Gene silencing by recombinant AAV-amiRNA in Alexander disease

Abstract

Aspects of the present disclosure relate to compositions (e.g., nucleic acids, rAAV vectors, rAAV, etc.) and methods for treating Alexander Disease (AxD). The present disclosure is based in part on nucleic acids encoding interfering nucleic acids (e.g., artificial microRNAs) that target glial fibrillary acidic protein (GFAP) RNA transcripts. In some embodiments, the interfering nucleic acids are encoded by rAAV vectors. Aspects of the present disclosure also provide methods of treating AxD by administering nucleic acids to a subject.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC 119(e) of the filing date of U.S. Provisional Application No. 63 / 331,710, entitled "Gene Silencing with Recombinant AAV-AMIRNA in Alexander Disease," filed April 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (U012070176WO00-SEQ-LGE.xml; size: 38,407 bytes; and creation date: April 14, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Alexander disease (AxD) is an autosomal dominant neurological disorder that affects approximately 1 in every 1 million live births. Approximately 95% of AxD patients are identified by heterozygous mutations in the glial fibrillary acidic protein (GFAP) gene, which maps to the long arm of chromosome 17q21.31. A distinctive feature of AxD is the presence of Rosenthal fibers in the cell body region of astrocytes, which is caused by the accumulation of GFAP in the cytoplasm. Currently, no treatment is available that can treat AxD. Summary of the Invention

[0004] overview Aspects of the present disclosure relate to compositions (e.g., nucleic acids, rAAV vectors, rAAV, etc.) and methods for treating Alexander disease (AxD). The present disclosure is based in part on a nucleic acid encoding an interfering nucleic acid (e.g., an artificial microRNA) that targets glial fibrillary acidic protein (GFAP) RNA transcripts. In some embodiments, the interfering nucleic acid is encoded by a rAAV vector. The inventors have surprisingly discovered that including an endogenous GFAP promoter in the nucleic acid described herein results in astrocyte-specific expression of the inhibitory nucleic acid and reduces off-target effects (e.g., cytotoxicity). Aspects of the present disclosure also provide a method of treating AxD by administering to a subject a nucleic acid or rAAV described herein.

[0005] Thus, in some aspects, the present disclosure provides a nucleic acid comprising a nucleic acid sequence encoding an artificial microRNA (amiRNA) that targets a glial fibrillary acidic protein (GFAP) RNA transcript flanked by an adeno-associated virus inverted terminal repeat (AAV ITR). In some embodiments, the AAV ITR is an AAV2 ITR or a variant thereof.

[0006] In some embodiments, the amiRNA comprises a nucleic acid sequence encoding a pri-miRNA scaffold, a nucleic acid sequence encoding a guide strand, and a nucleic acid sequence encoding a passenger strand. In some embodiments, the pri-miRNA scaffold is derived from a naturally occurring pri-miRNA and comprises at least one flanking sequence and a loop-forming sequence comprising at least four nucleotides.

[0007] In some embodiments, the pri-miRNA scaffold is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451.

[0008] In some embodiments, the nucleic acid sequence encoding the guide strand and / or passenger strand comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence represented by SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid sequence encoding the guide strand and / or passenger strand comprises or consists of the sequence represented by SEQ ID NO: 1 or 2.

[0009] In some embodiments, the nucleic acid further comprises a promoter operably linked to the nucleic acid sequence encoding the amiRNA. In some embodiments, the promoter comprises chicken beta actin (CB) promoter or GFAP promoter. In some embodiments, the endogenous GFAP promoter is a GfaABC1D promoter. In some embodiments, the GfaABC1D promoter comprises the sequence represented by SEQ ID NO:3.

[0010] In some embodiments, the nucleic acid comprises a self-complementary AAV (scAAV) vector (e.g., as described in U.S. Pat. No. 11,046,955, the entire contents of which are incorporated herein by reference). In some embodiments, the nucleic acid comprises the sequence represented by SEQ ID NO:4 or 5 or consists of the sequence represented by SEQ ID NO:4 or 5.

[0011] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising a nucleic acid comprising a nucleic acid sequence encoding an artificial microRNA (amiRNA) targeting a glial fibrillary acidic protein (GFAP) RNA transcript flanked by adeno-associated virus inverted terminal repeat sequences, and at least one AAV capsid protein. In some embodiments, the AAV ITRs are AAV2 ITRs or variants thereof.

[0012] In some embodiments, at least one capsid protein has a serotype selected from AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 capsid protein. In some embodiments, at least one capsid protein is an AAV9 capsid protein. In some embodiments, the rAAV is a self-complementary AAV (sCAAV).

[0013] In some aspects, the present disclosure provides a method for reducing glial fibrillary acidic protein (GFAP) in a cell or subject, the method comprises administering to a cell or subject the nucleic acid or rAAV described herein.In some embodiments, the administration reduces GFAP in the brain.In some embodiments, the administration reduces GFAP in the hippocampus and / or olfactory bulb.

[0014] In some aspects, the present disclosure provides a method for reducing the formation of Rosenthal fiber in a subject, the method comprises administering the nucleic acid or rAAV described herein to a cell or a subject.In some embodiments, the administration reduces the formation of Rosenthal fiber in the brain.In some embodiments, the administration reduces the formation of Rosenthal fiber in the hippocampus and / or olfactory bulb.

[0015] In some aspects, the disclosure provides a method for treating Alexander Disease (AxD) in a subject, the method comprising administering to the subject a nucleic acid or rAAV as described herein.

[0016] In some embodiments, the cell or subject is a mammalian cell or a mammalian subject. In some embodiments, the cell is a mouse, rat, or human cell. In some embodiments, the subject is a mouse, rat, or human subject.

[0017] In some embodiments, the cell or subject comprises one or more mutations in the GFAP gene. In some embodiments, the one or more mutations comprise heterozygous mutations in each copy of the GFAP gene. In some embodiments, the cell or subject has or is suspected of having Alexander Disease (AxD).

[0018] In some embodiments, the administration comprises systemic administration. In some embodiments, the administration comprises injection. In some embodiments, the injection comprises intravenous injection. In some embodiments, the administration results in reduced formation of Rosenthal fibers in the subject. [Brief description of the drawings]

[0019] [Figure 1A-1B] Figure 1A-1B shows a schematic diagram of rAAV vector for silencing GFAP. Figure 1A shows amiR-GFAP packaging in adeno-associated virus serotype 9 (for example, scAAV9) driven by ubiquitous beta-actin promoter with CMV enhancer (CMVen / CB). Figure 1B shows amiR-GFAP packaging in adeno-associated virus serotype 9 (for example, scAAV9) driven by endogenous GFAP promoter (GFaABC1D). rAAV vectors may be self-complementary AAV (scAAV) vectors or single-stranded AAV vectors.

[0020] [Figure 2A-2C] Figures 2A-2C show that the two scAAV9-CMVen / CB-amiR-GFAP vectors downregulated GFAP mRNA and protein in the brains of GFAP+ / R236H mice. Figure 2A, top panel, shows the reduction of GFAP protein levels from whole brain after 3 weeks of treatment with scAAV9-CMVen / CB-amiR-GFAP vectors; the bottom panel shows quantification of GFAP protein levels. Figure 2B shows qRT-PCR showing the reduction of GFAP mRNA after 3 weeks of treatment with scAAV9-CMVen / CB-amiR-GFAP vectors. Figure 2C shows that GFAP was reduced after 3 months of treatment with scAAV9-CB6-amiR-GFAP-2 (left panel); the right panel represents the quantification of a Western blot.

