Regulatory nucleic acid sequences

Abstract

In gene therapy, controlling the expression of exogenous nucleic acids introduced into cells is crucial for patient health and safety. Not only must the level of the expression product be within the therapeutic concentration range, but the expression must also be located within the tissue where it is needed or within a specific region within that tissue. Expression outside the therapeutic concentration range (i.e., lower or higher), or outside the therapeutic region, or even outside the specific region within the tissue where it is needed, may be therapeutically ineffective or even harmful. [Solution] The present invention relates to regulatory nucleic acid sequences, particularly CNS-specific promoters and their elements. The present invention also relates to expression constructs, vectors, virions, pharmaceutical compositions and cells containing such promoters, and methods of use thereof. Regulatory nucleic acid sequences are particularly useful for gene therapy applications.

Description

[Technical Field]

[0001] This invention relates to regulatory nucleic acid sequences, particularly CNS-specific promoters and their elements. The invention also relates to expression constructs, vectors, virions, pharmaceutical compositions and cells containing such promoters, and methods of using them. Regulatory nucleic acid sequences are particularly useful for gene therapy applications. [Background technology]

[0002] The following considerations are provided to assist the reader in understanding this disclosure and do not constitute any endorsement of the content or relevance of the prior art.

[0003] Following extensive research into the internal mechanisms of gene regulation within the body, the focus of research has recently shifted to regulating gene expression by introducing exogenous nucleic acid sequences into cells.

[0004] This is a conventional practice in research and bioprocessing, where the nucleic acid sequence of the desired expression product, operably linked to a promoter, is often introduced into the producing cell line in the form of a vector.

[0005] In the field of gene therapy, this has been of particular interest to single-gene disorders or Mendelian disorders caused by the presence of a defective gene in the patient's cells. Introducing the nucleic acid sequence of the wild-type allele of the defective gene, operably linked to a promoter, into the patient's cells is a favorable treatment option because, while conventional medicines can only address the symptoms, it can theoretically cure the condition.

[0006] In gene therapy, controlling the expression of exogenous nucleic acids introduced into cells is crucial for patient health and safety. Not only must the level of the expression product be within the therapeutic concentration range, but the expression must also be located within the tissue where it is needed or within a specific region within that tissue. Expression outside the therapeutic concentration range (i.e., lower or higher), or outside the therapeutic region, or even outside the specific region within the tissue where it is needed, may be therapeutically ineffective or even harmful.

[0007] Dopamine transporter deficiency syndrome, a type of childhood parkinsonism, is a candidate for gene therapy by introducing a replacement gene because it is caused by a loss-of-function mutation in a single gene, DAT1 / SLC6A3 (Kurian et al., 2009). DAT1 / SLC6A3 encodes a presynaptic dopamine transporter involved in the transfer of extraneuronal dopamine to dopaminergic neurons. The dopamine transporter uses the driving force of a sodium gradient across the cell membrane to transport dopamine, two sodium ions, and one chloride ion into the cell. As a result, DAT1 / SLC6A3 plays a role in regulating the duration and intensity of dopamine signaling (Ng et al., 2014), and its dysfunction is associated with various neuropsychiatric disorders, such as attention deficit hyperactivity disorder (ADHD) (Kurian et al., 2009).

[0008] A particular challenge in introducing the substituted DAT1 / SLC6A3 gene is that, as shown in Figure 1A, in non-disease states, DAT1 / SLC6A3 is specifically expressed in the midbrain. To best mimic the native expression of DAT1 / SLC6A3, it is desirable to ensure that the substituted DAT1 / SLC6A3 gene is expressed in the midbrain (where dopaminergic neurons are located), but it is also desirable that expression in other parts of the brain be minimal.

[0009] Therefore, among other CNS regions, promoters that drive expression in the midbrain, as well as promoters that specifically drive expression in dopaminergic neurons in the midbrain, are needed.

[0010] Angelman syndrome is also a candidate for gene therapy through the introduction of replacement genes. Angelman syndrome is most commonly caused by a mutation or absence of a single gene, UBE3A. UBE3A is involved in targeting proteins for degradation. In most neurons, only the maternal copy of the UBE3A gene is active, and the loss of the maternal UBE3A gene leads to Angelman syndrome.

[0011] A particular difficulty in introducing the substituted UBE3A gene is that, as shown in Figure 1B, UBE3A is widely expressed in the brain in non-disease states. To best mimic the native expression of the UBE3A gene, it is desirable that the substituted UBE3A gene be widely expressed in the brain.

[0012] Therefore, a promoter is needed that drives expression in many or all regions of the brain (e.g., pan-CNS).

[0013] Other CNS disorders are suitable targets for gene therapy, and in some of these disorders, targeted expression of therapeutic genes in specific CNS tissues may be desirable, while in others, more generalized disorders, nonspecific expression in the CNS may be appropriate. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] WO2014 / 144229 [Patent Document 2] WO2019 / 028306 [Patent Document 3] WO2000 / 28004

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[Non-Patent Document 51] Lagos-Quintana et al., 2003, RNA, 9, pp. 175-179 [Non-Patent Document 52] mousebrain.org / genesearch.html [Overview of the project] [Problems that the invention aims to solve]

[0016] One or more aspects of the present invention are intended to address one or more of the above-mentioned problems. [Means for solving the problem]

[0017] In a first aspect of the present invention, a synthetic central nervous system (CNS)-specific promoter is provided which includes or comprises a sequence or functional variant thereof that follows any one of sequence numbers 1-8, 21-26.

[0018] In some embodiments, the synthetic CNS-specific promoter includes or comprises sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 1-8, 21-26.

[0019] The present invention therefore provides various synthetic CNS-specific promoters and their functional variants. Promoters according to the present invention, which are variants of any one of SEQ ID NOs: 1-8, 21-26, are generally preferred to retain at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference promoter. The activity may be evaluated using examples as described herein, but other methods may also be used.

[0020] In some embodiments, the synthetic CNS-specific promoter includes SYNP_CRE151 (SEQ ID NO: 12) and at least one of the following CREs: - CRE0004_Lmx1b (Sequence ID 9), - CRE0003_Pitx3 (Sequence ID 10), - CRE0005_faf1_short (Sequence ID 28), - CRE0006_Pitx2_short(sequence number 29), - CRE0007_Pitx2_short (sequence number 30), and - CRE0008_Pitx2_short (sequence number 31).

[0021] In another aspect of the present invention, a CNS-specific cis-regulating element (CRE) is provided which includes or comprises a sequence or a functional variant thereof that follows any one of sequence numbers 9-11, 28-31. In some embodiments, the CNS-specific CRE includes a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 9-11, 28-31.

[0022] It is generally preferable that the CNS-specific CRE according to the present invention, which is a variant of any one of SEQ ID NOs: 9-11, 28-31, retains at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference CRE. The retention of activity can be evaluated by comparing the expression of a suitable reporter under the control of a reference promoter with that of a otherwise identical promoter containing a substituted CRE under equivalent conditions. The activity may be evaluated using examples such as those described herein, but other methods may also be used.

[0023] The CRE according to the present invention can be combined with additional CREs as appropriate to form a cis-regulating module (CRM). The additional CRE may be the CREs according to SEQ ID NOs. 9-11, 28-31 or their functional variants, or they may be other CREs. The additional CRE may be CNS-specific as appropriate.

[0024] In another aspect of the present invention, a synthetic CNS-specific promoter is provided that includes or comprises a CRE or a functional variant thereof according to any one of SEQ ID NOs: 9-11, 28-31. In some embodiments, the CRE can be operably coupled to a promoter element. In some embodiments, the promoter element may be a minimal or proximal promoter. Preferably, the proximal promoter is a CNS-specific proximal promoter.

[0025] In a further aspect of the present invention, a minimal or proximal promoter is provided that includes or comprises a sequence or a functional variant thereof that conforms to any one of SEQ ID NOs. 12-13. In another aspect of the present invention, a synthetic promoter comprising the minimal or proximal promoter is provided, preferably a synthetic CNS-specific promoter comprising the minimal or proximal promoter. The functional variant of the minimal or proximal promoter may include a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 12-13.

[0026] As appropriate, one of CNS-4, CNS-5_v2, CNS-6_v2, CNS-7_v2, or CNS-8_v2 (SEQ ID NOs. 4-8) can function as a minimal or proximal promoter. Thus, a synthetic CNS-specific promoter is provided that includes a minimal or proximal promoter following either SEQ ID NOs. 12-13 or SEQ ID NOs. The minimal or proximal promoter can be operably coupled to a CRE or CRM. The CRE may be the CRE according to the present invention or any other CRE. The CRM may include the CRE according to the present invention. The CRE or CRM is CNS-specific as appropriate.

[0027] The CRE, minimal / proximal promoter, or promoter of the present invention may be active in specific regions of the CNS, preferably in specific regions of the brain, or in specific cell types (one or more) of the brain, or in a combination of both.

[0028] The CRE, minimal / proximal promoter, or promoter of the present invention may be active in one or more of the various parts of the CNS. The CNS mainly consists of the brain and spinal cord. The retina, optic nerve, olfactory nerve, and olfactory epithelium may also be considered parts of the CNS alongside the brain and spinal cord. This is because they are directly connected to brain tissue without intermediate nerve fibers. As appropriate, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in the brain and spinal cord. As appropriate, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in the brain but not in the spinal cord or any other part of the CNS. As appropriate, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in the spinal cord but not in the brain. Preferably, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in the brain. As appropriate, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in one or more of the various regions within the brain.

[0029] Examples of brain regions that may be affected include, but are not limited to, the frontal lobe, parietal lobe, occipital lobe, temporal lobe (including the hippocampus and amygdala), cerebellum, midbrain, pons, medulla oblongata, and diencephalon (including the thalamus and hypothalamus). Examples of spinal cord regions that may be affected include the cervical, thoracic, lumbar, sacral, and coccygeal vertebrae. In some embodiments, it may be desirable for the CRE, minimal / proximal promoter, or promoter of the present invention to exhibit broad activity in the brain. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in all parts of the brain or CNS (pan-CNS), preferably in all regions of the brain. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in the brain but not in other parts of the CNS, such as the spinal cord. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the brain regions listed above. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in most of the brain regions, i.e., at least 5, at least 6, at least 7, at least 8, or all 9 of the nine brain regions listed above. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in 4 to 6 of the brain regions listed above. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in 2 to 4 of the brain regions listed above, e.g., the midbrain, temporal lobe, and diencephalon. In some embodiments, the CRE, minimal / proximal promoter, or synthetic promoter of the present invention may be active in the brain and spinal cord regions listed above. In some embodiments, the CRE, CRM, minimal / proximal promoter, or synthetic promoter of the present invention is active in the spinal cord but not in other parts of the CNS, e.g., the brain. In some embodiments, the CRE, CRM, minimal / proximal promoter, or synthetic promoter of the present invention is active in 1, 2, 3, 4, or 5 of the spinal cord regions listed above.In some embodiments, the CRE, CRM, minimal / proximal promoter, or promoter of the present invention is active in most regions of the spinal cord, i.e., at least three, at least four, or all five of the five spinal cord regions listed above.

[0030] In some embodiments, it may be desirable that the CRE, minimal / proximal promoter, or promoter of the present invention exhibits dominant activity in one region of the CNS, and optionally in one region of the brain. Alternatively, it may be desirable that the CRE, minimal / proximal promoter, or promoter of the present invention exhibits activity in one region of the brain, but no activity or minimal activity in the rest of the brain or CNS. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active only in one of the CNS regions of the brain listed above, for example, in the midbrain. In some preferred embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is specifically active in the midbrain (midbrain-specific). In one preferred embodiment, the CRE, minimal / proximal promoter, or promoter of the present invention is specifically active in the midbrain (midbrain-specific), but no activity or minimal activity in other regions of the brain.

[0031] The CRE, minimal / proximal promoter, or promoter of the present invention may be active in various cells of the CNS. The dominant cell types in the brain are neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells. Other cell types may also be present, especially in inflammatory conditions. In some embodiments, it may be desirable for the promoter to be active in a number of different cell types. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in substantially all cells of the CNS (e.g., neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells). In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in at least four CNS cell types from those listed above, e.g., neurons, astrocytes, microglia, and oligodendrocytes. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in at least three CNS cell types from those listed above, e.g., neurons, astrocytes, and oligodendrocytes.

[0032] In some embodiments, it may be desirable for the promoter to be active in a limited number of CNS cell types, or in one or fewer CNS cell types. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in 4, 3, 2, or one or fewer CNS cell types from the CNS cell types listed above. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in two or fewer CNS cell types from the CNS cell types listed above, for example, neurons and oligodendrocytes. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in only one CNS cell type from the CNS cell types listed above, for example, neurons.

[0033] In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in specific subtypes of CNS cells, such as dopaminergic neurons. In some specific preferred embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in dopaminergic neurons. In some preferred embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in dopaminergic neurons but not in other CNS cell types or other CNS cell subtypes. In some preferred embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in GABAergic or glutamatergic neurons. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in specific types of CNS cells or specific subtypes of CNS cells, and in specific regions of the brain.

