Y cells were plated on 96-well plates with 4x10 ⁴ cells per well. The next day, cells were subjected to two virus stocks (Intronic_eSIBR_MAPT_MiR615 Conserved vector) encoding inhibitory RNAs targeting MAPT (J00130 produced in mammalian cell-based systems, and baculovirus-based systems. The produced J00122; (shown in FIG. 75C) was transducted in a triplet with a MOI of ^2x105 in a medium containing 1 uM hex. Excipients alone were used as a negative control. After 72 hours, cells were harvested and stained with a probe to detect an AAV vector expressing inhibitory RNA for MAPT. The probe targets BGHpA. FIG. 75A shows that both virus stocks were successfully transduced into SY5Y cells.
SY5Y cells were plated on 96-well plates with 4x10 ⁴ cells per well. The next day, cells were subjected to 2x106 in medium containing 1 uM Hoechst using two viral stocks (Intronic_eSIBR_MAPT_MiR615 Conserved vector) (J00130 and ^J00122 ; shown in Figure 75C) encoding inhibitory RNA targeting MAPT. MOI was transducted in a triplet. Excipients alone were used as a negative control. 72 hours or 7 days after transduction, SY5Y cells were lysed for RNA extraction. cDNA was made from extracted RNA using the Invitrogen Power SYBR Green Cells-to-Ct Kit. qRT-PCR was performed on cDNA samples and performed in triplicate using primers for both human MAPT and GAPDH. FIG. 75B shows data for knockdown of MAPT expression by J00130 and J00122.

Equivalents The entire content of the following documents is incorporated by reference in this application: International PCT application PCT / US2018 / 0541225 filed October 3, 2018; International PCT filed October 3, 2018. Application PCT / US2018 / 054223; US Provisional Application No. 62 / 567,296 filed October 3, 2017; title "Gene Therapy for Lysozome Disease"; No. 62 / filed October 3, 2017. No. 567,311, title "Gene treatment of lysosome disease"; No. 62 / 567,319 submitted on October 3, 2017; title "Gene treatment of lysosome disease"; submitted on October 3, 2018. No. 62 / 567,301, title "Gene treatment of lysosome disease"; No. 62 / 567,310 submitted on October 3, 2017; title "Gene treatment of lysosome disease"; October 3, 2017 No. 62 / 567,303, entitled "Gene Therapy for Lysozome Disease", submitted on the day; and No. 62 / 567,305, entitled "Gene Therapy for Lysozome Disease", submitted October 3, 2017.

Although some aspects of at least one aspect of the invention have been described in this way, it should be appreciated that various changes, modifications, and improvements will be readily apparent to those of skill in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Therefore, the above description and drawings are for illustration purposes only.

Although some aspects of the invention have been described and illustrated herein, one of ordinary skill in the art will perform the functions described herein and / or obtain results and / or one or more advantages. Various other means and / or structures of, will be readily envisioned, and each such modification and / or modification is considered to be within the scope of the invention. More generally, one of ordinary skill in the art will mean that all parameters, dimensions, materials, and configurations described herein are exemplary, and the actual parameters, dimensions, materials, and / or configurations are the book. It will be easily understood that the teachings of the invention depend on the particular application (s) used. One of ordinary skill in the art will be aware of many equivalents for a particular aspect of the invention described herein, or will be able to confirm using only routine experimentation. Accordingly, the aforementioned embodiments are presented by way of example only, and it is understood that, within the scope of the appended claims and their equivalents, the invention can be practiced in ways other than those specifically described and claimed. sea bream. The present invention is directed to the individual features, systems, articles, materials, and / or methods described herein. Further, any combination of two or more such features, systems, articles, materials, and / or methods is within the scope of the invention if such features, systems, articles, materials, and / or methods are consistent with each other. include.

As used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly reversed.
As used herein and in the claims, the phrase "and / or" is "either or both" of such combined elements, i.e., in some cases, in combination and in the other. In some cases, it should be understood to mean elements that exist separately. Unless explicitly reversed, any other element may be present, whether or not it is related to the specifically identified element, in addition to the specifically identified element in the "and / or" clause. .. Thus, as a non-limiting example, reference to "A and / or B" when used in combination with an open-ended language such as "contains", in one aspect, refers to A without B (optionally). Can include elements other than B; in another aspect can point to B without A (optionally to include elements other than A); in yet another aspect point to both A and B (includes elements other than A). Can optionally include other elements; etc.