[0021] [Figure 3A-3B] Figures 3A-3B show representative data of weight growth and survival curves in female (Figure 3A) and male (Figure 3B) GFAP mice treated with the scAAV9-CMVen / CB-amiR-GFAP vector. [Figure 4A-4B] Figures 4A-4B show representative data of body weight and survival curves of female (Figure 4A) and male (Figure 4B) GFAP+ / R236H mice treated with scAAV9-CMVen / CB-amiR-GFAP vectors; data shown are unpaired two-tailed t-tests comparing AAV9 empty as control.

[0022] [Figure 5A-5B]Figures 5A-5B show representative data of scAAV-GFaABC1D-amiR-GFAP vector-mediated GFAP silencing in vivo. Figure 5A, upper panel shows reduced GFAP protein levels after 3 weeks of treatment with scAAV-GFaABC1D-amiR-GFAP vector in GFAP+ / R236H mice; lower panel represents quantification of GFAP protein levels. Figure 5B shows quantification of GFAP transcripts in GFAP+ / R236H mice treated with scAAV-GFaABC1D-amiR-GFAP vector for 3 weeks by qRT-PCR analysis.

[0023] [Figure 6A-6B] Figures 6A-6B show representative data of body weight and survival curves of female (Figure 6A) and male (Figure 6B) GFAP+ / R236H mice treated with scAAV-GFaABC1D-amiR-GFAP vector; data are representative of unpaired two-tailed t-tests comparing with AAV9 empty as control. [Figure 7] Figure 7 shows representative data of scAAV-GFaABc1D-amiR-GFAP vector-mediated GFAP silencing in vivo. Figure 7, upper panel, shows decreased GFAP protein levels after 3 months of treatment with scAAV9-GFaABc1D-amiR-GFAP-2 vector in GFAP+ / R236H mice; lower panel represents quantification of GFAP protein levels.

[0024] [Figure 8]Figure 8 shows representative fluorescence imaging data of scAAV-GFaABc1D-amiR-GFAP vector-mediated silencing of GFAP in the brain. Figure 8, upper left panel, shows the reduction in the levels of GFAP protein in the hippocampus and olfactory bulb after 3 weeks of treatment with scAAV9-GFaABc1D-amiR-GFAP-2 vector in GFAP+ / R236H mice; lower left panel shows the reduction in the levels of GFAP protein in the hippocampus and olfactory bulb after 3 months of treatment with scAAV9-GFaABc1D-amiR-GFAP-2 vector in GFAP+ / R236H mice; right panel shows a schematic diagram of the mouse brain identifying the hippocampus and olfactory bulb.

[0025] [Figure 9] FIG. 9 shows representative images of scAAV-GFaABc1D-amiR-GFAP vector-mediated reduction of Rosenthal fibers (arrows) in the hippocampus and olfactory bulb after 3 weeks of treatment. [Figure 10] FIG. 10 shows representative images of scAAV-GFaABc1D-amiR-GFAP vector-mediated reduction of Rosenthal fibers (arrows) in the hippocampus and olfactory bulb after 3 months of treatment.

[0026] Detailed Description Aspects of the present invention relate to certain interfering RNAs (e.g., miRNAs, such as artificial miRNAs) that are effective to reduce the expression of glial fibrillary acidic protein (GFAP) in a subject when delivered to the subject. Accordingly, in some embodiments, the methods and compositions described by the present disclosure are useful in some embodiments for the treatment of Alexander Disease (AxD).

[0027] Isolated Nucleic Acids In some aspects, the disclosure provides nucleic acids or isolated nucleic acids useful for reducing (e.g., inhibiting) the expression and / or activity of GFAP. A "nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of the disclosure are isolated. As used herein, the term "isolated" means artificially produced. As used herein, the term "isolated" with respect to a nucleic acid means: (i) amplified in vitro, for example, by polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, for example, by cleavage and gel separation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is one that can be readily manipulated by recombinant DNA techniques that are well known in the art. Thus, a nucleotide sequence contained in a vector with known 5' and 3' restriction sites, or a nucleotide sequence for which a polymerase chain reaction (PCR) primer sequence is disclosed, is considered isolated, but a nucleic acid sequence that exists natively in a natural host is not considered isolated. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid isolated within a cloning vector or an expression vector is not pure in that it may comprise only a small percentage of the material in the cell in which it resides.However, such a nucleic acid, as the term is used herein, is isolated because it can be easily manipulated by standard techniques known to those skilled in the art.As used herein with respect to a protein or peptide, the term "isolated" refers to a protein or peptide that is isolated from its natural environment or that is artificially produced (e.g., by chemical synthesis, recombinant DNA technology, etc.).

[0028] Those skilled in the art will also recognize that conservative amino acid substitutions may be made to provide functionally equivalent variants or homologs of capsid proteins. In some aspects, the present disclosure encompasses sequence changes that result in conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Mutants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, and can be found in references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include those made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus, conservative amino acid substitutions can be made to the amino acid sequences of the proteins and polypeptides disclosed herein.

[0029] The isolated nucleic acid of the present invention may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid described in this disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. The isolated nucleic acid (e.g., a recombinant rAAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. A "recombinant AAV (rAAV) vector" typically consists, at a minimum, of a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise one or more regions encoding one or more inhibitory RNAs (e.g., miRNAs) including nucleic acids targeting endogenous mRNAs of the subject, as disclosed elsewhere herein. The transgene may also comprise a region encoding, for example, a protein and / or an expression control sequence (e.g., a polyA tail), as described elsewhere in this disclosure.

[0030] Generally, the ITR sequences are about 145 bp in length. Preferably, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, for example, texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing a transgene, in which a selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., a rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.

[0031] In some embodiments, the isolated nucleic acid further comprises a region (e.g., second region, third region, fourth region, etc.) that comprises a second AAV ITR. In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the second ITR is a mutant ITR that lacks a functional terminal release site (TRS). The term "lack of a terminal release site" can refer to an AAV ITR that contains a mutation (e.g., a sense mutation, such as a non-synonymous mutation or a missense mutation) that destroys the function of the terminal release site (TRS) of the ITR, or a truncated AAV ITR that lacks a nucleic acid sequence that encodes a functional TRS (e.g., ΔTRS ITR). Without wishing to be bound by any particular theory, rAAV vectors containing ITRs that lack a functional TRS generate self-complementary rAAV vectors, as described, for example, in McCarthy (2008) Molecular Therapy 16(10):1648-1656.

[0032] In addition to the major elements identified above for recombinant AAV vectors, the vector also includes conventional control elements operably linked to the transgene elements in a manner that allows transcription, translation and / or expression in cells transfected with the vector produced by the present invention or in cells infected with the virus. As used herein, "operably linked" sequences include both expression control sequences contiguous with the gene of interest and expression control sequences acting in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product, if desired. Numerous expression control sequences, including promoters that are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0033] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be "operably" linked when they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. If it is desired that the nucleic acid sequence be translated into a functional protein, the induction of the promoter in the 5'regulatory sequence results in the transcription of the coding sequence, and the nature of the linkage between the two DNA sequences is said to be operably linked if it (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if it is capable of effecting the transcription of that DNA sequence such that the resulting transcript may be translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked when they are linked such that their transcription from a common promoter results in the expression of two or more proteins translated in frame. In some embodiments, the operably linked coding sequences produce a fusion protein. In some embodiments, the operably linked coding sequences produce a functional RNA (eg, miRNA).