[0034] The CRE, minimal / proximal promoter, or promoter of the present invention may or may not be active in tissues outside the CNS. Examples of tissues outside the CNS include the heart, liver, kidneys, skeletal muscle, and spleen. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention may not be active, or only minimally active, in tissues or cells outside the CNS. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in ICV delivery in tissues 1, 2, 3, 4 or less among the above-mentioned tissues outside the CNS. In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in IV delivery in tissues 1, 2, 3, 4 or less among the above-mentioned tissues outside the CNS.

[0035] In some embodiments, it may be desirable for the CRE, minimal / proximal promoter, or promoter of the present invention to be active in the CNS but also in other tissues outside the CNS. The CRE, minimal / proximal promoter, or promoter of the present invention may be active in at least one, two, three, four, or five of the above-mentioned tissues outside the CNS during ICV delivery. The CRE, minimal / proximal promoter, or promoter of the present invention may be active in at least one, two, three, four, or five of the above-mentioned tissues outside the CNS during IV delivery.

[0036] In some embodiments, the CRE, minimal / proximal promoter, or synthetic promoter of the present invention may be active in the central nervous system (CNS) and the peripheral nervous system (PNS). When the CRE, minimal / proximal promoter, or synthetic promoter of the present invention is active in the CNS and PNS, it may be referred to as NS-specific. The PNS refers to the part of the nervous system outside the brain and spinal cord. Not limited examples of the peripheral nervous system include the cranial nerves, brachial plexus, thoracogastric nerves, lumbar plexus, sacral plexus, and neuromuscular junction. In some embodiments, it may be desirable for the CRE, CRM, minimal / proximal promoter, or promoter of the present invention to exhibit broad activity in the PNS. In some embodiments, the CRE, CRM, minimal / proximal promoter, or synthetic promoter of the present invention is active in 1, 2, 3, 4, 5, or 6 of the PNS regions listed above. In some embodiments, the CRE, CRM, minimal / proximal promoter, or synthetic promoter of the present invention is active in most of the regions in the PNS, i.e., in at least four, at least five, or all six of the six regions of the PNS listed above.

[0037] In some embodiments, synthetic promoters CNS-5 and CNS-5_v2 are active in the CNS and in most of the regions in the PNS, at least four, at least five, or all six of the six PNS regions listed above. In some embodiments, synthetic promoters CNS-2, CNS-3, and CNS-4 are active in the CNS and at least one of the PNS regions listed above. In some embodiments, synthetic promoters CNS-2, CNS-3, and CNS-4 are active in the CNS and in PNS sympathetic neurons.

[0038] CNS-specific promoters can be expressed in other non-CNS cells. However, they exhibit higher levels of expression in CNS cells, such as neurons in the brain and spinal cord, as well as in non-neuronal or nerve-supporting cells located in the brain and spinal cord. For example, CNS-specific promoters express genes at least 25%, at least 35%, at least 45%, at least 55%, at least 65%, at least 75%, at least 80%, at least 90%, at least 95%, or any integer percentage between 25% and 95% higher in cells located within the CNS, including neurons and non-neuronal cells in the brain and spinal cord, compared to cells located outside the CNS.

[0039] Expression driven by the promoter of the present invention in a desired tissue or cell is observed for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, It could be 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or a period longer than 10 years. Onset may occur between 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

[0040] In a further aspect of the present invention, an expression cassette is provided comprising a synthetic CNS-specific promoter of any aspect of the present invention operably linked to a sequence encoding an expression product. The expression product is, as appropriate, a gene, e.g., a transgene. In some embodiments, the expression product is a therapeutic expression product.

[0041] In further embodiments, vectors comprising a synthetic CNS-specific promoter or expression cassette according to the present invention are provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a gene therapy vector, optionally an AAV vector, an adenovirus vector, a retrovirus vector, a herpes simplex vector, or a lentiviral vector. Lentiviral vectors are widely used as gene transfer tools in the CNS and are known to be able to successfully transduce neurons, astrocytes, and oligodendrocytes (Jakobsson and Lundberg, 2006). They are beneficial because they have relatively high cloning capacity and because viral genes are not expressed. A particularly preferred lentiviral vector system is one based on HIV-1 (Jakobsson and Lundberg, 2006). Herpes simplex virus vectors and adenovirus vectors also show potential for use as gene transfer tools in the CNS because they demonstrate successful transduction of CNS cells, but are less preferred due to their toxicity.

[0042] AAV vectors have been widely discussed in the art. AAV vectors are particularly interesting because they are not typically integrated into the genome and do not induce an immune response. AAV serotypes 1, 2, 4, 5, 8, 9, rh10, DJ8, and 2g9 (AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAVrh10, AAVDJ8, and AAV2g9) have been noted for achieving efficient transduction in the CNS. Therefore, AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAVrh10, AAVVDJ8, AAV2g9, and their derivatives are particularly preferred AAV serotypes. In some embodiments, AAV9 is a particularly preferred AAV vector. In other embodiments, AAV2g9 is a particularly preferred AAV vector (WO2014 / 144229). In yet another embodiment, AAVDJ8 is a particularly preferred AAV vector. In some embodiments, AAVrh10 is a particularly preferred AAV vector. The AAV vector may optionally include a viral genome containing the nucleic acid sequence of the present invention located between two reverse terminal repeat sequences (ITRs). WO2019 / 028306 discloses various wild-type and modified AAV vectors, for example, for use in the CNS. In one embodiment, the AAV vector is capable of crossing the blood-brain barrier after delivery. In one embodiment, the AAV vector of the present invention is a replication-deficient recombinant AAV viral vector lacking sequences encoding functional Rep and Cap proteins within its viral genome. These defective AAV vectors may lack most or all of the parental coding sequences and essentially retain only one or two AAV ITR sequences and the nucleic acid of interest for delivery to cells, tissues, organs, or organisms. The AAV vector for use herein may optionally include a virus reduced to the minimum components necessary for transduction of the nucleic acid payload or cargo of interest. In this method, the AAV vector is manipulated as a medium for specific delivery while lacking the harmful replication and / or integration mechanisms found in wild-type viruses. In one embodiment, the AAV particle of the present invention is scAAV. In another embodiment, the AAV particle of the present invention is ssAAV.Methods for generating and / or modifying AAV particles are widely disclosed in the Art (see, for example, WO2000 / 28004, WO2001 / 23001, WO2004 / 112727, WO2005 / 005610 and WO2005 / 072364, which are incorporated herein by reference). In one embodiment, the AAV vector comprises a capsid that enables translocation across the blood-brain barrier after intravascular (e.g., intravenous or intra-arterial) administration (see, for example, WO2014 / 144229, which discusses capsids or peptide inserts including, for example, capsids manipulated for efficient translocation across the blood-brain barrier, e.g., VOY101, VOY201, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S and their variants).

[0043] Methods for producing AAV vectors are well known in the art, for example, U.S. Patents US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948, or international publications WO1996039530, WO1998010088, WO1999014354, WO1999 / 015685, WO1999 / 047691, WO2000 / 055342, WO2000 / 075353 and WO2001 / 023597; Methods in Molecular Biology, edited by Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J Fir. 63: pp. 3822-38 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: pp. 4646-45 (1991); Ruffing et al., J. Vir. 66: pp. 6922-693 (1992); Kimbauer et al., Vir., 219: pp. 37-44 (1996); Zhao et al., Vir. 272: pp. 382-393 (2000). The disclosures of each of these publications are incorporated herein by reference. Commonly used viral replication cells for generating recombinant AAV virus particles include, but are not limited to, HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.

[0044] In some embodiments, the vector is a non-viral vector using, for example, a cationic polymer or cationic lipid, as is known in the art. Various non-viral vectors are discussed by Selene Ingusci et al. (Gene Therapy Tools for Brain Diseases. Front. Pharmacol. 10:724. doi: 10.3389).

[0045] In further embodiments, vectors according to the present invention, and optionally virions (viral particles) containing a viral vector, are provided. In some embodiments, the virions are AAV virions.

[0046] In a further embodiment, a pharmaceutical composition comprising a synthetic CNS-specific promoter, expression cassette, vector, or virion according to the present invention is provided.

[0047] For example, AAV vector particles can be prepared as pharmaceutical compositions. It will be understood that such compositions will always contain one or more active ingredients, most often pharmaceutically acceptable excipients.

[0048] Pharmaceutical compositions relating to this disclosure may be prepared, packaged, and / or sold in bulk as single-dose units and / or multiple single-dose units. In this specification, “unit dose” means a distinct amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to a favorable proportion of the active ingredient and / or such dose to be administered to a subject, for example, half or one-third of such dose.

[0049] In a further embodiment, synthetic CNS-specific promoters, expression cassettes, vectors, virions, or pharmaceutical compositions according to the present invention for use as pharmaceuticals are provided.

[0050] In a further embodiment, synthetic CNS-specific promoters, expression cassettes, vectors, virions, or pharmaceutical compositions according to the present invention are provided for use in therapy, i.e., in the prevention or treatment of a medical condition or disease.

[0051] Where appropriate, the medical condition or disease is associated with abnormal gene expression, optionally, with abnormal gene expression in CNS tissue or cells. Where appropriate, the use is for gene therapy, preferably for the treatment of diseases involving abnormal gene expression. Where appropriate, the medical condition or disease involving abnormal gene expression may be a disease of the CNS. Where appropriate, the medical condition or disease may be a single gene disorder of the CNS. Where appropriate, gene therapy includes the expression of a therapeutic expression product in CNS cells or tissues. Exemplary medical conditions or diseases relevant to this embodiment are discussed below.

[0052] In further embodiments, cells comprising the synthetic CNS-specific promoter, expression cassette, vector, or virion of the present invention are provided. In some embodiments, the cells are mammalian cells, optionally human cells. Optionally, the cells are CNS cells. Optionally, the cells may be neurons, astrocytes, oligodendrocytes, ependymal cells, or microglial cells. Optionally, the cells may be human neurons, astrocytes, oligodendrocytes, ependymal cells, or microglial cells. The synthetic CNS-specific promoter may be episomal or present in the cell's genome.

[0053] In further embodiments, synthetic CNS-specific CREs, synthetic CNS-specific promoters, expression cassettes, vectors, virions, or pharmaceutical compositions as described herein are provided for use in the manufacture of pharmaceutical compositions for the treatment of medical conditions or diseases. Exemplary medical conditions or diseases relevant to these embodiments are discussed below.

[0054] In a further embodiment, a method is provided for generating an expression product, comprising providing a synthetic CNS-specific expression cassette, vector, or virion of the present invention in CNS cells or tissues, and expressing a target gene present in the synthetic CNS-specific expression cassette, vector, or virion. The method may be in vitro or ex vivo, or may be in vivo.

[0055] In a further embodiment, a method for expressing a therapeutic transgene in CNS cells is provided, comprising the steps of introducing a synthetic CNS-specific expression cassette, vector, or virion as described herein into CNS cells and expressing an expression product (e.g., the gene of interest) present in the synthetic CNS-specific expression cassette, vector, or virion. The CNS cells may be, for example, neurons, astrocytes, oligodendrocytes, ependymal cells, or microglial cells.

[0056] In a further embodiment, a method of therapy for a subject requiring it, preferably a human being, - A step of administering to a target an expression cassette, vector, virion, or pharmaceutical composition as described herein, which includes a sequence encoding a therapeutic product operably linked to a promoter according to the present invention; and - A method is provided which includes the step of expressing a therapeutic amount of the therapeutic product in the target CNS.

[0057] Where appropriate, the method is for the treatment, prevention, relief, or remission of neurological disorders and / or disabilities. Illustrative medical conditions or diseases relevant to this embodiment are discussed below.

[0058] Suitable methods of administration may be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection), including intravenous, intra-arterial, intracranial, intramuscular, subcutaneous, intra-articular, subarachnoid, and intradermal injections. Preferred methods of administration are intravenous, intra-arterial, intracranial, and subarachnoid injections.

[0059] In some embodiments, the method involves introducing an expression cassette, vector, virion, or pharmaceutical composition, as described herein, containing a gene encoding a therapeutic product, into the target CNS. A particular difficulty with respect to introducing an expression cassette, vector, virion, or pharmaceutical composition into the CNS is the blood-brain barrier. The blood-brain barrier is a semipermeable boundary of endothelial cells that prevents certain chemicals and molecules in the bloodstream from entering the extracellular fluid of certain nervous systems. In animal studies, this barrier has been overcome by direct injection into the brain of animals, e.g., intracranial injection, and, where appropriate, intraventricular (ICV) injection (see, e.g., Keiser et al., Curr Protoc Mouse Biol. 2018 Dec;8(4):e57). This method of administration can be difficult to perform and may be dangerous to the subject, and therefore may be disadvantageous for gene therapy in humans.

[0060] Alternatively, in human gene therapy settings, it is preferable that the expression cassette, as described herein, is introduced into the CNS by intravenous or intra-arterial (e.g., intracarotid) administration of a viral vector containing the expression cassette. The viral vector is, as appropriate, an AAV vector. Intravenous or intra-arterial administration of several serotypes of AAV enables the AAV vector to penetrate into the brain. Minimal expression in non-CNS tissues and cells is predicted due to the CNS specificity of the synthetic CNS-specific promoter according to the present invention. Furthermore, the development of improved AAV capsids for CNS penetration is predicted to improve the penetration of the AAV vector. Intravenous or intra-arterial administration is safer and less invasive than intracranial administration while still allowing penetration across the blood-brain barrier.