As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is comprehensive, that is, includes at least one of the number of elements or the list, and also more than one, and is not arbitrarily listed. Interpreted as containing additional items. When the exact opposite term is given, for example "only one" or "exactly one", or "consisting of" when used in the claims, is the number or list of elements. Refers to including exactly one of. In general, the term "or" as used herein is preceded by a term of exclusivity, such as "any", "one" or "only one", or "exactly one". , Should be interpreted as referring to exclusive choices (ie, one or the other, but not both). When used in the claims, "becomes essential" shall have the usual meaning as used in the field of patent law.

As used herein and in the claims, the phrase "at least one" with respect to a list of one or more elements is at least one element selected from any one or more elements in the list of elements. However, it is not necessary to include at least one of all the elements specifically listed in the list of elements, and exclude any combination of elements in the list of elements. There is no such thing. By this definition, any element other than the specifically identified element in the list of elements referenced by the phrase "at least one" is present, regardless of whether it is related to the specifically identified element. It is possible. Thus, as a non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") , In one embodiment, at least one, optionally comprising a plurality of A's, and B is absent (and optionally including an element other than B); in another embodiment, at least one, optionally comprising a plurality of B's. A is absent (optionally contains elements other than A); in yet another embodiment, it contains at least one, optionally more than one A, and at least one, optionally more than one B (and optionally others). Includes elements of); etc.

In the claims and in the above specification, "comprising", "including", "carrying", "having", "containing", "involving", It should be understood that all transitional phrases such as "holding" are meant to be open-ended, that is, to include without limitation. Only the transitional phrases "consisting of" and "consisting of essentially" are closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the US Patent Office's Patent Examination Procedures Manual.
The use of terms such as "first,""second,""third," etc. in a claim to modify a claim element is itself a priority, precedence, or, in itself, to another of one claim element. It does not indicate the order, or the temporal order in which the act of the method is performed, but distinguishes certain claim elements with a particular name from other elements with the same name (but only the ordering term is different). Used only as a label for distinguishing claim elements.
Unless expressly reversed, in any method claimed herein, including one or more steps or actions, the order of the steps or actions of the method does not necessarily indicate the steps or actions of the method. It should also be understood that the order is not limited.

Sequence In some embodiments, the expression cassette encoding one or more gene products (eg, first, second and / or third gene product) is set forth in any one of SEQ ID NOs: 1-149. Contains or consists of a sequence (or encodes a peptide having that sequence). In some embodiments, the gene product is encoded by a portion (eg, a fragment) of any one of SEQ ID NOs: 1-149.

Claims (15)

A method for treating a subject who has or is suspected of having a central nervous system (CNS) disease, the method of which is:
(I) An expression construct comprising an introductory gene encoding one or more inhibitory nucleic acids targeting one or more gene products listed in Table 1 and / or one or more gene products listed in Table 1. (Ii) Two adeno-associated virus (AAV) reverse terminal repeats (ITRs) flanking the expression construct,
The method comprising administering to a subject an isolated nucleic acid comprising. The method of claim 1, wherein the transgene encodes one or more proteins selected from GBA1, GBA2, PGRN, TREM2, PSAP, SCARB2, GALC, SMPD1, CTSB, RAB7L, VPS35, GCH1, and IL34. The method of claim 1 or 2, wherein the transgene encoding one or more gene products comprises a codon-optimized protein coding sequence. The method of any one of claims 1-3, wherein the transgene encodes one or more inhibitory nucleic acids targeting SNCA, MAPT, RPS25 and / or TMEM106B. The method according to any one of claims 1 to 4, wherein the AAV ITR is an AAV2 ITR. The method of any one of claims 1-5, wherein the isolated nucleic acid is packaged in a recombinant adeno-associated virus (rAAV). The method of claim 6, wherein the rAAV comprises an AAV9 capsid protein. The method according to any one of claims 1 to 6, wherein the subject is a mammal and optionally the subject is a human. The method according to any one of claims 1 to 8, wherein the CNS disease is a neurodegenerative disease, a synuclein disease, tauopathy and / or lysosomal storage disease (LSD). The method of claim 9, wherein the CNS disease is listed in Table 2, Table 3, Table 4, or Table 5. Dosage comprises direct injection into the subject's CNS, and optionally, direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intratubal injection or any combination thereof. The method according to any one of 10. 11. The method of claim 11, wherein the intracisterna injection is an occipital injection into the cisterna magna. The method of claim 11 or 12, wherein the direct injection into the subject's CNS comprises convection enhanced delivery (CED). The method according to any one of claims 1 to 13, wherein the administration comprises a peripheral injection and optionally the peripheral injection is an intravenous injection. The method of any one of claims 6-14, wherein the subject is administered an rAAV of about 1 × 10 ¹⁰ vg to about 1 × 10 ¹⁶ vg.

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