[0034] In some embodiments, the inhibitory nucleic acid targeting GFAP provided herein is a small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA. siRNA is a class of double-stranded RNA molecule, typically about 20-25 base pairs in length, that targets a nucleic acid (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule may be determined by the binding of the guide strand of the molecule to its target RNA. Although longer siRNAs may also be effective, effective siRNA molecules are generally less than 30-35 base pairs in length to avoid triggering non-specific RNA interference pathways in cells via the interferon response. In some embodiments, the RNAi molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more bases in length. In some embodiments, the siRNA molecule is 8-30 base pairs in length, 10-15 base pairs in length, 10-20 base pairs in length, 15-25 base pairs in length, 19-21 base pairs in length, 21-23 base pairs in length. In some embodiments, the RNAi molecule is an siRNA, shRNA, or miRNA. In some embodiments, the RNAi molecule is an artificial microRNA (AmiRNA or amiR).

[0035] Following selection of an appropriate target RNA sequence, an RNAi (e.g., siRNA, shRNA, miRNA, AmiRNA) molecule comprising a nucleotide sequence complementary to all or a portion of the target sequence, i.e., the antisense strand or guide strand, can be designed and prepared using methods known in the art (see, e.g., PCT Publication No. WO2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791; each of which is incorporated herein by reference).

[0036] RNAi (e.g., siRNA, shRNA, miRNA, AmiRNA) molecules can be double-stranded (i.e., dsRNA molecules that include a guide strand and a complementary passenger strand) or single-stranded (i.e., ssRNA molecules that include only a guide strand). RNAi (e.g., siRNA, shRNA, miRNA, AmiRNA) molecules can include duplex (i.e., annealed sense strand and guide strand with 3' overhang), asymmetric duplex (i.e., duplex with 3' and 5' antisense overhang), hairpin (i.e., nucleotide sequences are normally complementary when read in opposite directions, and two regions of the same strand base pair to form a double helix that ends in an unpaired loop), or asymmetric hairpin (i.e., hairpin with strand overhang) secondary structure, with self-complementary passenger strand and guide strand. In some embodiments, the GFAP target inhibitory nucleic acid described herein is an AmiRNA that includes a guide strand (i.e., antisense strand) and a passenger strand (i.e., sense strand). The double-stranded RNAi molecule (for example, siRNA or miRNA) described herein may comprise RNA strands of the same length or different lengths.Double-stranded siRNA molecule can also be related to a single oligonucleotide of stem-loop structure, where the self-complementary sense and antisense regions of siRNA molecule are linked using nucleic acid-based or non-nucleic acid-based linker(s), and a circular single-stranded RNA with two or more loop structures and a stem that comprises self-complementary sense and antisense strands, where the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule that can mediate RNAi.

[0037] In some embodiments, small hairpin RNA (shRNA) molecules are also contemplated herein. These molecules contain a specific antisense sequence in addition to a reverse complementary (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides the single-stranded RNA molecule and its reverse complement so that they can anneal to form a dsRNA molecule (optionally with additional processing steps that may result in the addition or removal of one, two, three or more nucleotides from the 3'-end and / or (for example and) the 5'-end of one or both strands). The spacer can be of sufficient length to allow the antisense sequence and the sense sequence to anneal to form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally with subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3'-end and / or (for example and) the 5'-end of one or both strands). The spacer sequence may be an unrelated nucleotide sequence placed between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise the shRNA.

[0038] In some embodiments, the guide strand of an RNAi molecule described herein (e.g., an siRNA, shRNA, miRNA, or AmiRNA targeting GFAP) is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the sense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, 21-23 nucleotides in length.

[0039] In some embodiments, the passenger strand of the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA targeting GFAP) is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the sense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, 21-23 nucleotides in length.

[0040] In some aspects, the present disclosure provides inhibitory miRNAs that specifically bind (e.g., hybridize) to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive bases of GFAP (e.g., human or mouse GFAP). In some embodiments, the inhibitory nucleic acid targets a conserved region in human and mouse GFAP. As used herein, "consecutive bases" refers to two or more nucleotide bases (e.g., as part of a nucleic acid molecule) that are covalently linked to each other (e.g., by one or more phosphodiester bonds, etc.). In some embodiments, at least one miRNA is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% identical to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleotide bases of human GFAP NCBI sequences NM_002055.5, NM_001131019.3, NM_001242376.3, or NM_001363846.2, SEQ ID NOs: 6-9. In some embodiments, the inhibitory RNA is an miRNA that includes or is encoded by a sequence represented by any one of SEQ ID NOs: 1 or 2.

[0041] In some embodiments, at least one miRNA is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% identical to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleotide bases of mouse GFAP NCBI sequence NM_001131020.1 or NM_010277.3 SEQ ID NOs: 10-11. In some embodiments, the inhibitory RNA is a miRNA that includes or is encoded by a sequence represented by any one of SEQ ID NOs: 1 or 2.

[0042] In some embodiments, the RNAi molecules (e.g., siRNA, shRNA, miRNA, AmiRNA) described herein comprise a guide strand that comprises a region that is complementary to a target region in GFAP mRNA. In some embodiments, the region of complementarity is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region on GFAP mRNA. In some embodiments, the target region is a region of consecutive nucleotides on GFAP mRNA. In some embodiments, a complementary nucleotide sequence does not need to be 100% complementary to its target nucleotide sequence in order to be specifically hybridizable or specific to GFAP mRNA.

[0043] In some embodiments, the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) comprise a guide strand that includes a region of complementarity to a GFAP mRNA sequence, where the region of complementarity ranges from 8-15, 8-30, 8-40, or 10-50, or 5-50, or 5-40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a GFAP mRNA (e.g., a GFAP coding sequence represented by any one of SEQ ID NOs: 6-11). In some embodiments, the region of complementarity comprises a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of a GFAP mRNA (e.g., a GFAP coding sequence represented by any one of SEQ ID NOs: 6-11). In some embodiments, the region of complementarity comprises a nucleotide sequence with up to 3 mismatches over 15 bases, up to 2 mismatches over 10 bases, or up to 1 mismatch over 5 bases.

[0044] In some embodiments, the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) include a guide strand that includes a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to a target sequence represented by any one of SEQ ID NOs: 6 to 11. In some embodiments, the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) include a guide strand that includes a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to a target sequence represented by SEQ ID NO: 16 or 17. In some embodiments, the siRNA molecule includes a guide strand that includes a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to a sequence represented by SEQ ID NO: 12 or 14. In some embodiments, the siRNA molecule comprises a guide strand that comprises at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the sequence represented by SEQ ID NO:12 or 14.

[0045] In some embodiments, the RNAi molecules (e.g., siRNA, shRNA, miRNA, AmiRNA) targeting GFAP described herein include a guide strand that is 18-25 nucleosides in length (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) and includes a region of complementarity to a sequence represented by any one of SEQ ID NOs: 6-11, where the region of complementarity is at least 13 nucleotides in length (e.g., 13, 14, 15, 16, 17, 18, or 19 nucleotides). In some embodiments, the region of complementarity is fully complementary to all or a portion of its target sequence. In some embodiments, the region of complementarity encompasses one, two, three, or more mismatches.