[0061] Where appropriate, medical condition or disease refers to a medical condition or disease of the CNS, such as neurological disorders and / or disorders. Depending on the context, medical conditions or diseases may be selected from, for example, dopamine transporter deficiency syndrome, attention deficit / hyperactivity disorder (ADHD), bipolar disorder, epilepsy, multiple sclerosis, tauopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Krabbe disease, adrenoleukodystrophy, motor neuron disease, cerebral palsy, Batten disease, Gaucher disease, Tay-Sachs disease, Rett syndrome, Sandhoff disease, Charcot-Marie-Tooth disease, Angelman syndrome, Canavan disease, late-onset childhood neuronal ceroid lipofuscinosis, mucopolysaccharidosis IIIA, mucopolysaccharidosis IIIB, metachromatic leukodystrophy, hereditary lysosomal storage disorders, such as Niemann-Pick disease type C1 and / or neuronal ceroid lipofuscinosis, such as Batten disease, progressive supranuclear palsy, corticobasal syndrome and brain cancer (including astrocytoma and glioblastoma).

[0062] The nucleic acid encoding the expression product may be one of the genes selected from the group consisting of NPC1, EAAT2, NPY, CYP46A1, GLB1, APOE (or APOE2), HEX, CLN1, CLN2, CLN3, CLN4, CLN5, CLN6, SUMF1, DCTN1, PRPH, SOD1, NEFH, GBA, IDUA, NAGLU, GUSB, ARSA, MANB, AADC, GDNF, NTN, ASP, MECP2, PTCHD1, GJB1, UBE3A, HEXA, FXN, and MOG.

[0063] Furthermore, or alternatively, the expression product may be an antibody, an antibody fragment, or an antibody-like scaffold protein.

[0064] Furthermore, the expression product may be a gene editing system directed at the disease allele (e.g., CRISPR-Cas9 system, TALEN, ZFN, etc.).

[0065] Furthermore, or alternatively, the expression product may be one or more regulatory polynucleotides, such as RNA or DNA molecules as therapeutic agents. For example, the regulatory polynucleotide may be miRNA or siRNA. The target gene may be any gene associated with any neurological disease, for example, but not limited to those listed herein. For example, siRNA double-stranded or dsRNA encoding a target gene may reduce or silence the expression of the target gene in CNS cells, thereby alleviating the symptoms of the neurological disease. In one example, the target gene is huntingtin (HTT). In another example, the target gene is microtubule-associated protein tau (MAPT).

[0066] In a further embodiment, a synthetic CNS-specific promoter comprising or comprising SEQ ID NO: 1 or SEQ ID NO: 21 is provided. The synthetic CNS-specific promoter may, when administered by ICV injection, promote broad intracranial expression of an expression product operably linked to the CNS-specific promoter. The synthetic CNS-specific promoter may, when, be active in at least six regions of the brain. The synthetic CNS-specific promoter may, when administered by ICV injection, promote CNS-specific expression of synapsin in the brain at levels of at least 100%, 150%, or 200% compared to synapsin-1 (SEQ ID NO: 14). The synthetic CNS-specific promoter may, when administered by ICV injection, promote expression in the cortex and hippocampus.

[0067] In a further embodiment, a method for expressing an expression product in the CNS is provided, comprising introducing an expression cassette into CNS cells containing a synthetic CNS-specific promoter comprising or operably linked to the expression product, either SEQ ID NO: 1 or SEQ ID NO: 21. The expression cassette is optionally introduced into the CNS by ICV injection, and the expression product is widespread in the brain. The expression of the expression product in the brain is optionally in at least six brain regions. The synthetic CNS-specific promoter can optionally promote CNS-specific expression of the expression product at levels of at least 100%, 150%, or 200% compared to synapsin-1 (SEQ ID NO: 14) in the brain. The expression cassette is optionally introduced into the CNS by ICV injection, and the expression product is expressed in the cortex and hippocampus.

[0068] In further embodiments, synthetic CNS-specific promoters are provided that include or comprise functional variants thereof, such as SEQ ID NO: 2, SEQ ID NO: 25, or SEQ ID NO: 7, as discussed above. Optionally, such synthetic CNS-specific promoters can promote broad brain expression of an expression product from a nucleic acid operably linked to the CNS-specific promoter when administered by ICV injection. Optionally, the synthetic CNS-specific promoter is active in at least six brain regions. Optionally, synthetic CNS-specific promoters that include or comprise SEQ ID NO: 2 or a functional variant thereof can promote broad intracranial expression of an expression product operably linked to the CNS-specific promoter when administered by IV injection. Optionally, synthetic CNS-specific promoters that include or comprise SEQ ID NO: 2 or a functional variant thereof do not express promoter in the midbrain. Optionally, synthetic CNS-specific promoters that include or comprise SEQ ID NO: 7, SEQ ID NO: 25, or a functional variant thereof can promote expression in the cortex, hippocampus, and midbrain when administered by IV injection.

[0069] In a further embodiment, a method for expressing an expression product in the CNS is provided, comprising introducing an expression cassette into CNS cells, the cassette comprising a synthetic CNS-specific promoter comprising or comprising SEQ ID NO: 2 or a functional variant thereof, SEQ ID NO: 25 or a functional variant thereof, or SEQ ID NO: 7 or a functional variant thereof, operably linked to a nucleic acid encoding the expression product. The expression cassette is optionally introduced into the CNS by ICV injection, and the expression of the expression product is widespread in the brain. Optionally, the expression of the expression product in the brain is in at least six brain regions as discussed above. Optionally, an expression cassette comprising or comprising SEQ ID NO: 2 or a functional variant thereof is introduced into the CNS by IV injection, and the expression of the expression product is widespread in the brain but not widespread in the midbrain. Optionally, an expression cassette comprising or comprising SEQ ID NO: 7 or SEQ ID NO: 25 or a functional variant thereof is introduced into the CNS by IV injection, and the expression of the expression product is expressed in the cortex, hippocampus and midbrain, but not in the midbrain.

[0070] In a further embodiment, synthetic CNS-specific promoters are provided that include or comprise functional variants thereof of SEQ ID NO: 3, SEQ ID NO: 22, or SEQ ID NO: 4, as discussed above. The synthetic CNS-specific promoter may, as appropriate, promote expression in the cortex and hippocampus when administered by ICV injection. The synthetic CNS-specific promoter may, as appropriate, be inactive or minimally active in other areas of the brain. The synthetic CNS-specific promoter, as appropriate, may include or comprise SEQ ID NO: 3 or its functional variant, SEQ ID NO: 22 or its functional variant, or SEQ ID NO: 4 or its functional variant, when administered by IV injection, promote expression in the cortex, striatum, and hippocampus. The synthetic CNS-specific promoter, as appropriate, may include or comprise SEQ ID NO: 4 or SEQ ID NO: 22 or its functional variants, further promote expression in the midbrain.

[0071] In a further embodiment, a method for expressing an expression product in the CNS is provided, comprising introducing an expression cassette into CNS cells, the cassette comprising a synthetic CNS-specific promoter comprising or consisting of SEQ ID NO: 3 or a functional variant thereof, SEQ ID NO: 22 or a functional variant thereof, or SEQ ID NO: 4 or a functional variant thereof, operably linked to a nucleic acid encoding the expression product. The expression cassette is optionally introduced into the CNS by ICV injection, and the expression product is expressed in the cortex and hippocampus. The expression is optionally minimal in other areas of the brain. The expression cassette is optionally introduced into the CNS by ICV injection, and the expression product is expressed in the cortex and hippocampus. The expression cassette is optionally introduced into the CNS by IV injection, and the expression product is expressed in the cortex, striatum, and hippocampus.

[0072] In a further embodiment, synthetic CNS-specific promoters are provided that include or consist of SEQ ID NO: 5 or SEQ ID NO: 23 or functional variants thereof, as discussed above. The synthetic CNS-specific promoters may, as appropriate, promote expression in the cortex, striatum, hippocampus, and midbrain. The synthetic CNS-specific promoters may, as appropriate, be inactive or minimally active in other areas of the brain. The synthetic CNS-specific promoters may, as appropriate, be administered by ICV injection.

[0073] In a further embodiment, a method for expressing an expression product in the CNS is provided, comprising introducing an expression cassette into CNS cells, the cassette comprising a synthetic CNS-specific promoter comprising SEQ ID NO: 5 or SEQ ID NO: 23 or a functional variant thereof, operably linked to a nucleic acid encoding the expression product, or comprising such a promoter. The expression cassette is optionally introduced into the CNS by ICV injection. The expression product is optionally expressed in the cortex, striatum, hippocampus, and midbrain. Optionally, expression is minimal in other areas of the brain.

[0074] In further embodiments, synthetic CNS-specific promoters are provided that include or comprise SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 8, or their functional variants, as discussed above. The synthetic CNS-specific promoters may, as appropriate, promote expression in the hippocampus, cortex, and midbrain when administered by ICV injection. The synthetic CNS-specific promoters may, as appropriate, include or comprise SEQ ID NO: 6, SEQ ID NO: 24, or their functional variants, and promote expression in the hippocampus, midbrain, and cerebellum when administered by IV injection. The synthetic CNS-specific promoters may, as appropriate, include or comprise SEQ ID NO: 8, SEQ ID NO: 26, or their functional variants, and promote expression in the hippocampus and midbrain when administered by IV injection. The synthetic CNS-specific promoters may, as appropriate, be inactive or minimally active in other areas of the brain. The synthetic CNS-specific promoters may, as appropriate, include or comprise SEQ ID NO: 6 or its functional variants, SEQ ID NO: 24 or its functional variants, SEQ ID NO: 26 or its functional variants, or SEQ ID NO: 8 or its functional variants, and are primarily active in neurons. Synthetic CNS-specific promoters, which may include or consist of SEQ ID NO: 8 or SEQ ID NO: 26 or their functional variants, are primarily active in dopaminergic neurons.

[0075] In a further embodiment, a method for expressing an expression product in the CNS is provided, comprising introducing an expression cassette into CNS cells containing a synthetic CNS-specific promoter comprising or comprising SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 8, operably linked to a nucleic acid encoding the expression product. The expression cassette is optionally introduced into the CNS by ICV injection, and the expression product is expressed in the hippocampus, cortex, and midbrain. Alternatively, an expression cassette comprising or comprising SEQ ID NO: 6 or SEQ ID NO: 24 or functional variants thereof is introduced into the CNS by IV injection, and the expression product is expressed in the hippocampus, midbrain, and cerebellum. Alternatively, an expression cassette comprising or comprising SEQ ID NO: 8 or SEQ ID NO: 26 or functional variants thereof is introduced into the CNS by IV injection, and the expression product is expressed in the hippocampus and midbrain. Alternatively, the expression is minimal in other areas of the brain.

[0076] In a further embodiment, a method for expressing an expression product in dopaminergic neurons is provided, comprising introducing a synthetic CNS-specific expression cassette into the dopaminergic neurons by IV injection, wherein the CNS-specific expression cassette comprises SEQ ID NO: 8 or SEQ ID NO: 26 or a functional variant thereof. [Brief explanation of the drawing]

[0077] [Figure 1A] This figure shows the expression pattern of the DAT1 / SLC6A3 gene in coronal sections obtained from adult mouse brains (obtained from the Alan mouse brain atlas; mouse.brain-map.org). DAT1 / SLC6A3 is highly expressed in the midbrain. [Figure 1B] This figure shows the expression pattern of the UBE3A gene in coronal sections obtained from adult mouse brains (taken from the Alan mouse brain atlas; mouse.brain-map.org). UBE3A is widely expressed in the brain. [Figure 2A]This figure shows the intracranial distribution in sagittal section of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4) delivered by ICV and IV, as well as the transgene GFP under the control of the control promoter hSyn1. The scale bar is 1 mm. [Figure 2B] This figure shows the intracranial distribution of GFP, a transgene regulated by CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26), delivered via ICV and IV, in a sagittal section. The scale bar represents 1 mm. [Figure 3A] This figure shows the intracranial distribution of transgene GFP under the control of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4) delivered by ICV in a coronal section. The scale bar is 1 mm. [Figure 3B] This figure shows the intracranial distribution in the coronal section of the transgene GFP delivered by ICV: CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26), as well as the transgene GFP under the control of the control promoter hSyn1. The scale bar represents 1 mm. [Figure 4A] This figure shows the intracranial distribution of transgenes GFP under the control of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4) delivered by IV in a coronal section. The scale bar is 1 mm. [Figure 4B] This figure shows the intracranial distribution of GFP, a transgene regulated by CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26), delivered via IV, in a coronal section. The scale bar represents 1 mm. [Figure 5A]This figure shows the intracranial distribution of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4), delivered by ICV, and the transgene GFP under the control of the control promoter hSyn1, in various parts of the brain at higher magnification. The scale bar is 100 μm. [Figure 5B] This figure shows the intracranial distribution of CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26), delivered by ICV, and the transgene GFP under the control of the control promoter hSyn1, in various parts of the brain at higher magnification. The scale bar is 100 μm. [Figure 6A] This figure shows the higher-magnification intracranial distribution of the transgene GFP in various brain regions under the control of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4) delivered by IV, as well as in uninjected control. The scale bar is 100 μm. [Figure 6B] This figure shows the higher-magnification intracranial distribution of transgene GFP in various brain regions under the control of CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26) delivered by IV, as well as in uninjected control. The scale bar is 100 μm. [Figure 7A] This figure shows the in vivo distribution in the midbrain of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4) delivered by ICV, as well as the transgene GFP under the control of the control promoter hSyn1. The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows two overlays with the nuclear dye DAPI. The scale bar is 25 μm. [Figure 7B]This figure shows the in vivo distribution of the regulated transgenes GFP in the midbrain, delivered by ICV to CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26). The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows two overlays with the nuclear dye DAPI. The scale bar is 25 μm. [Figure 8A] This figure shows the in vivo distribution of the transgene GFP in the midbrain under control of CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), and CNS-4 (SEQ ID NO: 4) delivered by IV, as well as in uninjected control cells. The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows two overlays with the nuclear dye DAPI. The scale bar is 25 μm. [Figure 8B] This figure shows the in vivo distribution of the regulated transgenes GFP in the midbrain, delivered via IV to CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), and CNS-8 (SEQ ID NO: 26). The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows two overlays with the nuclear dye DAPI. The scale bar is 25 μm. [Figure 9] This figure shows the in vivo distribution of the transgene GFP in various tissues under the control of CNS-1~8 (SEQ ID NOs: 1~4, 23~26) and the control promoter synapsin 1 (SEQ ID NO: 14), delivered by ICV or IV. RNA extracted from organs throughout the body was converted to RNA and quantified by qPCR. Various promoters of CNS-1~8 (SEQ ID NOs: 1~4, 23~26) showed off-target expression in the liver, kidney, heart, skeletal muscle, or spleen. [Figure 10]This figure shows the percentage of GFP immunoreactivity in various brain regions after ICV or IV delivery of GFP driven by CNS 1-8 (SEQ ID NOs: 1-4, 23-26) or synapsin-1 (SEQ ID NO: 14). Data were obtained by quantitative measurement of 10 non-overlapping RGB images of GFP staining intensity by threshold analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at ×40 magnification across separate brain regions while maintaining constant settings. Foreground immunostaining was defined by averaging the highest and lowest signals. Data were expressed as the mean area percentage of immunoreactivity per field for each region of interest (n=3). With ICV delivery, expression is highest in the cortical and hippocampal brain regions. CNS 1-8 (SEQ ID NOs: 1-4, 23-26) show higher expression in the hippocampus than hSyn1 control. CNS-1 (SEQ ID NO: 1) shows higher expression in the hippocampus, midbrain, and cerebellum compared to hSyn1 with ICV delivery. [Figure 11] This figure shows GFP expression under the control of CNS-1 (SEQ ID NO: 1) during ICV delivery. The magnification is ×40. NeuN is a marker for neuronal nuclei. GFAP is a marker for astrocytes, and IBA1 is a marker for microglia. GFP expression driven by the CNS-1 (SEQ ID NO: 1) promoter is primarily from neurons. [Figure 12] This figure shows the intracranial distribution of CNS-8 (SEQ ID NO: 26) delivered by ICV and IV, and the transgene GFP under the control of the control promoter hSyn1, in a sagittal section. The scale bar is 1 mm. [Figure 13A] This figure shows the intracranial distribution of CNS-8 (SEQ ID NO: 26) delivered by ICV and the transgene GFP under the control of the control promoter hSyn1 in a coronal section. On the left, the scale bar is 1 mm. On the right, brain regions are shown at a higher magnification. The scale bar is 100 μm. [Figure 13B]This figure shows the intracranial distribution of CNS-8 (SEQ ID NO: 26) delivered by IV and the transgene GFP under the control of the control promoter hSyn1 in a coronal section. On the left, the scale bar is 1 mm. On the right, brain regions are shown at a higher magnification. The scale bar is 100 μm. [Figure 14A] This figure shows the in vivo distribution of the transgene GFP in the midbrain under the control of CNS-8 (SEQ ID NO: 26) delivered by ICV (upper) and IV (lower). The left column shows TH+ positive cells (dopaminergic neurons), the middle column shows GFP expression, and the right column shows two overlays with the nuclear dye DAPI. The scale bar is 25 μm. [Figure 14B] This figure shows the quantification of the percentage of dopaminergic neurons expressing GFP (TH+GFP+ cells) out of all dopaminergic neurons. The left portion of the graph shows the percentage of dopaminergic neurons expressing GFP under the control of the control promoter Syn-1 during ICV and IV delivery. The middle portion of the graph shows the quantification of the percentage of dopaminergic neurons expressing GFP under the control of CNS-8 (SEQ ID NO: 26) when low doses were administered during ICV and IV delivery (Example 1). The right portion of the graph shows the percentage of dopaminergic neurons expressing GFP under the control of CNS-8 (SEQ ID NO: 26) when high doses were administered during ICV and IV delivery (Example 2). [Figure 15] This figure compares the in vivo distribution of CNS-8 (SEQ ID NO: 26)-regulated transgene GFP in various tissues after low and high dose administration. The left side shows the in vivo distribution of CNS-8 (SEQ ID NO: 26)-regulated GFP after low dose administration, and the right side shows the in vivo distribution of CNS-8 (SEQ ID NO: 26)-regulated GFP after high dose administration. The data for the in vivo distribution of CNS-8 (SEQ ID NO: 26)-regulated GFP after low dose administration is the same as the data shown in Figure 9. RNA extracted from organs throughout the body was converted to RNA and quantified by qPCR. [Figure 16A]This figure shows the expression pattern of the faf1 gene in mouse PNS neurons obtained from single-cell transcriptome data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. faf1 is expressed in a large number of PNS neurons. [Figure 16B] This figure shows the expression pattern of the pitx3 gene in PNS neurons obtained from single-cell transcriptome data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. pixt3 is expressed in sympathetic PNS neurons. [Modes for carrying out the invention]

[0078] Various CREs and their functional variants that can be used in the construction of CNS-specific promoters are disclosed herein. Where appropriate, the CREs are CNS-specific. These CREs generally originate from genome promoter and enhancer sequences, but are used herein in contexts quite different from their natural genomic environment. Generally, CREs constitute small parts of fairly large genomic regulatory domains that control the expression of the genes they normally associate with. Surprisingly, we have found that many of these CREs are extremely small, can be isolated from their normal environment, and retain their CNS-specific regulatory activity. This is surprising because the recovery of regulatory sequences from the complex "three-dimensional" native state of the genome often results in a significant loss of activity, and therefore there is no reason to expect a given CRE to retain the observed level of activity once recovered from its natural environment. It is even more surprising when CREs retain CNS-specific activity in AAV vectors. This is especially true when AAV vectors contain reverse-terminal repeats (ITRs), have a different DNA structure compared to the genome, and both ITRs and DNA structure are known to affect CRE activity.

[0079] It is noteworthy that the sequence of CRE in this invention can be modified without causing a substantial loss of activity. Functional variants of CRE can be prepared by modifying the sequence of CRE, provided that modifications that are significantly detrimental to the activity of CRE are avoided. Considering the information provided in this disclosure, modifications of CRE to provide functional variants are straightforward. Furthermore, this disclosure provides a methodology for easily evaluating the functionality of any given CRE variant.

[0080] The relatively small size of certain CREs according to the present invention is advantageous because it allows the CRE, more specifically the promoter containing it, to be provided in the vector while occupying only a minimal amount of the vector's payload. This is particularly important when the CRE is used in a vector with limited capacity, such as an AAV-based vector.

[0081] The CRE of this invention includes certain CNS-specific TFBSs. Generally, in functional variants of CRE, it is desirable that these CNS-specific TFBSs retain their functionality. Those skilled in the art are well aware that TFBS sequences can change while retaining functionality. In consideration of this, the sequences of TFBSs are usually exemplified by consensus sequences, which typically exhibit some degree of variation. Further information about the variation occurring in TFBSs can be exemplified using a frequency-based position-weight matrix (PWM), which is used to determine that a given nucleotide is typically found at a given position in the consensus sequence. Details of TF consensus sequences and associated position-weight matrices can be found, for example, in the Jaspar or Transfac databases (http: / / jaspar.genereg.net / and http: / / gene-regulation.com / pub / databases.html). This information allows those skilled in the art to modify the sequences in any given TFBS of CRE in a way that retains, and in some cases even increases, the functionality of the CRE. With this in mind, those skilled in the art have ample guidance on how to modify a TFBS for any given TF while maintaining its ability to bind to a desired TF; the Jaspar system, for example, scores estimated TFBS based on their similarity to a given PWM. Furthermore, all TFBS can be identified / analyzed by scanning the CRE for all PWMs from the JASPAR database. Those skilled in the art can, of course, find additional guidance in the literature and, furthermore, use routine experimental methods to confirm TF binding to estimated TFBS in any variant CRE. It will become clear that significant sequence modifications in the CRE are possible, even within the TFBS in the CRE, while retaining functionality.

[0082] The CRE of the present invention can be used in combination with various suitable minimal promoters or CNS-specific proximal promoters.

[0083] Functional variants of CRE include sequences that differ from the reference CRE element but substantially retain its activity as a CNS-specific CRE. It will be apparent to those skilled in the art that it is possible to alter the sequence of CRE while retaining its ability to recruit suitable CNS-specific transcription factors (TFs) and thereby enhance their expression. Functional variants of CRE may include substitutions, deletions, and / or insertions compared to the reference CRE, provided that the CRE is not substantially rendered non-functional.

[0084] In some embodiments, a functional variant of a CRE can be considered a CRE that substantially retains its activity when substituted in place of a reference CRE in a promoter. For example, a CNS-specific promoter containing a given functional variant of a CRE preferably retains at least 80% of its activity, more preferably at least 90%, more preferably at least 95%, and even more preferably 100% of its activity (compared to a reference promoter containing an unmodified CRE).

[0085] Where appropriate, the functional variant of the CRE maintains a significant level of sequence identity with respect to the reference CRE. Where appropriate, the functional variant includes sequences that are at least 70% identical to the reference CRE, more preferably at least 80%, 90%, 95%, or 99% identical to the reference CRE.

[0086] The retention of activity can be assessed by comparing the expression of a suitable reporter under the control of a reference promoter with that of an otherwise identical promoter containing a substituted CRE under equivalent conditions. Suitable assays for evaluating CNS-specific promoter activity are disclosed herein, for example, in the examples.

[0087] In some embodiments, a CRE can be combined with one or more additional CREs to create a cis-regulation module (CRM). Additional CREs may be provided upstream or downstream of a CRE according to the present invention. The additional CREs may be any of the CREs disclosed herein, or they may be other CREs. The additional CREs may, as appropriate, be CNS-specific.

[0088] A CRE according to the present invention or a CRM containing a CRE according to the present invention may include one or more additional regulatory elements. For example, they may include inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, and splicing elements, provided that they do not substantially render the CRE or CRM non-functional.

[0089] The inclusion of a CRE according to the present invention may include a spacer between the CRM and the least or proximal promoter and / or between the CRE. Furthermore, or alternatively, the spacer may be present at the 5' end of the CRM.

[0090] It will be apparent that, in order to provide a synthetic CNS-specific promoter according to the present invention, a CRE according to the present invention or a CRM containing a CRE according to the present invention or a functional variant thereof can be combined with any suitable promoter element. The promoter element is preferably a CNS-specific proximal promoter.

[0091] In many cases, shorter promoter sequences are preferred, especially for use in situations where the vector (e.g., a viral vector such as AAV) has a limited capacity. Therefore, in some embodiments, synthetic CNS-specific CRMs comprising at least one CRE following SEQ ID NOs. 9-11, 28-31 or their functional variants have a length of 1000 or fewer nucleotides, for example, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50 or fewer nucleotides.

[0092] Synthetic CNS-Specific Promoters and Their Functional Variants Various synthetic CNS-specific promoters are disclosed herein. A functional variant of a reference synthetic CNS-specific promoter is a promoter that differs from the reference synthetic CNS-specific promoter but substantially retains CNS-specific promoter activity. It will be understood by those skilled in the art that the sequence of a synthetic CNS-specific promoter can be modified while retaining the ability to recruit a suitable CNS-specific transcription factor (TF) and RNA polymerase II to provide CNS-specific expression of a operably linked sequence (e.g., an open reading frame). Functional variants of a synthetic CNS-specific promoter may include substitutions, deletions and / or insertions compared to the reference promoter, provided that such substitutions, deletions and / or insertions do not render the synthetic CNS-specific promoter substantially non-functional compared to the reference promoter.

[0093] Therefore, in some embodiments, a functional variant of a synthetic CNS-specific promoter can be considered as a variant that substantially retains the CNS-specific promoter activity of a reference promoter. For example, a functional variant of a synthetic CNS-specific promoter preferably retains at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 100% of the activity of the reference promoter.

[0094] Functional variants of synthetic CNS-specific promoters often retain a significant level of sequence similarity to a reference synthetic CNS-specific promoter. In some embodiments, the functional variant includes a sequence that is at least 70% identical to the reference synthetic CNS-specific promoter, and more preferably at least 80%, 90%, 95%, or 99% identical to the reference synthetic CNS-specific promoter.