[0046] In some embodiments, the GFAP-targeting RNAi molecules (e.g., siRNA, shRNA, miRNA, AmiRNA) described herein comprise a guide strand that comprises at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) of any one of the sequences set forth in SEQ ID NOs: 12 or 14. In some embodiments, the GFAP-targeting siRNA or shRNA further comprises a passenger strand that comprises at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) that are complementary to any one of the sequences set forth in SEQ ID NOs: 13 or 15.

[0047] In some embodiments, the GFAP-targeting RNAi molecules described herein are AmiRNAs comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1 or 2.

[0048] The total length of the RNAi molecule described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) can vary from about 14 nucleotides to about 100 nucleotides, depending on the type of siRNA molecule designed. Generally, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., they constitute the specific antisense sequence of the RNAi molecule described herein (e.g., siRNA, shRNA, miRNA, AmiRNA). For example, when the siRNA is the double-stranded or single-stranded RNAi molecule described herein (e.g., siRNA, miRNA), the length can vary from about 14 nucleotides to about 50 nucleotides, while when the siRNA is an shRNA, AmiRNA, or circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.

[0049] The RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) may include a 3' overhang at one end of the molecule, and the other end may be blunt-ended or have an overhang (5' or 3'). When the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) include an overhang at both ends of the molecule, the length of the overhang may be the same or different. In one embodiment, the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) include a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule. In some embodiments, the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) include a 3' overhang of about 1 to about 3 nucleotides on the passenger strand. In some embodiments, the RNAi molecules described herein (e.g., siRNA, shRNA, miRNA, AmiRNA) include a 3' overhang of about 1 to about 3 nucleotides on the guide strand. In some embodiments, the RNAi molecules described herein (eg, siRNA, shRNA, miRNA, AmiRNA) comprise a 3' overhang of about 1 to about 3 nucleotides on both the passenger and guide strands.

[0050] It should be understood that in some embodiments, an isolated nucleic acid or vector (e.g., an rAAV vector) comprises a nucleic acid sequence encoding more than one (e.g., a plurality, such as 2, 3, 4, 5, 10, or more) miRNA. In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds to) the same target gene (e.g., an isolated nucleic acid encoding three unique miRNAs, each of which targets the GFAP gene). In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds to) a different target gene.

[0051] In some aspects, the disclosure provides isolated nucleic acids and vectors (e.g., rAAV vectors) encoding one or more artificial miRNAs. As used herein, "artificial miRNA," "amiRNA," or "amiR" refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA backbone that is a precursor miRNA capable of producing a functional mature miRNA), where miRNA and miRNA * The sequence (e.g., the passenger strand of the miRNA duplex) directs highly efficient RNA silencing of the target gene and the corresponding amiRNA / amiRNA duplex, as described, for example, in Eamens et al. (2014), Methods Mol. Biol. 1062:211-224. * In some embodiments, the miRNA described by the present disclosure (e.g., the artificial miRNA that targets GFAP) comprises a miR-155 backbone sequence, a miR-30 backbone sequence, a mir-64 backbone sequence, a miR-122 backbone sequence, a pri-MIR-21, a pri-MIR-22, a pri-MIR-26a, a pri-MIR-30a, a pri-MIR-33, a pri-MIR-122, a pri-MIR-375, a pri-MIR-199, a pri-MIR-99, a pri-MIR-194, a pri-MIR-155, or a pri-MIR-451.

[0052] The region containing the transgene (e.g., the second region, third region, fourth region, etc.) may be located in any suitable location of the isolated nucleic acid. The region may be located in any untranslated portion of the nucleic acid, such as, for example, an intron, a 5' or 3' untranslated region, etc.

[0053] In some cases, it may be desirable to position a region (e.g., a second region, a third region, a fourth region, etc.) upstream of the first codon of a nucleic acid sequence (e.g., a protein coding sequence) that encodes a protein. For example, the region may be positioned between the first codon of the protein coding sequence and 2000 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein coding sequence and 1000 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein coding sequence and 500 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein coding sequence and 250 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein coding sequence and 150 nucleotides upstream of the first codon.

[0054] In some cases (e.g., when the transgene lacks a protein coding sequence), it may be desirable to position a region (e.g., a second region, a third region, a fourth region, etc.) upstream of the poly-A tail of the transgene. For example, the region may be positioned between the first base of the poly-A tail and 2000 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 1000 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 500 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 250 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 150 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 100 nucleotides upstream of the first base. The region may be positioned between the first base of the poly-A tail and 50 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 20 nucleotides upstream of the first base. In some embodiments, the region is located between the last nucleotide base of the promoter sequence and the first nucleotide base of the poly-A tail sequence.

[0055] In some cases, the region may be located downstream of the last base of the polyA tail of the transgene. The region may be between the last base of the polyA tail and a position 2000 nucleotides downstream of the last base. The region may be between the last base of the polyA tail and a position 1000 nucleotides downstream of the last base. The region may be between the last base of the polyA tail and a position 500 nucleotides downstream of the last base. The region may be between the last base of the polyA tail and a position 250 nucleotides downstream of the last base. The region may be between the last base of the polyA tail and a position 150 nucleotides downstream of the last base.

[0056] It should be understood that when a transgene encodes multiple miRNAs, each miRNA may be located at any suitable location within the transgene. For example, a nucleic acid encoding a first miRNA may be located in an intron of the transgene, and a nucleic acid sequence encoding a second miRNA may be located in another untranslated region (e.g., between the last codon of the protein-coding sequence and the first base of the polyA tail of the transgene).

[0057] In some embodiments, the transgene further comprises a nucleic acid sequence that codes for one or more expression control sequences (such as, for example, a promoter). Expression control sequences include appropriate transcription initiation sequences, termination sequences, promoter sequences and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (for example, Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of encoded products, if desired. Numerous expression control sequences, including promoters that are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0058] "Promoter" refers to a DNA sequence that is recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate specific transcription of a gene. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct location and orientation relative to the nucleic acid to control the initiation of RNA polymerase and expression of the gene.

[0059] In the case of nucleic acids encoding proteins, polyadenylation sequences are generally inserted following the transgene sequence and before the 3'AAVITR sequence. The rAAV constructs useful in this disclosure may also contain an intron, desirably located between the promoter / enhancer sequence and the transgene. One viable intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences will be used to produce proteins that contain more than one polypeptide chain. The selection of these and other common vector elements is conventional, and many such sequences are available [see, e.g., Sambrook et al., and references cited therein, e.g., pages 3.18 3.26 and 16.17 16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, the foot and mouth disease virus 2A sequence is included in the polyprotein, which is a small peptide (approximately 18 amino acids long) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459).The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0060] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In some embodiments, the promoter is a chicken β-actin promoter. In some embodiments, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is a U6 promoter.

[0061] Inducible promoters allow the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of certain physiological conditions, such as acute phase, certain differentiation states of cells, or only in replicating cells. Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters controlled by an exogenously supplied promoter include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline inducible system (Gossen et al., Science, 268:1766-1769 (1995), Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), and the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)). (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin.Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0062] In another embodiment, the native promoter of the transgene will be used. If it is desired that the expression of the transgene should mimic native expression, the native promoter may be preferred. The native promoter may be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In further embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression. In some embodiments, the native promoter is the GFAP promoter. In some embodiments, the GAFP promoter is the GfaABC1D promoter.