[0095] The activity of a functional variant can be evaluated by comparing the expression of a suitable reporter under the control of a reference synthetic CNS-specific promoter with that of the putative functional variant under equivalent conditions. Suitable assays for evaluating CNS-specific promoter activity are disclosed herein, for example, in the examples.

[0096] A given functional variant of a synthetic CNS-specific promoter may include functional variants of CRE present in the reference synthetic CNS-specific promoter. A given functional variant of a synthetic CNS-specific promoter may include functional variants of promoter elements or different promoter elements compared to the reference synthetic CNS-specific promoter.

[0097] A given functional variant of a synthetic CNS-specific promoter may include one or more additional CREs (Chemical Enzymes) compared to those present in the reference synthetic CNS-specific promoter. These additional CREs may be provided, for example, upstream of or downstream of the CREs present in the reference synthetic CNS-specific promoter. These additional CREs may be the CREs disclosed herein, or they may be other CREs.

[0098] A given functional variant of a synthetic CNS-specific promoter may include additional spacers between adjacent elements (CRE, CRM, or promoter elements), or, if one or more spacers are present in the reference synthetic CNS-specific promoter, those spacers may be longer or shorter than those in the reference synthetic CNS-specific promoter.

[0099] It will become clear that the synthetic CNS-specific promoter of the present invention may include the CRE of the present invention or a CRM containing the CRE of the present invention and additional regulatory sequences. For example, they may include one or more additional CREs, inducible or repressible elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, and splicing elements, provided that they do not substantially render the promoter non-functional.

[0100] In some embodiments, the CNS-specific promoter described above is operably ligated to one or more additional regulatory sequences. These additional regulatory sequences can enhance expression compared to, for example, a CNS-specific promoter that is not operably ligated to additional regulatory sequences. Generally, it is preferable that the additional regulatory sequences do not substantially reduce the specificity of the CNS-specific promoter.

[0101] For example, a CNS-specific promoter according to the present invention can be operably linked to a sequence encoding a UTR (e.g., 5' and / or 3'UTR) and / or an intron or the like.

[0102] In some embodiments, a CNS-specific promoter is operably ligated to a sequence encoding a UTR, for example, a 5'UTR. The 5'UTR may contain various elements capable of regulating gene expression. In native genes, the 5'UTR begins at the transcription start site and ends one nucleotide before the start codon of the coding region. It should be noted that the 5'UTR referred to herein may be the entire naturally occurring 5'UTR or a portion of the naturally occurring 5'UTR. The 5'UTR may also be partially or entirely synthetic. In eukaryotes, the 5'UTR has a median length of approximately 150 nucleotides, but can be considerably longer in some cases. Regulatory sequences that can be found in the 5'UTR include, but are not limited to: - Protein binding sites that may affect mRNA stability or translation, - Riboswitch, - Sequences that promote or inhibit translation initiation, and - Introns within the 5'UTR are associated with the regulation of gene expression and mRNA export.

[0103] When a regulatory sequence contains both a 5'UTR and an intron, it is sometimes referred to as a 5'UTR and intron.

[0104] In some embodiments, the synthetic CNS-specific promoter described above is operably ligated to sequences encoding the 5'UTR and introns. In some embodiments, the 5'UTR and introns are derived from the CMV major immediate gene (CMV-IE gene). For example, the 5'UTR and introns derived from the CMV-IE gene may include CMV-IE gene exon 1 and CMV-IE gene exon 1 or a portion thereof.

[0105] In some embodiments, the promoter element can be modified to accommodate linking to the 5'UTR, for example, by removing the sequence downstream of the transcription start site (TSS) in the promoter element (e.g., replacing it with the 5'UTR).

[0106] The CMV-IE 5'UTR and intron are described by reference in Simari et al., Molecular Medicine 4: pp. 700-706, 1998, "Requirements for Enhanced Transgene Expression by Untranslated Sequences from the Human Cytomegalovirus Immediate-Early Gene," which is incorporated herein by reference. Variants of the CMV-IE 5'UTR and intron sequences discussed by Simari et al. are also described by reference in WO2002 / 031137, which also uses the regulatory sequences disclosed therein.

[0107] Other regulatory elements that can be used in combination with the promoter, such as other UTRs, are known in the Art of this, for example, in Leppek, K., Das, R. & Barna, M., "Functional 5'UTR mRNA structures in eukaryotic translation regulation and how to find them," Nat Rev Mol Cell Biol 19, pp. 158-174 (2018), which is incorporated herein by reference.

[0108] In some embodiments, one of the CNS-specific promoters or variants thereof described herein is ligated to a sequence encoding the 5'UTR and / or the 5'UTR and an intron.

[0109] In some embodiments, the sequence encoding the 5'UTR and intron includes SEQ ID NO: 27 or a functional variant thereof. In some embodiments, the functional variant may have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. SEQ ID NO: 27 encodes the CMV-IE 5'UTR and intron.

[0110] [ka]

[0111] In some embodiments, the CNS-specific promoter CNS-1 (SEQ ID NO: 1) is operably coupled to the CMV-IE 5'UTR and an intron (SEQ ID NO: 27) to provide SEQ ID NO: 21.

[0112] In some embodiments, the CNS-specific promoter CNS-4 (SEQ ID NO: 4) is operably coupled to the CMV-IE 5'UTR and intron (SEQ ID NO: 27) to provide SEQ ID NO: 22.

[0113] In some embodiments, any of the CNS-specific promoters CNS-2, CNS-3, CNS-5, CNS-5_v2, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are operably linked to the CMV-IE 5'UTR and intron (SEQ ID NO: 27).

[0114] The preferred synthetic CNS-specific promoters of the present invention exhibit CNS-specific promoter activity that is at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity shown by the synapsin-1, Camk2a, or NSE promoter in CNS cells. While higher levels of promoter activity are often preferred, this is not always the case, and therefore, in some instances, more moderate levels of expression may be preferable. In some cases, it is desirable to have a variety of promoters with different levels of activity available to allow for matching the level of expression to specific requirements, and this disclosure provides promoters with such a variety of activities. The activity of a given synthetic CNS-specific promoter of the present invention, compared to Syn-1, can be evaluated by comparing the CNS-specific expression of a reporter gene under the control of the synthetic CNS-specific promoter with the expression of the same reporter gene under the control of the Syn-1 promoter, provided that the two promoters are otherwise provided in equivalent expression constructs and under equivalent conditions.

[0115] In addition to different activity levels, it is desirable in some cases to have various promoters that are active in different regions of the brain. Therefore, it may be desirable to have various promoters that have different activity levels across different regions of the brain in order to enable the level of expression to be tailored to the requirements, and this disclosure provides promoters with such diverse activity. In some cases, expression in a specific region of the brain is desired. In some embodiments, expression in a specific region of the brain is desired, and there is little to no expression in the rest of the brain. This may be the case, for example, in the treatment of diseases such as dopamine transporter deficiency syndrome in which expression is desired in the midbrain. In some preferred embodiments, the CNS-specific promoter according to the present invention is active in the midbrain. In some preferred embodiments, the CNS-specific promoter according to the present invention is active in the midbrain and has little to no activity in other regions of the brain. In some preferred embodiments, the CNS-specific promoter according to the present invention is active in dopaminergic neurons. In some embodiments, the CNS-specific promoter according to the present invention is active in dopaminergic neurons and has little to no expression in other CNS cell types or CNS subtypes. The preferred synthetic CNS-specific promoters of the present invention exhibit dopaminergic neuron-specific promoter activity that is at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity exhibited by tyrosine hydroxylase in dopaminergic neurons. The activity of a given synthetic CNS-specific promoter of the present invention compared to tyrosine kinase can be evaluated by comparing the dopaminergic neuron-specific expression of a reporter gene under the control of the synthetic CNS-specific promoter with the expression of the same reporter under the control of a tyrosine hydroxylase promoter in dopaminergic neurons, provided that the two promoters are otherwise provided in equivalent expression constructs and under equivalent conditions.In some embodiments, the synthetic CNS-specific promoter of the present invention can increase the expression of a gene (e.g., a therapeutic gene or target gene) in a target dopaminergic neuron by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300%, at least 500%, at least 1000%, or more, compared to a known dopaminergic neuron-specific promoter, and optionally a tyrosine hydroxylase promoter.

[0116] Alternatively, widespread expression in all or almost all areas of the brain may be preferable. This may be the case, for example, in the treatment of diseases such as Angelman syndrome, where widespread expression throughout the entire brain is necessary.

[0117] In some embodiments, the synthetic CNS-specific promoters of the present invention can increase the expression of a gene (e.g., a therapeutic gene or a gene of interest) in the target CNS or in CNS cells by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300%, at least 500%, at least 1000%, or more, compared to a known CNS-specific promoter, optionally Syn1, Camk2a, or NSE promoter.

[0118] The preferred synthetic CNS-specific promoter of the present invention exhibits activity in non-CNS cells (e.g., Huh7 and HEK293 cells) at a level of 50% or less compared to CMV-IE, preferably 25% or less of CMV-IE, more preferably 10% or less of CMV-IE, and in some cases, at a level of 5% or less of CMV-IE or 1% or less of CMV-IE.

[0119] In many cases, shorter promoter sequences are preferred, especially when used in situations where the vector (e.g., a viral vector such as AAV) has a limited capacity. Therefore, in some embodiments, the synthetic CNS-specific promoter has a length of 1000 or fewer nucleotides, for example, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100 or fewer nucleotides.

[0120] Particularly preferred synthetic CNS-specific promoters are those that are both short and exhibit high levels of activity.

[0121] It is known that the ITR and DNA structure of AAV vectors, which differ from those of the genome, affect promoter activity. Often, the ITR and DNA structure negatively impact promoter activity, so it is surprising that a CNS-specific promoter retains CNS-specific activity in an AAV vector.

[0122] Synthetic CNS-Specific Expression Cassette The present invention also provides a synthetic CNS-specific expression cassette comprising the synthetic CNS-specific promoter of the present invention operably linked to a sequence encoding an expression product, optionally a gene (e.g., a transgene).

[0123] When a gene codes for a protein, it can be essentially any type of protein. Examples, though not limited to, include enzymes, antibodies or antibody fragments (e.g., monoclonal antibodies), viral proteins (e.g., REP-CAP, REV, VSV-G, or RD114), therapeutic proteins, or toxic proteins (e.g., caspases 3, 8, or 9).

[0124] In some preferred embodiments of the present invention, the gene optionally encodes a therapeutic expression product, preferably a therapeutic polypeptide, suitable for use in the treatment of diseases or conditions associated with abnormal gene expression in the CNS.

[0125] In some embodiments, therapeutic expression products include those useful in the treatment of CNS diseases. The term "CNS disease" is, in principle, understood by those skilled in the art. This term relates to diseases suitable for treatment and / or prevention by administration of active compounds to the CNS, and in particular to CNS cells. In some embodiments, CNS disease is a neurological disease and / or disorder.