[0063] In some embodiments, GfaABC1D is a promoter that selectively targets astrocytes, e.g., as described in Griffin, JM, Fackelmeier, B., Fong, DM et al. Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction in rat spinal cord following injury. Gene Ther 26, 198-210 (2019). In some embodiments, the promoter is a GfaABC1D promoter. In some embodiments, the nucleic acid sequence of the GfaABC1D promoter is represented by SEQ ID NO:3. In some embodiments, the nucleic acid encoding an artificial microRNA (amiRNA or amiRNA) targeting GFAP comprises a promoter that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3.

[0064] In some embodiments, the regulatory sequence confers tissue-specific gene expression capability. In some cases, the tissue-specific regulatory sequence binds to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: astrocyte-specific promoter (GfaABC1D), liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, alpha-myosin heavy chain (a-MHC) promoter, or cardiac troponin T (cTnT) promoter.Other exemplary promoters include the beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)), the immunoglobulin heavy chain promoter, the T cell receptor alpha chain promoter, and neuronal promoters such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others, which will be apparent to one of skill in the art. In some embodiments, the tissue-specific promoter is an astrocyte-specific promoter. In some embodiments, the astrocyte-specific promoter is the GfaABC1D promoter.

[0065] Aspects of the present disclosure relate to isolated nucleic acids that include more than one promoter (e.g., 2, 3, 4, 5, or more promoters). For example, in the context of a construct having a transgene that includes a first region that encodes a protein and a second region that encodes an inhibitory RNA (e.g., an AmiRNA that targets GFAP), it may be desirable to use a first promoter sequence (e.g., a first promoter sequence operably linked to the protein coding region) to drive expression of the protein coding region, and a second promoter sequence (e.g., a second promoter sequence operably linked to the inhibitory RNA coding region) to drive expression of the inhibitory RNA coding region. In general, the first promoter sequence and the second promoter sequence can be the same promoter sequence or different promoter sequences. In some embodiments, the first promoter sequence (e.g., the promoter that drives expression of the protein coding region) is an RNA polymerase III (polIII) promoter sequence. Non-limiting examples of polIII promoter sequences include the U6 promoter sequence and the H1 promoter sequence. In some embodiments, the second promoter sequence (e.g., the promoter sequence driving the expression of an inhibitory RNA) is an RNA polymerase II (polII) promoter sequence. Non-limiting examples of polII promoter sequences include T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the polIII promoter sequence drives the expression of an inhibitory RNA (e.g., miRNA) coding region. In some embodiments, the polII promoter sequence drives the expression of a protein coding region.

[0066] Reporter sequences (eg, nucleic acid sequences encoding reporter proteins) that may be provided in the transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other sequences well known in the art. When associated with regulatory elements that drive their expression, reporter sequences provide signals that are detectable by conventional means, including enzymatic, radioactive, colorimetric, fluorescent or other spectroscopic analyses, fluorescence-activated cell sorting analyses, and immunological analyses, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assaying β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be visually measured by color or light production in a luminometer. Such reporters can be useful, for example, in verifying tissue-specific targeting capabilities of nucleic acids and tissue-specific promoter regulatory activity.

[0067] Recombinant adeno-associated virus (rAAV) In some aspects, the present disclosure provides an isolated AAV. As used herein, the term "isolated" in relation to AAV refers to AAV that is artificially produced or obtained. Isolated AAV may be produced using recombinant methods. Such AAV is referred to herein as "recombinant AAV". Recombinant AAV (rAAV) preferably has tissue-specific targeting capability, thus allowing the nuclease and / or transgene of rAAV to be specifically delivered to one or more predefined tissues. AAV capsid is an important factor in determining these tissue-specific targeting capabilities. Therefore, rAAV with a capsid suitable for the tissue to be targeted can be selected.

[0068] Methods for obtaining recombinant AAV with desired capsid proteins are well known in the art. (See, e.g., US2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell containing nucleic acid sequences encoding AAV capsid proteins; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to allow packaging of the recombinant AAV vector into AAV capsid proteins. In some embodiments, the capsid proteins are structural proteins encoded by the cap gene of AAV. In some embodiments, AAV contains three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are about 87 kDa, about 72 kDa and about 62 kDa, respectively. In some embodiments, upon translation, capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the function of capsid protein is to protect the viral genome, deliver the genome, and interact with the host. In some aspects, capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0069] In some embodiments, the AAV capsid protein is of an AAV serotype selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAV9, and AAV 10. In some embodiments, the AAV capsid protein is of the AAV9 serotype.

[0070] The components to be cultured in the host cell to package the rAAV vector into an AAV capsid may be provided in trans to the host cell. Alternatively, any one or more of the necessary components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the necessary components using methods known to those of skill in the art. Most preferably, such a stable host cell will contain the necessary components under the control of an inducible promoter. However, the necessary components may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of suitable regulatory elements for use with the transgene. In yet another alternative, the selected stable host cell may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a stable host cell may be generated that is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but contains rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be generated by those of skill in the art.

[0071] In some embodiments, the present disclosure relates to a composition comprising the host cell described above. In some embodiments, the composition comprising the host cell further comprises a cryopreservation agent.

[0072] The recombinant AAV vectors, rep sequences, cap sequences, and helper functions useful for producing the rAAV of the present disclosure may be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limitation to the present invention. For example, see K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0073] In some embodiments, recombinant AAV may be produced using a triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (containing a transgene) for packaging into AAV particles, an AAV helper function vector, and an auxiliary function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions that contain functional rep and cap genes). Non-limiting examples of vectors suitable for use in the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650, and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, both of which are incorporated herein by reference in their entirety. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and / or cellular functions (e.g., "accessory functions") on which AAV depends for replication. Accessory functions include those functions required for AAV replication, including, but not limited to, those portions involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and activation of AAV capsid ad assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0074] In some aspects, the present disclosure provides a transfected host cell. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

[0075] "Host cell" refers to any cell that carries or is capable of carrying a substance of interest. Often, the host cell is a mammalian cell. The host cell may be used as a recipient of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other introduced DNA related to the production of recombinant AAV. The term encompasses the progeny of the original transfected cell. Thus, as used herein, "host cell" may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genome, or in total DNA complement, to the original parent due to natural, accidental, or deliberate mutations.

[0076] As used herein, the term "cell line" refers to a cell population capable of continuous or sustained growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. Furthermore, it is known in the art that spontaneous or induced changes in karyotype may occur during storage or transfer of such clonal populations. Thus, cells derived from the cell line referred to may not be exactly the same as the ancestral cell or culture, and the cell line referred to includes such variants.

[0077] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

[0078] As used herein, the term "vector" encompasses any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication and transfer of genetic sequences between cells when associated with the correct control elements. Thus, the term encompasses cloning and expression vehicles, as well as viral vectors. In some embodiments, it is contemplated that a useful vector is a vector in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence that is recognized by the cell's synthetic machinery or introduced synthetic machinery necessary to initiate specific transcription of a gene. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct location and orientation relative to the nucleic acid to control the initiation of RNA polymerase and the expression of the gene. The term "expression vector or construct" refers to any type of genetic construct that contains a nucleic acid that can transcribe part or all of the nucleic acid coding sequence. In some embodiments, expression encompasses transcription of a nucleic acid, for example, to produce a biologically active polypeptide product or functional RNA (e.g., guide RNA) from the transcribed gene. The above methods of packaging a recombinant vector in a desired AAV capsid to produce a rAAV of the present disclosure are not meant to be limiting, and other suitable methods will be apparent to those of skill in the art.