[0126] Examples of CNS disorders that are not limited to these include: septal pellucidum defect, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupil, Addie syndrome, adrenoleukodystrophy, corpus callosum agenesis, agnosia, Eicardi syndrome, Eicardi-Gutierre syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, and alternating hemiplegia. Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, telangiectatic ataxia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic nervous system Dermatitis, back pain, Birth syndrome, Batten disease, Becker myotonia, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhard Roth syndrome, Binswanger's disease, blepharospasm, Bloch-Salzberger syndrome, brachial plexus injury during childbirth, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysm, brain injury, Brown-Séquard syndrome, globus Spinal muscular atrophy, autosomal dominant cerebral arteriovenous disease with subcortical infarction and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cabernoma, cavernous hemangioma, cavernous vascular malformation, central cervical syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelin disintegration, cranial disorders, ceremidase deficiency, cerebellar degeneration, cerebellar dysplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation (Cerebral Cavemous malformation, cerebral gigantism, hypoxic encephalopathy, cerebral palsy, cerebro-ocular-facial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disorder, chorea, acanthocyanotic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic dysregulation, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colposphecy, coma, complex regional pain syndrome, congenital bilateral facial palsy, congenital myasthenia gravis, congenital myopathy, congenital cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis,Creutzfeldt-Jakob disease, cumulative traumatic injury, Cushing's syndrome, giant cell inclusion body disease, cytomegalovirus infection, dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, Demorsia syndrome, Dejurine-Klumpke palsy, dementia, multiple stroke dementia, semantic dementia, subcortical dementia, Lewy body dementia, dentate nucleocerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental apraxia, Devic syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, autonomic nervous system disorders, dysgraphia, dyslexia, dysphagia, dysphagia, dyscokinetic disorders, myoclonus cerebellar synergy disorder, progressive cerebellar synergy disorder, dystonia, early infantile epileptic encephalopathy, empty cell syndrome, encephalitis, encephalitis lethargica, brain herniation, encephalopathy, encephalopathy (familial neonatal), trigeminal nerve hemangioma, epilepsy, epilepsy Hemiplegia, Herb's palsy, Herb-Duchenne and Dessulin-Klumpke palsy, essential tremor, extrapleural myelin disintegration, Fabry disease, Fahl's syndrome, syncope, familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Faber's disease, febrile seizures, fibromuscular dysplasia, Fisher syndrome, hypotonia syndrome, foot drop, Friedreich's ataxia, frontal palsy Dementia palsy, Gaucher disease, systemic gangliosidosis, Gerstmann syndrome, Gerstmann-Streussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Haller-Volden-Spatz disease, head injury, headache, persistent hemicalcemia, hemifacial spasm, alternating hemiplegia Alterans), hereditary neuropathy, hereditary spastic paraplegia, polyneuroneurotic hereditary ataxia, herpes zoster, herpes zoster otophylaxis, Hirayama syndrome, Holmes-Addie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, hydrocephalus anencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelopathy, adrenocortical hypertonia, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, pediatric hypotonia, infantile neuroaxonal dystrophy, infantile phytanate storage, infantile Refsum disease, infantile seizures, inflammatory myopathy, foramen occipital encephalopathy, enteric steatosis, intracranial cyst, intracranial hypertension, Isaacs syndrome,Joubert syndrome, Kaens-Sayer syndrome, Kennedy disease, Kinsborne syndrome, Klein-Levin syndrome, Klippel-Fail syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesia syndrome, Krabbe disease, Kugelberg-Wellander disease, Kuru disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral myelitis syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levin-Kritley syndrome, Lewy body dementia, lipid storage disease, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus - neurological sequelae, Lyme disease - Neurological complications, Machado-Joseph disease, cerebral encephalopathy, megacephaly, Melkerson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease, paresthesia femoral neuralgia, metachromatic leukodystrophy, microcephaly, migraine, Miller-Fischer syndrome, mild stroke, mitochondrial myopathy, Moebius syndrome, unilateral muscular atrophy, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidosis, multiple cerebral infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, orthostatic hypotension Multiple system atrophy, muscular dystrophy, congenital myasthenia gravis, myasthenia gravis, diffuse myelodestructive sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, congenital myopathy, thyroid-toxic myopathy, myotonia, congenital myotonia, narcolepsy, neuroacanthocytosis, neurodegenerative diseases with intracerebral iron deposition, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological outcomes of cytomegalovirus infection, neurological symptoms of Pompe disease, neurological outcomes of lupus Sequelae, neuromyelitis optica, neurogenic myotonia, neuronal ceroid lipofuscinosis, neuronal cell migration disorders, hereditary neuropathy, neurosarcoidosis, neurosyphilis, neurotoxicity, spongiform nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenate kinase-associated neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal chorea athetosis, paroxysmal hemiparesis,Parry Romberg disease, Pelizaeus-Merzbacher disease, Pena-Shocker syndrome type II, nerve root cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage, Pick's disease, nerve compression, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, porencephaly, post-polio syndrome, postherpetic neuralgia, post-infectious encephalomyelitis, postural hypotension, postural orthostatic tachycardia syndrome, postural orthostatic tachycardia syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive ataxia, progressive Multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, pseudotorch syndrome, pseudotoxoplasmosis, pseudocerebral tumor, psychogenic movement disorders, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Rasmussen encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, infantile Refsum disease, repetitive movement disorder, repetitive strain injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rheumatic encephalitis, Riley-Day syndrome, sacral nerve radiculoma, St. Vitus chorea, salivary gland diseases, Sandhoff disease, Schilder's disease, schizencephaly, Zytelberger's disease, seizure disorder, semantic dementia, septal-optic dysplasia, severe myoclonic epilepsy of infants (SMEI), shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, convulsions, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olsewski syndrome, generalized rigidity syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerosis Syndrome, short-duration persistent hemiglinal headache (SUNCT), dysphagia, Sydenham's chorea, syncope, syphilitic myelosclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, spinal fistula, tardive dyskinesia, Tarlov's cyst, Tay-Sachs disease, temporal arteritis, tethered cord syndrome, Thomsen's myotonic syndrome, thoracic outlet syndrome, thyroid toxic myopathy, painful tics, Todd's palsy, Tourette's syndrome, transient ischemic attack, transmissible hereditary spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraplegia, Troyer's syndrome, tuberous sclerosis, vascular erectile neoplasm,The following conditions may be selected: central and peripheral vasculitis syndromes, von Economo disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash, Wipple disease, Williams syndrome, Wilson's disease, Wolmann disease, and X-linked spinal and bulbar muscular atrophy.

[0127] In some embodiments, CNS disorders are selected from a list consisting of dopamine transporter deficiency syndrome, attention deficit / hyperactivity disorder (ADHD), bipolar disorder, epilepsy, multiple sclerosis, tauopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Krabbe disease, adrenoleukodystrophy, motor neuron disease, cerebral palsy, Batten disease, Gaucher disease, Tay-Sachs disease, Rett syndrome, Sandhoff disease, Charcot-Marie-Tooth disease, Angelman syndrome, Canavan disease, late-onset childhood neuronal ceroid lipofuscinosis, mucopolysaccharidosis IIIA, mucopolysaccharidosis IIIB, metachromatic leukodystrophy, hereditary lysosomal storage disorders, e.g., Niemann-Pick disease type C1 and / or neuronal ceroid lipofuscinosis, e.g., Batten disease, progressive supranuclear palsy, corticobasal syndrome, and brain cancer (including astrocytoma and glioblastoma).

[0128] Various expression products suitable for treating the above conditions have been described in the Art. The nucleic acid encoding the CRE, minimal / proximal promoter, or expression product operably linked to a promoter according to the Invention may be one of the genes selected from the group consisting of NPC1, EAAT2, NPY, CYP46A1, GLB1, APOE (e.g., ApoE2, ApoE3, or ApoE4), HEX, CLN1, CLN2, CLN3, CLN4, CLN5, CLN6, SUMF1, DCTN1, PRPH, SOD1, NEFH, GBA, IDUA, NAGLU, GUSB, ARSA, MANB, AADC, GDNF, NTN, ASP, MECP2, PTCHD1, GJB1, UBE3A, HEXA, and MO. Furthermore, or alternatively, the CRE, minimal / proximal promoter, or expression product operably linked to a promoter according to the Invention may be miRNA / CRISPR-Cas9 directed to a disease allele.

[0129] CYP46A1 is the rate-limiting enzyme in cholesterol breakdown and is known to play a beneficial role in several CNS diseases. CYP46A1 inhibition may contribute to the induction and / or exacerbation of Alzheimer's disease by increasing viral cholesterol levels, as described in Djelti et al., 2015 (incorporated herein by reference). CYP46A1 is also known to be neuroprotective in Huntington's disease, as described in Boussicault et al., 2016 (incorporated herein by reference). Therefore, the CYP46A1 gene is a particularly preferred nucleic acid encoding an expression product. In some preferred embodiments, the CYP46A1 gene is operably ligated to a CRE, minimal / proximal promoter, or promoter according to the present invention. Where appropriate, the CYP46A1 gene is operably ligated to a (pan-CNS) synthetic promoter that is active in all regions of the CNS or to a promoter that is active in more than 5, 6, 7, 8, or 9 of the brain regions listed above. Expression of CYP45A1 in all regions of the CNS or in more than 5, 6, 7, 8, or 9 of the brain regions listed above may be beneficial, as CYP46A1 expression via the ubiquitous promoter CMV or CAG has been shown to be beneficial in a mouse Huntington's disease model (Kacher et al., 2019). Where appropriate, the CYP46A1 gene is operably ligated to a synthetic promoter consisting of or including SEQ ID NO: 1, SEQ ID NO: 21, or SEQ ID NO: 2.

[0130] In some embodiments, useful expression products include dystrophin (including micro-dystrophin), beta-1,4-n-acetylgalactosamine galactosyltransferase (GALGT2), carbamoyl synthetase I, alpha-1 antitrypsin, ornithine v transcarbamylase, arginosuccinate synthetase, arginosuccinate triase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, and por Examples include phobilinogen deaminase, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl-coA carboxylase, methylmalonyl-coA mutase, glutaryl-coA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, and cystic fibrosis transmembrane regulator (CFTR).

[0131] Other useful expression products include enzymes useful in enzyme replacement therapy, which are beneficial for various conditions resulting from enzyme deficiency. For example, enzymes containing mannose-6-phosphate can be used in the treatment of lysosomal storage disorders (suitable genes include those encoding β-glucuronidase (GUSB), for instance).

[0132] In some embodiments, exemplary polypeptide expression products include neuroprotective polypeptides and anti-angiogenic polypeptides. Suitable polypeptides, but not limited to these, include glial neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), nurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growth factor (EGF), pigment epithelial factor (PEDF), Wnt polypeptide, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibody, soluble VEGFR, factor VIII (FVIII), factor IX (FIX), and members of the hedgehog family (e.g., sonic hedgehog, Indian hedgehog, and desert hedgehog).

[0133] In some embodiments, useful therapeutic expression products include insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFa, activin, inhibin, or bone morphogenetic protein (BMP). Examples of hormones and growth and differentiation factors include, but are not limited to, any one of the transforming growth factor alpha superfamily including BMP1-15, any one of the Neu differentiation factor (NDF) family such as heregluin / neuregluin / ARIA / growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), any one of the families of neuruturin, agrin, semaphorin / colapsin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0134] In some embodiments, useful expression products include, but are not limited to, cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 to IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemoattractant proteins, leukemia suppressor factors, granulocyte-macrophage colony-stimulating factor, Fas ligands, tumor necrosis factor alpha and beta, interferons (alpha, beta, and gamma), stem cell factors, and proteins that modulate the immune system, including flk-2 / flt3 ligands. Gene products generated by the immune system are also useful in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. Other useful gene products include complement regulatory proteins, such as complement regulatory proteins (MCP), complement dysintegration factors (DAF), CR1, CF2, and CD59.

[0135] In some embodiments, useful expression products include any one of the following: receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. Other useful heterogeneous nucleic acid sequences include receptors for cholesterol regulation and / or lipid regulation, and include low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors, and scavenger receptors. The present invention also encompasses the use of gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. Furthermore, useful gene products include jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MyoD and myogenin, ETS-box-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT-box-binding proteins, interferon regulator (IRF-1), Wilms tumor protein, ETS-binding proteins, STAT, GATA-box-binding proteins, and transcription factors such as GATA-3 and the forkhead family of wing helix proteins.

[0136] In some embodiments, useful expression products include polypeptides that do not exist in nature, such as chimeric or hybrid polypeptides having amino acid sequences that do not exist in nature, including insertions, deletions, or amino acid substitutions.

[0137] More suitable expression products include microRNAs (miRNAs), interfering RNAs, antisense RNAs, ribozymes, and aptamers.

[0138] In some embodiments of the present invention, the synthetic CNS-specific expression cassette comprises a gene useful for gene editing, e.g., a gene encoding a site-specific nuclease, e.g., a meganuclease, a zinc finger nuclease (ZFN), a transcriptional activator-like effector-based nuclease (TALEN), or a clustered regularly interspaced short palindromic repeat system (CRISPR-Cas). The site-specific nuclease is optionally adapted to edit a desired target genomic locus by performing a cleavage (usually a site-specific double-strand break) and then repairing it via non-homologous end joining (NHEJ) or homology-dependent repair (HDR) to obtain the desired edit. The edit may be a partial or complete repair of a gene that is a dysfunctional or functional gene knockdown or knockout. Alternatively, the edit may be via base editing or prime editing using a suitable system known in the art.

[0139] The synthetic CNS-specific expression cassette may optionally include a sequence that provides or codes for one or more, preferably all, of the following: a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. The expression cassette may optionally include a nucleic acid encoding a post-transcriptional regulatory element. The expression cassette may optionally include a nucleic acid encoding a poly(A) element.

[0140] Vectors and Virus Particles The present invention further provides vectors comprising a synthetic CNS-specific promoter or expression cassette according to the present invention.

[0141] In some embodiments of the present invention, the vector is a plasmid. Such a plasmid may contain various other functional nucleic acid sequences, such as one or more selection markers, one or more origins of replication, multiple cloning sites, etc. In some embodiments of the present invention, the vector is a viral vector.

[0142] In some embodiments of the present invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic cell expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXTl, and pSG, available from Stratagene; pSVK3, pBPV, pMSG, and pSVL, available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP, available from Clontech. Numerous other vectors are known and commercially available. Among mammalian cell adenovirus vectors, the pSV and pCMV series vectors are particularly well known but not limited to. Numerous well-known yeast expression vectors exist, including, but are not limited to, yeast integration plasmids (YIp) and yeast replication plasmids (YRp). For plants, the Agrobacterium Ti plasmid is an exemplary expression vector, and plant viruses also provide suitable expression vectors, such as tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

[0143] In some preferred embodiments, the vector is a gene therapy vector. Various gene therapy vectors are known in the art and include AAV vectors, adenovirus vectors, retrovirus vectors, and lentivirus vectors. When the vector is a gene therapy vector, the vector preferably comprises a nucleic acid sequence operably linked to the synthetic CNS-specific promoter of the present invention, which optionally encodes a therapeutic product, or therapeutic protein. The therapeutic protein may be a secretible protein. Not limited examples of secretible proteins have been discussed above, and exemplary secretible therapeutic proteins include coagulation factors, e.g., factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, and toxic proteins.