[0079] In some embodiments, any one or more thymidine (T) or uridine (U) nucleotides in the sequences provided herein, including those provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing with an adenosine nucleotide (e.g., via Watson-Crick base pairing). For example, in some embodiments, any one or more thymidine (T) nucleotides in the sequences provided herein, including those provided in the sequence listing, may be suitable for replacement with a uridine (U) nucleotide, or vice versa.

[0080] Methods for Treating Alexander Disease The present disclosure provides a method for delivering a transgene (e.g., an inhibitory RNA, such as miRNA) to a subject. The method typically involves administering to a subject an effective amount of an isolated nucleic acid encoding an interfering RNA capable of reducing the expression of glial fibrillary acidic protein (GFAP), or an rAAV comprising a nucleic acid described herein for expressing an inhibitory RNA capable of reducing the expression and / or activity of GFAP. In some embodiments, administration of a nucleic acid or rAAV described herein reduces GFAP in the brain. In some embodiments, administration of a nucleic acid or rAAV described herein reduces GFAP activity in the brain. In some embodiments, administration of a nucleic acid or rAAV described herein reduces GFAP expression and / or activity in the hippocampus and / or olfactory bulb. In some embodiments, the method typically involves administering to a subject an effective amount of a nucleic acid encoding an interfering RNA targeting GFAP, or an rAAV thereof, which results in a reduced level of Rosenthal fibers. In some embodiments, administration of a nucleic acid or rAAv reduces Rosenthal fibers in the brain. In some embodiments, administration of the nucleic acid or rAAv reduces Rosenthal fibers in the hippocampus and / or olfactory bulb.

[0081] As used herein, "Alexander disease" or "AxD" is an autosomal dominant neurological disorder and a type of leukodystrophy. Leukodystrophy is a neurological disorder caused by abnormalities in myelin, which insulates and protects nerve fibers in the brain. AxD affects approximately 1 in every 1 million live births. Approximately 95% of AxD patients are identified by heterozygous mutations in the glial fibrillary acidic protein (GFAP) gene, which is located on the long arm of chromosome 17q.31.

[0082] A unique feature of AxD is the appearance of Rosenthal fibers in the cell body region of astrocytes, caused by the accumulation of GFAP in the cytoplasm and the destruction of myelin. Rosenthal fibers are abnormal clumps of proteins that accumulate in astrocytes, impairing the formation of the cytoskeleton and the survival and function of astrocytes, leading to demyelination and destruction of the white matter of the brain. Myelin allows the efficient transmission of electrical impulses in the brain.

[0083] GFAP is a type III intermediate filament protein expressed in astrocytes as well as many other cells of the central nervous system (CNS). The concentration of GFAP varies depending on the region of the brain, with the highest concentrations found in the hippocampus, olfactory bulb, medulla oblongata, and cervical spinal cord. GFAP plays a role in cell communication in the brain, proper functioning of the blood-brain barrier, and maintaining the mechanical strength and cell shape of astrocytes. Under normal conditions, GFAP protein forms filaments that support the nervous system, however, when produced in excess, such as in AxD, GFAP kills cells and damages myelin. The disease is most often caused by sporadic mutations, although it can be inherited. AxD is most common in infancy or early childhood, but has been found to occur at any age. There is no cure for AxD, and it is often fatal.

[0084] In some embodiments, the subject has Rosenthal fibers in the hippocampus and / or olfactory bulb, as described, for example, in Hagemann et al., Alexander disease-associated glial fibrillary acidic protein mutations in mice induce Rosenthal fiber formation and a white matter stress response. J Neurosci. 2006;26(43):11162-11173. In some embodiments, the treatments disclosed herein are effective in reducing the formation of Rosenthal fibers. In some embodiments, the treatments disclosed herein are effective in reducing the formation of Rosenthal fibers in the hippocampus and / or olfactory bulb.

[0085] In some embodiments, the subject has increased GFAP levels in the hippocampus and / or olfactory bulb (see, e.g., Jany et al., GFAP Expression as an Indicator of Disease Severity in Mouse Models of Alexander Disease. ASN Neuro. 2013;5(2)). In some embodiments, the treatments disclosed herein are effective in reducing GFAP expression and / or activity. In some embodiments, the treatments disclosed herein are effective in reducing GFAP expression and / or activity in the hippocampus and / or olfactory bulb.

[0086] An "effective amount" of a substance is an amount sufficient to produce a desired effect. In some embodiments, an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or infect in the context of rAAV-mediated delivery) a sufficient number of target cells of a target tissue of a subject. In some embodiments, the target tissue is a central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, an effective amount of an isolated nucleic acid (e.g., may be delivered via rAAV) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to reduce expression of a pathogenic gene or protein (e.g., GFAP), to reduce activity of a pathogenic protein (e.g., GFAP) to extend the lifespan of the subject, to ameliorate one or more symptoms of a disease in the subject (e.g., symptoms of Alexander's disease), etc. The effective amount depends on various factors, such as, for example, the species, age, weight, health status, and tissue targeted by the subject, and may vary between subjects and tissues, as described elsewhere in this disclosure.

[0087] The terms "treating," "treat," or "treatment" as used herein refer to the application or administration of a composition (e.g., an isolated nucleic acid or rAAV as described herein) to a subject with a disease or disorder associated with a genetic mutation in the gene for GFAP, increased GFAP expression, and increased formation of Rosenthal fibers, including leukodystrophies (e.g., Alexander Disorder), for the purpose of curing, curing, alleviating, mitigating, altering, treating, ameliorating, improving, or affecting the disorder, symptoms of the disease, or pre-deposition of the disease. In some embodiments, the subject has a mutation in the GFAP gene. In some embodiments, the subject has increased expression of GFAP compared to subjects without AxD. In some embodiments, the subject has increased formation of Rosenthal fibers compared to subjects without AxD. In some embodiments, the subject has increased GFAP expression in the brain compared to subjects without AxD. In some embodiments, the subject has increased GFAP expression in the hippocampus and / or olfactory bulb compared to subjects without AxD. In some embodiments, the subject has increased Rosenthal fiber expression in the brain compared to subjects without AxD. In some embodiments, the subject has increased Rosenthal fiber formation in the hippocampus and / or olfactory bulb compared to subjects without AxD.

[0088] In some embodiments, the methods provided herein result in decreased GFAP expression and / or activity (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%) in the brain (e.g., hippocampus and / or olfactory bulb) of a subject with AxD compared to the subject prior to administration. GFAP expression and / or activity can be measured by any suitable method known in the art, e.g., by immunoassay (e.g., enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay (CLIA)) or real-time polymerase chain reaction (RT-PCR).

[0089] In some embodiments, the methods provided herein result in reduced Rosenthal fiber formation in the brain (e.g., hippocampus and / or olfactory bulb) of a subject with AxD compared to the subject prior to administration (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% difference). The formation of Rosenthal fibers can be measured by suitable methods known in the art, e.g., magnetic resonance imaging (MRI), immunostaining of brain biopsies (e.g., immunohistochemistry, immunofluorescence, or Western blot).

[0090] Mode of Administration The rAAV of the present disclosure may be delivered to a subject in a composition according to any suitable method known in the art.For example, the rAAV, preferably suspended in a physiologically compatible carrier (i.e., in a composition), may be administered to a subject, i.e., a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque).In some embodiments, the host animal does not include a human.