[0144] In some embodiments of the present invention, the vector is a viral vector, such as a retrovirus, lentivirus, adenovirus, herpes simplex, or adeno-associated virus (AAV) vector. In some preferred embodiments, the vector is a lentiviral vector, optionally an HIV-1-based lentiviral vector. In some preferred embodiments, the vector is an AAV vector. In some preferred embodiments, the AAV has a serotype suitable for or specifically optimized for CNS transduction. In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAVrh10, AAVDJ8, and AAV2g9 or derivatives thereof.

[0145] AAV vectors are preferably used as self-complementary, double-stranded AAV vectors (scAAV) to overcome one of the limiting steps in AAV transduction (i.e., conversion from single-stranded to double-stranded AAV), although the use of single-stranded AAV vectors (ssAAV) is also encompassed herein. In some embodiments of the present invention, the AAV vector is a chimeric vector, meaning that it contains components derived from at least two AAV serotypes, e.g., the ITR of AAV2 and the capsid protein of AAV5. AAV9 is known to transduce efficiently into CNS cells and particularly efficiently into tissues, and therefore AAV9 and its derivatives are of particular interest for targeting CNS cells and tissues. AAV2g9 is known to transduce efficiently into CNS cells and particularly efficiently into tissues, and therefore AAV2g9 and its derivatives are of particular interest for targeting CNS cells and tissues. AAVrh10 is known to efficiently transduce CNS cells and particularly efficiently into tissues; therefore, AAVrh10 and its derivatives are of particular interest for targeting CNS cells and tissues. Systemic or intravenous delivery of AAVrh10 is known to provide high transgene expression in the central nervous system, as described in (Tanguy et al., 2015), incorporated herein by reference; therefore, AAVrh10 and its derivatives are of particular interest for targeting CNS cells and tissues. AAVDJ8 is preferred because it has been shown to efficiently target multiple regions of the brain and efficiently target astrocytes, as described in (Hammond et al., 2017), incorporated herein by reference. AAV1, AAV2, AAV4, AAV5, and AAV8 are also known to target CNS cells and tissues, and therefore these AAV serotypes and their derivatives are of particular interest for targeting CNS cells and tissues.

[0146] The present invention further provides recombinant virions (viral particles) containing the above-described vector.

[0147] Pharmaceutical Compositions: The vector or virion of the present invention can be formulated into pharmaceutical compositions using pharmaceutically acceptable excipients, i.e., one or more pharmaceutically acceptable carriers and / or additives, such as buffers, carriers, excipients, stabilizers, etc. Pharmaceutical compositions may be provided in kit form. AAV vectors and / or suitable pharmaceutical compositions and delivery systems for the method and use thereof are known in the art.

[0148] Therefore, further aspects of the present invention provide pharmaceutical compositions comprising vectors or virions as described herein.

[0149] The relative amounts of the active ingredient (e.g., AAV vector particles), pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to this disclosure may vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route through which the composition is to be administered. For example, a composition may contain between 0.1 percent and 99 percent (w / w) of the active ingredient. For example, a composition may contain between 0.1 percent and 100 percent, for example, between 5 and 50 percent, between 1 and 30 percent, between 5 and 80 percent, or at least 80 percent (w / w) of the active ingredient.

[0150] The pharmaceutical composition may be formulated with one or more excipients or diluents to (1) increase stability, (2) increase cell transfection or transduction, (3) enable sustained or delayed release of the payload, (4) alter in vivo distribution (e.g., targeting viral particles to specific tissues or cell types), (5) increase translation of the encoded protein, (6) alter the release profile of the encoded protein, and / or (7) enable regulated expression of the payload of the present invention. In some embodiments, pharmaceutically acceptable excipients may be at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or 100 percent pure. In some embodiments, the excipients are approved for human and veterinary use. In some embodiments, the excipients may be approved by the U.S. Food and Drug Administration. In some embodiments, the excipients may be pharmaceutical grade. In some embodiments, the excipients may meet the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia and / or International Pharmacopoeia. Excipients, as used herein, include, but are not limited to, any solvent, dispersion medium, diluent or other liquid vehicle, dispersion or suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, etc., suitable for a desired specific dosage form. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, AR Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006, incorporated herein by reference in its entirety). The use of conventional excipient media may be contemplated within the scope of this disclosure, except where any conventional excipient media may be incompatible with the substance or its derivatives by causing any undesirable biological effect or by interacting with any other component of the pharmaceutical composition in a harmful manner.

[0151] Therapeutic agents, other methods and uses: The present invention also provides synthetic CNS-specific promoters, expression cassettes, vectors, virions or pharmaceutical compositions according to various embodiments of the present invention for use in the treatment of diseases, preferably, optionally, diseases associated with abnormal gene expression in the CNS (e.g., hereditary CNS diseases). The relevant conditions, diseases and therapeutic expression products are discussed above.

[0152] The present invention also provides synthetic CNS-specific promoters, expression cassettes, vectors, and virions according to various embodiments of the present invention for use as pharmaceuticals.

[0153] The present invention also provides synthetic CNS-specific promoters, expression cassettes, vectors, and virions according to various embodiments of the present invention for use in the manufacture of pharmaceutical compositions for the treatment of any condition or disease described herein.

[0154] The present invention further provides cells comprising synthetic CNS-specific promoters, expression cassettes, vectors, and virions according to various embodiments of the present invention. The cells are, as appropriate, eukaryotic cells. The eukaryotic cells may, as appropriate, be animal (metazoan) cells (e.g., mammalian cells). The cells are, as appropriate, human cells.

[0155] In some embodiments of the present invention, the cells are ex vivo, for example, in cell culture. In other embodiments of the present invention, the cells may be part of a tissue or a multicellular organism.

[0156] In a preferred embodiment, the cells are CNS cells, which may be ex vivo or in vivo. The CNS cells may be primary neurons, astrocytes, oligodendrocytes, microglial cells, or ependymal cells. Alternatively, the CNS cells may be CNS-derived cell lines, such as immortalized cell lines.

[0157] Cells may reside within the CNS tissue environment (e.g., within the CNS of an animal), or they may be isolated from CNS tissue, for example, in cell culture. Where appropriate, the primary cells or cell lines are human cells.

[0158] The synthetic CNS-specific promoters, expression cassettes, or vectors according to the present invention can be inserted into the cellular genome or may be episomal (for example, present in an episomal vector).

[0159] In a further embodiment, the present invention provides a method for producing an expression product, comprising providing a synthetic CNS-specific expression cassette (preferably in a vector as described above) in cells, preferably CNS cells, and expressing a gene present in the synthetic CNS-specific expression cassette. The method may optionally include maintaining the CNS cells under conditions suitable for gene expression. In culture, this may include incubating cells or tissues containing cells under suitable culture conditions. Expression may, of course, occur in vivo, for example, in one or more cells in the CNS of interest.

[0160] The method may include, as appropriate, the step of introducing a synthetic CNS-specific expression cassette into CNS cells. A wide range of methods for transfecting CNS cells are well known in the art. A preferred method for transfecting CNS cells is to transduce cells with a viral vector containing a synthetic CNS-specific expression cassette, such as an AAV vector.

[0161] It will be apparent to those skilled in the art that synthetic CNS-specific promoters, expression cassettes, vectors, or virions according to various embodiments of the present invention can be used for gene therapy. Therefore, the use of such nucleic acid constructs in gene therapy forms part of the present invention.

[0162] The present invention therefore provides, in some embodiments, expression cassettes, vectors, or virions according to the present invention for use in gene therapy, preferably in gene therapy by CNS-specific expression of therapeutic genes, in a target area. Therapies may include the treatment of diseases by secretion of therapeutic products from CNS cells, and, as appropriate, diseases involving abnormal gene expression in the CNS, as discussed above.

[0163] The present invention also provides a method for expressing a therapeutic transgene in CNS cells, comprising introducing an expression cassette or vector according to the present invention into the CNS cells. The CNS cells may be in vivo or ex vivo.

[0164] The present invention also relates to a method of gene therapy for a subject requiring it, preferably a human, - The present invention provides a method comprising administering to a target a synthetic CNS-specific expression cassette, vector, virion, or pharmaceutical composition of the present invention, which contains a gene encoding a therapeutic product (or, as appropriate, introducing it into the target's CNS).

[0165] The method includes, as appropriate, expressing a therapeutic dose of the therapeutic product from a gene in the target CNS. Various conditions and diseases that can be treated are discussed above. Suitable genes encoding the therapeutic product are discussed above.

[0166] The method may include, as appropriate, administering a vector or virion according to the present invention to a target. The vector may, as appropriate, be a viral gene therapy vector, such as an AAV vector.

[0167] In some embodiments, the method includes administering the gene therapy vector systemically. Systemic administration may be enteral (e.g., orally, sublingually, and rectally) or parenteral (e.g., by injection). Preferred injection routes include intravenous, intramuscular, subcutaneous, intra-arterial, intra-articular, subarachnoid, and intradermal injections. In one embodiment, the gene therapy vector may be delivered into the CSF pathway by injection. Examples of delivery into the CSF pathway that are not limited to this include subarachnoid and intraventricular administration.

[0168] A particularly preferred route of administration for AAV vectors or virions containing a synthetic CNS-specific promoter or expression cassette according to the present invention is intravascular. Optionally, AAV vectors or virions containing a synthetic CNS-specific promoter or expression cassette according to the present invention may be administered via a vein in the back of the hand or a vein in the forearm. Suitable veins in the forearm are the cephalic vein, median vein, or ulnar cephalic vein. This is because this route of administration is generally safe for the patient while still allowing some permeability into the CNS.

[0169] In some embodiments, the viral gene therapy vector may be administered simultaneously or sequentially with one or more additional therapeutic agents or one or more saturators designed to prevent vector clearance by the reticular endothelial system.

[0170] If the vector is an AAV vector, the dose of the vector is 1 × 10⁻⁶ 10 gc / kg ~ 1 × 10 15 gc / kg or more, as appropriate, 1 × 10 12 gc / kg ~ 1 × 10 14 gc / kg, as appropriate, 5×10 12 gc / kg ~ 5 × 10 13 It could be gc / kg.

[0171] Generally, the subjects requiring it are mammals, preferably primates, and more preferably humans. Typically, the subjects requiring it exhibit symptoms characteristic of a disease. The method typically involves alleviating the symptoms exhibited by the subject requiring it by expressing the therapeutic effect of the therapeutic product. In one embodiment, the therapeutic method of the present invention can be used to reduce declines in functional ability and activities of daily living, as measured by standard assessment systems such as the Total Functional Capacity (TFC) scale, but not limited to these. In one embodiment, the method of the present invention can be used to improve performance in any assessment used to measure symptoms of neurological disorders.Such assessments are not limited to these, but include ADAS-cog (Alzheimer's Disease Assessment Scale - Cognitive Assessment), MMSE (Mini-Mental State Examination), GDS (Geriatric Depression Scale), FAQ (Functional Activity Questionnaire), ADL (Activities of Daily Living), GPCOG (General Practitioner Assessment of Cognition), Mini-Cog, AMTS (Abbreviated Mental Test Score), Clock Drawing Test, 6-CIT (6-Item Cognitive Impairment Test), TYM (Test Your Memory), MoCa (Montreal Cognitive Assessment), ACE-R (Addenbrookes Cognitive Assessment), MIS (Memory Impairment Screening), BADLS (Bristol Activities of Daily Living Scale), Barthel Index, Functional Independence Measure, Instrumental Activities of Daily Living, and IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly). Examples include the Elderly)), neuropsychiatric symptom assessment, the Cohen-Mansfield Agitation Inventory, BEHAVE-AD, EuroQol, Short Form-36 and / or the MBR Caregiver Strain Instrument, or any other test as incorporated herein in whole by reference, such as those described in Sheehan B (Ther Adv Neurol Disord. 5(6):349-358 (2012)).

[0172] Genetherapy protocols for therapeutic gene expression in target cells in vitro and in vivo are well known in the art and will not be discussed in detail here. Briefly, they include intravenous or intra-arterial administration (e.g., intra-corotid artery, intrahepatic artery, intrahepatic vein), intracranial administration, intramuscular injection, interstitial injection, drip infusion in the airway, and application into the endothelium and liver parenchyma of plasmid DNA vectors (naked or in liposomes) or viral vectors. Various devices have been developed to enhance the availability of DNA to target cells. A simple approach is to physically bring target cells into contact with a catheter or implantable material containing the relevant vector, while more complex approaches can utilize jet injection devices and similar devices. Gene transfer into mammalian CNS cells is carried out using both ex vivo and in vivo procedures. Ex vivo approaches typically require the collection of CNS cells, in vitro transduction using a suitable expression vector, and subsequent reintroduction of the transduced CNS cells into the CNS. This approach is generally undesirable due to the difficulties and risks involved in collecting and reintroducing CNS cells in the brain. In vivo gene transfer has been achieved, for example, by directly injecting DNA or viral vectors into the CNS via intracranial injection of viral vectors, or by intravenous or intra-arterial injection.