[0091] Delivery of rAAV to a mammalian subject may be, for example, by intramuscular injection of the mammalian subject or by administration to the bloodstream. Administration to the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream by isolated limb perfusion, a technique well known in the surgical arts, which essentially allows one of skill in the art to isolate a limb from the systemic circulation prior to administration of rAAV virions. A variation of isolated limb perfusion, described in U.S. Pat. No. 6,177,403, can also be employed by one of skill in the art to administer viral particles to the vasculature of an isolated limb to potentially enhance transduction of muscle cells or tissues. Moreover, in some cases, it may be desirable to deliver virions to the CNS of a subject. By "CNS" is meant all cells and tissues of the vertebrate brain and spinal cord. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and the like. Recombinant AAV may be delivered directly to the CNS or brain using neurosurgical techniques known in the art, such as stereotactic injection, with a needle, catheter, or related device, for example, by injection into the ventricular region, as well as the striatum (for example, the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule (for example, Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In some embodiments, the rAAV described in this disclosure is administered by intravenous injection. In some embodiments, the rAAV is administered by intracerebral injection. In some embodiments, the rAAV is administered by intraspinal injection. In some embodiments, the rAAV is administered by intrastriatal injection.In some embodiments, the rAAV is delivered by intracranial injection. In some embodiments, the rAAV is delivered by cisternal injection. In some embodiments, the rAAV is delivered by lateral ventricle injection.

[0092] Aspects of the present disclosure relate to compositions comprising recombinant AAVs comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs. In some embodiments, each miRNA comprises a sequence represented by any one of SEQ ID NOs: 1 or 2. In some embodiments, the nucleic acid further comprises an AAV ITR. In some embodiments, the rAAV comprises a rAAV vector represented by a sequence represented by any one of SEQ ID NOs: 4 or 5, or a portion thereof. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier.

[0093] The compositions of the present disclosure may include rAAV alone or in combination with one or more other viruses (e.g., a second rAAV carrying and encoding one or more different transgenes). In some embodiments, the composition includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rAAVs, each carrying one or more different transgenes.

[0094] Suitable carriers can be easily selected by those skilled in the art, taking into consideration the indications for which rAAV is intended.For example, one suitable carrier includes saline, which can be formulated with various buffers (e.g., phosphate-buffered saline).Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water.The selection of carriers is not a limitation of the present disclosure.

[0095] Optionally, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers.Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

[0096] rAAV is administered in an amount sufficient to transfect the cells of desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects.Conventional pharmacologic acceptable administration routes include, but are not limited to, direct delivery to selected organ (e.g., intraportal delivery to liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, intrathecal, intracranial, intraventricular, and other parenteral administration routes.If desired, routes of administration may be combined.

[0097] The dose of rAAV virions required to achieve a particular "therapeutic effect", e.g., in units of dose in genome copies per kilogram of body weight (GC / kg), will vary based on several factors, including, but not limited to, the route of administration of the rAAV virions, the level of gene or RNA expression required to achieve the therapeutic effect, the particular disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine the dose range of rAAV virions for treating a patient with a particular disease or disorder based on the factors described above, as well as other factors well known in the art.

[0098] The effective amount of rAAV is sufficient to target and infect animals and target tissue of interest.In some embodiments, the effective amount of rAAV is sufficient to produce a stable somatic transgenic animal model.The effective amount mainly depends on factors such as the subject's species, age, weight, health status, and target tissue, and may vary between animals and tissues.For example, the effective amount of rAAV is generally about 10 9 ~10 16 The range is from about 1 ml to about 100 ml of solution containing genome copies. In some cases, about 10 11 ~10 13 A dosage of between 10 and 20 rAAV genome copies is suitable. 12 or 10 13 100 copies of the rAAV genome are effective in targeting CNS tissues, and in some cases, stable transgenic animals are produced by multiple doses of rAAV.

[0099] In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., 24 hours). In some embodiments, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once every 2 weeks (e.g., once in a 2 calendar week period). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once every 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per 6 calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0100] In some embodiments, the rAAV composition is particularly effective when high rAAV concentrations are present (e.g., about 10 13GC / mL or more) and formulated to reduce aggregation of AAV particles in the composition. Methods for reducing aggregation of rAAV are well known in the art and include, for example, adding detergents, adjusting pH, adjusting salt concentrations, etc. (See, e.g., Wright et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)

[0101] The formulation of pharma- ceutically acceptable excipient and carrier solutions is well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for use with the particular compositions described herein in the various treatment regimens.

[0102] Typically, these preparations may contain at least about 0.1% of the active compound, although of course the percentage of active ingredient may vary and may conveniently be from about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of active compound in each therapeutically useful composition may be prepared so that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, as well as other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical preparations. Various dosages and treatment regimes may be desired therefor.

[0103] In some circumstances, it may be desirable to deliver the rAAV-based therapeutic construct in the appropriately formulated pharmaceutical composition disclosed herein either subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, intracranially, intracerebroventricularly, or orally, intraperitoneally, or by inhalation.In some embodiments, the administration modalities as described in U.S. Patent Nos. 5,543,158; 5,641,515 and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety) may be used to deliver rAAV.In some embodiments, the preferred administration mode is by portal vein injection.

[0104] Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0105] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that may be used will be known to those skilled in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the proposed site of injection (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0106] Sterile injectable solution is prepared by incorporating the required amount of active rAAV into a suitable solvent with various other components as listed herein as necessary, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains basic dispersion medium and other necessary components from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technique, which produces powder of active component plus any additional desired components from its solution that has been previously sterilized and filtered.

[0107] The rAAV compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

[0108] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

[0109] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, transgenes delivered by rAAV vectors may be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like.

[0110] Such formulations may be preferred for the introduction of pharma- ceutically acceptable formulations of nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-time have been developed (U.S. Patent No. 5,741,516). In addition, various methods of using liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0111] Liposomes have been used successfully in a number of cell types that are normally resistant to transfection by other procedures. Furthermore, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed testing the efficacy of liposome-mediated drug delivery.

[0112] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å, containing aqueous solution in their core.

[0113] Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (size of about 0.1 μm) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0114] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering rAAV compositions to a host. Sonophoresis (i.e., ultrasound) is used as a device to enhance the rate of penetration and effectiveness of drugs into the circulatory system and is described in U.S. Patent No. 5,656,016. Other contemplated drug delivery alternatives are intraosseous injections (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0115] Kits and Related Compositions The agents described herein may in some embodiments be assembled into pharmaceutical kits or diagnostic kits or research kits to facilitate their use in therapeutic, diagnostic or research applications. The kit may include one or more containers that contain the components of the present disclosure and instructions. Specifically, such kits may include one or more agents together with instructions that describe the intended use and proper use of these agents. In some embodiments, the agents in the kit may be in pharmaceutical formulations and dosages suitable for a particular use and method of administration of the agents. Kits for research purposes may contain components in concentrations or amounts suitable for carrying out various experiments.

[0116] In some embodiments, the disclosure relates to a kit for producing rAAV, the kit comprising a container housing an isolated nucleic acid comprising an miRNA comprising or encoded by a sequence represented by any one of SEQ ID NOs: 1 or 2. In some embodiments, the kit further comprises a container housing an isolated nucleic acid encoding an AAV capsid protein, e.g., an AAV9 capsid protein.

[0117] The kit may be designed to facilitate the use of the methods described herein by researchers and may take many forms. Each composition of the kit may be provided in liquid form (e.g., solution) or in solid form (e.g., dry powder), if applicable. In some cases, some of the compositions may be configurable (e.g., to an active form) or otherwise processable by adding appropriate solvents or other species (e.g., water or cell culture medium), for example, which may or may not be provided in the kit. As used herein, "instructions" may define an instruction and / or promotion component, typically accompanied by written instructions on or associated with the packaging of the present disclosure. Instructions may also encompass, for example, any oral or electronic instructions provided in any manner that a user would clearly recognize that instructions should be associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communication, etc. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of a drug or biological product, and the instructions may also reflect approval by the agency of the manufacture, use, or sale for administration to animals.

[0118] The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or for isolating and mixing a sample and applying it to a subject. The kit may include a container that contains an agent described herein. The agent may be in liquid, gel or solid (powder) form. The agent may be sterilely prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage. A second container may have another agent that is sterilely prepared. Alternatively, the kit may include an active agent that is premixed and shipped in a syringe, vial, tube, or other container.

[0119] Exemplary embodiments of the present invention are described in more detail by the following examples. These embodiments are illustrative of the present invention, and those skilled in the art will recognize that they are not limited to the exemplary embodiments.

[0120] example This example describes artificial mRNAs (amiRNAs or amiRs) designed to target selected GFAP sequences in mice, rats, and humans. Multiple amiR-GFAP constructs that can efficiently silence GFAP expression in all three species were tested in vitro. Self-complementary rAAV vectors encoding one amiR-GFAP design (designated #1 and #2) were packaged into AAV serotype 9 (AAV9) under the control of the universal chicken beta actin promoter with a CMV enhancer (CMVen / CB) (Figure 1A) and transfected into 5-week-old Alexander Disease (AxD) mice (GFAP + / R236H ) 2x10 14 The mice were injected intravenously at a dose of 1000 genome copies (GC) / kg. After 3 weeks, we observed that both scAAV9-CMVen / CB-amiR-GFAP vectors downregulated GFAP mRNA and protein in the brain by more than 50% compared to the control vector (Figures 2A-2C). In addition, histopathological analysis of the brains of treated mice showed a reduction in Rosenthal fibers.

[0121] To monitor long-term therapeutic efficacy and safety, treatment was extended to 3 months. We observed that mice treated with scAAV9-CMVen / CB-amiR-GFAP#1 began to die from digestive diseases approximately 3-4 weeks after treatment (Figures 3A-3B). Interestingly, mice treated with scAAV9-CMVen / CB-amiR-GFAP#2 died of gastrointestinal disease. + / R236HMice showed improved body weight by 10 weeks after injection (FIGS. 4A-4B). During this period, animals began to lose weight, hunch, and breathe abnormally fast. Necropsy of these animals revealed enlarged hearts, as well as reduced GFAP expression in the brain, compared to mice treated with a control vector.

[0122] We observed that expression of amiR-GFAP in non-astrocytic cells may have led to the aforementioned side effects. To reduce off-target silencing, the CMVen / CB promoter was replaced with the endogenous GFAP promoter (e.g., GFaABC1D) in the amiR-GFAP vector to drive astrocyte-specific expression. The second generation of the amiR-GFAP#1 vector not only eliminated early death, but also improved weight gain during development, as early as 3 weeks after injection. Treatment with the second generation amiR-GFAP#2 vector also improved the expression of GFAP + / R236H The body weight of the mice improved (Figure 6A-6B). Western blot and qRT-PCR analysis revealed that GFAP protein and mRNA levels were reduced by 30% in mice treated with the amiR-GFAP#2 vector at 3 weeks after injection (Figure 5A-5B). Long-term monitoring of animals receiving the second generation vector continued for 3 months. Western blot analysis revealed that GFAP protein levels were reduced by 50% in mice treated with the amiR-GFAP#2 vector compared at 3 months after injection, returning to GFAP protein levels comparable to healthy littermates (Figure 7).

[0123] Next, GFAP immunofluorescence staining was performed at 3 weeks and 3 months after injection to see which specific brain regions were affected. Treatment with the second generation amiR-GFAP#2 vector reduced GFAP expression in the hippocampus and olfactory bulb, which are crucial for AxD pathology, at 3 weeks and 3 months after injection (Figure 8). In addition, histopathological analysis of the brains of second generation mice treated with amiR-GFAP#2 showed a reduction in Rosenthal fibers in the hippocampus and olfactory bulb after 3 weeks and 3 months of treatment (Figures 9 and 10). Together, these results indicate that treatment with amiR-GFAP#2 reduces GFAP expression and Rosenthal fiber expression in both the hippocampus and olfactory bulb, which are hallmarks of AxD.

[0124] Representative sequence >amiR-GFAP-2 (SEQ ID NO: 1) (the guide strand is in bold, the passenger strand is underlined) [ka] >amiR-GFAP-1 (SEQ ID NO:2) (the guide strand is in bold, the passenger strand is underlined) [ka]

[0125] >GfaABC1D promoter (SEQ ID NO:3) [ka] [ka]

[0126] >rAAV_amiR-GFAP-2 (SEQ ID NO: 4) [ka]

[0127] >rAAV_amiR-GFAP-1 (SEQ ID NO:5) [ka] [ka]

[0128] Human GFAP NM_002055.5 (SEQ ID NO: 6) (amiR-GFAP-1 target region underlined; amiR-GFAP-2 target region bold) [ka] [ka] [ka]

[0129] Human GFAP NM_001131019.3 (SEQ ID NO: 7) (amiR-GFAP-1 target region underlined; amiR-GFAP-2 target region bold) [ka] [ka]

[0130] Human GFAP NM_001242376.3 (SEQ ID NO: 8) (amiR-GFAP-1 target region underlined; amiR-GFAP-2 target region bold) [ka]

[0131] Human GFAP NM_001363846.2 (SEQ ID NO: 9) (amiR-GFAP-1 target region underlined; amiR-GFAP-2 target region bold) [ka] [ka]

[0132] Mouse GFAP NM_001131020.1 (SEQ ID NO: 10) (amiR-GFAP-1 target region underlined; amiR-GFAP-2 target region bold) [ka] [ka]

[0133] Mouse GFAP NM_010277.3 (SEQ ID NO: 11) (amiR-GFAP-1 target region underlined; amiR-GFAP-2 target region bold) [ka] [ka]

[0134] amiR-GFAP-2 guide strand (SEQ ID NO: 12) [ka] amiR-GFAP-2 passenger strand (SEQ ID NO: 13) [ka] amiR-GFAP-1 guide strand (SEQ ID NO: 14) [ka] amiR-GFAP-1 passenger strand (SEQ ID NO: 15) [ka] amiR-GFAP-2 target sequence (SEQ ID NO: 16) [ka] amiR-GFAP-1 target sequence (SEQ ID NO: 17) [ka]

Claims

1. An isolated nucleic acid comprising a nucleic acid sequence encoding an artificial microRNA (amiRNA) that targets a glial fibrillary acidic protein (GFAP) RNA transcript, wherein the nucleic acid sequence is positioned alongside an adeno-associated virus inverted terminal repeat (ITR).

2. The following: (i) an isolated nucleic acid according to claim 1; and (ii) a recombinant adeno-associated virus (rAAV) comprising at least one AAV capsid protein.

Images