[0173] In one embodiment, a gene therapy vector may be administered to a subject (e.g., the subject's CNS) in a therapeutically effective dose to reduce the symptoms of the neurological disorder in question (determined, for example, using a known assessment method). In some embodiments, the gene therapy vector and the composition comprising the gene therapy vector may be administered in a manner that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0174] Gene therapy vectors may be used in combination with one or more other therapeutic, prophylactic, research, or diagnostic agents. The term "in combination with" is not intended to imply that the agents must be administered simultaneously and / or formulated for joint delivery, but these delivery methods are within the scope of the invention. The composition may be administered simultaneously with, before, or after one or more other desired therapeutic or medical treatments.Compounds that can be used in combination with the AAV particles described herein include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists, e.g., memantine, antipsychotics, antidepressants, anticonvulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, tau aggregation inhibitors, e.g., methylene blue, phenothiazine, anthraquinone, n-phenylamine or rhodamine, microtubule stabilizers, e.g., NAP, Taxol or This includes paclitaxel, kinase or phosphatase inhibitors, e.g., those targeting GSK3 (lithium) or PP2A; immunization using beta-peptides or tau phosphoepitopes; anti-tau or anti-amyloid antibodies; dopamine depletion agents (e.g., tetrabenazine for chorea); benzodiazepines (e.g., clonazepam for myoclonus, chorea, dystonia, rigidity and / or spasticity); amino acid precursors of dopamine (e.g., levodopa for rigidity); skeletal muscle relaxants (e.g., baclofen, tizanidine for rigidity and / or spasticity); and acetylcholine release at the neuromuscular junction causing muscle paralysis. Examples include inhibitors of choline release (e.g., botulinum toxin for bruxism and / or dystonia), atypical neuroleptics (e.g., olanzapine and quetiapine for psychosis and / or irritability, risperidone, sulpiride and haloperidol for psychosis, chorea and / or irritability, clozapine for treatment-resistant psychosis, and aripiprazole for psychosis with marked negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, miltrazapine, and venlafaxine for depression, anxiety disorders, obsessive-compulsive behavior and / or irritability), hypnotics (e.g., zopiclone and / or zolpidem for altering the sleep-wake cycle), anticonvulsants (e.g., sodium valproate and carbamazepine for mania or hypomania), and mood stabilizers (e.g., lithium for mania or hypomania).

[0175] According to some preferred embodiments, the above method can be used for the treatment of subjects with CNS-related disorders such as those discussed above, for example, dopamine transporter deficiency syndrome.

[0176] Definitions and General Points: While the preparation and use of various embodiments of the present invention will be discussed in detail below, it should be noted that the present invention provides a number of applicable inventive concepts that can be embodied in a wide variety of specific situations. The specific embodiments discussed herein are merely illustrative of specific methods for preparing and using the present invention and do not limit the scope of the present invention.

[0177] To facilitate understanding of the present invention, several terms are defined below. Terms as defined herein have meanings that are generally understood by those skilled in the art in the field relating to the present invention. Terms such as “a,” “an,” and “the” are not intended to refer only to singular entities, but include general classes for which specific examples may be used. Technical terms used herein are used to describe specific embodiments of the present invention, but their use does not limit the present invention unless outlined in the claims.

[0178] The background discussion of the invention in this specification is included to illustrate the context of the invention. This should not be construed as an acknowledgment that any of the materials referred to were part of known, common general knowledge that was published in any country as of the priority date of any of the claims.

[0179] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by specific references. All documents cited herein are incorporated herein in their entirety by reference. In particular, teachings or sections of such documents specifically referenced herein are incorporated by reference.

[0180] The implementation of this invention utilizes, unless otherwise specified, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the scope of the art. Such techniques are well described in the literature. For example, *Current Protocols in Molecular Biology* (Ausubel, 2000, Wiley and Son Inc., Library of Congress, USA); *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); *Oligonucleotide Synthesis* (MJ Gait, ed., 1984); U.S. Patent No. 4,683,195; *Nucleic Acid Hybridization* (Harries and Higgins, eds., 1984); *Transcription and Translation* (Hames and Higgins, eds., 1984); *Culture of Animal Cell* (Freshney, Alan R. Liss, Inc., 1987); *Immobilized Cell and Enzymes* (IRL Press, 1986); *Perbal, A Practical Guide to Molecular Cloning* (1984); *Methods in Enzymology* series (Abelson and Simon, editors-in-chief, Academic Press, Inc.).See, in particular, Volumes 154 and 155 (edited by Wu et al.) and 185, "Gene Expression Technology" (edited by Goeddel); Gene Transfer Vectors For Mammalian Cell (edited by Miller and Calos, 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (edited by Mayer and Walker, Academic Press, London, 1987); Handbook of Experimental Immunology, Volumes I-IV (edited by Weir and Blackwell, 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0181] The terms “central nervous system” or “CNS” are well understood by those skilled in the art. The CNS consists of the brain and spinal cord. Preferably, the synthetic CNS-specific promoter is active in the brain. The promoter of the present invention may be active in the brain and / or spinal cord. Preferably, the CNS is a mammalian, and more preferably a human, CNS.

[0182] "CNS cells(singular or plural)" refers to cells found in or derived from the CNS (CNS tissue). CNS cells may be primary cells or cell lines (e.g., SH-Sy5y, Neuro2A, U87-MG). CNS cells may be in vivo (e.g., in CNS tissue) or in vitro (e.g., in cell cultures). CNS cells consist of neurons, astrocytes, oligodendrocytes, microglial cells, and ependymal cells. Neurons, as found in CNS tissue, include a cell body, a long axon, and a synaptic terminal. Neurons transmit electrical signals received in the cell body to other cells near their synaptic terminals via their long axon. Oligodendrocytes are a type of glial cell in the CNS that produce a myelin sheath that surrounds the neuron's axon for faster electrical signal conduction. Astrocytes are star-shaped and are the most abundant cell type in the brain. They have multiple roles and neural functions that assist and regulate the transmission of electrical impulses. Microglia are commensal macrophage cells in the brain that are involved in immune defense. Ependymal cells form the inner lining of the ventricles. The term "CNS cells(s)" as used herein includes neurons, astrocytes, oligodendrocytes, microglial cells and / or ependymal cells. The promoters of the present invention may be active in any of the CNS cells (e.g., neurons). The promoters of the present invention may be active in more than one type of CNS cell (e.g., neurons and astrocytes). The promoters of the present invention may be active in all types of CNS cells (neurons, astrocytes, oligodendrocytes, microglial cells and ependymal cells). Furthermore, the synthetic CNS-specific promoters of the present invention may be active in one subtype of CNS cell, e.g., dopaminergic neurons or mature oligodendrocytes. In some embodiments, the synthetic CNS-specific promoter of the present invention may be active only in one subtype of CNS cell, such as dopaminergic neurons or mature oligodendrocytes.The CRE, proximal / minimal promoter, and promoter of the present invention may be active in specific regions of the CNS, in specific CNS cells or CNS cell subtypes, or both. In some embodiments, the CRE, proximal / minimal promoter, and promoter of the present invention may be active in all regions of the CNS in a specific CNS cell type, e.g., a neuron. In other embodiments, the CRE, proximal / minimal promoter, and promoter of the present invention may be active in one or fewer regions of the CNS in a specific CNS cell type, e.g., a neuron, e.g., in the midbrain. In some embodiments, the CRE, proximal / minimal promoter, and promoter of the present invention may be active in all CNS cells, e.g., in all regions of the CNS. In some embodiments, the CRE, proximal / minimal promoter, and promoter of the present invention may be active in all CNS cells, e.g., in one or fewer regions of the CNS, e.g., in the midbrain.

[0183] The terms “cis-regulatory element” or “CRE” are well known to those skilled in the art and refer to nucleic acid sequences, such as enhancers, promoters, insulators, or silencers, that can regulate or modulate the transcription of an adjacent gene (i.e., in cis). CREs are found near the gene they regulate. CREs typically regulate gene transcription by binding to TFs; i.e., they include TFBSs. A single TF may bind to multiple CREs, thereby regulating the expression of multiple genes (pleomorphism). CREs are usually, but not always, located upstream of the transcription start site (TSS) of the gene they regulate. In this context, “enhancer” refers to a CRE that is operably associated with the gene it regulates, and can be found upstream, downstream, or even within its introns, that enhances (i.e., upregulates) the transcription of that gene. Multiple enhancers may act in a cooperative manner to regulate the transcription of a single gene. In this context, “silencer” refers to a CRE that binds to a TF called a repressor and acts to inhibit or downregulate the transcription of a gene. The term “silencer” can also refer to a region in the 3' untranslated region of messenger RNA that binds to a protein that suppresses the translation of that mRNA molecule, but this use differs from its use in the description of CRE. Generally, the CRE of the present invention is a CNS-specific enhancer element (often referred to as CNS-specific CRE or CNS-specific CRE enhancer or so). In this context, the CRE is preferably located 2500 nucleotides or less from the transcription start site (TSS), more preferably 2000 nucleotides or less from the TSS, more preferably 1500 nucleotides or less from the TSS, and optionally 1000, 750, 500, 250, 200, 150 or 100 nucleotides or less from the TSS. The CREs of the present invention are preferably relatively short in length, preferably 1000 nucleotides or less. For example, they may be 800, 700, 600, 500, 400, 300, 200, 175, 150, 90, 80, 70, 60, or 50 nucleotides or less.The CRE of the present invention is typically provided in combination with an operably coupled promoter element, which may be a minimal promoter or a proximal promoter, and the CRE of the present invention can enhance the CNS-specific activity of the promoter element.

[0184] The terms “cis-regulatory module” or “CRM” typically refer to a functional regulatory nucleic acid module containing two or more CREs, where in this invention, CREs are usually CNS-specific enhancers, and therefore, CRMs are synthetic CNS-specific regulatory nucleic acids. A CRM may contain multiple CNS-specific CREs. Optionally, at least one of the CREs contained in a CRM is a CRE or a functional variant thereof according to SEQ ID NOs. 9-11, 28-31. Typically, multiple CREs in a CRM act together (e.g., additively or synergistically) to enhance the transcription of genes to which the promoter containing the CRM is operably associated. There is considerable room to shuffle (i.e., rearrange), invert (i.e., reverse the orientation) and alter the spacing of CREs within a CRM. Therefore, functional variants of CRMs in this invention include, among other things, variants of the mentioned CRMs in which the CREs within them are shuffled and / or inverted and / or the spacing between CREs is altered.

[0185] As used herein, the term “promoter” generally refers to a region of DNA located upstream of a nucleic acid sequence to be transcribed, where transcription is necessary, i.e., where transcription is initiated. Promoters enable the appropriate activation or repression of transcription of the coding sequence under their control. Promoters typically contain specific sequences that are recognized and bound by multiple TFs. TFs bind to the promoter sequence, resulting in the recruitment of RNA polymerase, the enzyme that synthesizes RNA from the coding region of a gene. A wide variety of promoters are known in the art.

[0186] The term “synthetic promoter,” as used herein, refers to a promoter that does not occur naturally. In this context, it typically includes the CRE and / or CRM of the present invention operably ligated to a minimal (or core) promoter or a CNS-specific proximal promoter (promoter element). The CRE and / or CRM of the present invention work to enhance CNS-specific transcription of a gene operably ligated to a synthetic promoter. Some synthetic promoters may occur naturally (e.g., a minimal promoter or one or more CREs in a promoter), but synthetic promoters as entities do not occur naturally. Alternatively, a synthetic promoter may be a shorter, truncated version of a naturally occurring promoter.

[0187] As used herein, a “minimal promoter” (also known as a “core promoter”) typically refers to a short DNA segment that is inactive or nearly inactive on its own but can mediate transcription when combined with other transcriptional regulatory elements. Minimal promoter sequences can originate from a variety of different sources, including prokaryotic and eukaryotic genes. An example of a minimal promoter is SYNP_CRE151 (SEQ ID NO: 12). Other examples of minimal promoters include the dopamine beta-hydroxylase gene minimal promoter, the cytomegalovirus (CMV) pre-early gene minimal promoter (CMV-MP), and the herpesthymidine kinase minimal promoter (MinTK). Minimal promoters typically include a transcription start site (TSS) and elements immediately upstream, an RNA polymerase II binding site, and a common transcription factor binding site (often a TATA box). Minimal promoters may also include some elements downstream of the TSS, which are usually of little functionality without additional regulatory elements.

[0188] As used herein, “proximal promoter” refers to the proximal sequence upstream of a gene, which typically tends to contain a minimal promoter and at least some additional regulatory sequences, usually the main regulatory elements. Often extends approximately 250 base pairs upstream of the TSS and includes specific TFBSs. The proximal promoter may also contain one or more regulatory elements downstream of the TSS, e.g., UTRs or introns. In this case, the proximal promoter may, as appropriate, be a short, terminally cleaved version of a naturally occurring CNS-specific proximal promoter. The proximal promoters of the present invention may be combined with one or more CREs or CRMs of the present invention. However, the proximal promoter may also be synthetic.

[0189] As used herein, “promoter element” refers to either a minimal promoter or a proximal promoter as defined above. In the context of the present invention, a promoter element can be combined with one or more CREs to provide the synthetic CNS-specific promoter of the present invention.

[0190] In the context of the present invention, a “functional variant” of a CRE, CRM, promoter element, promoter, or other regulatory nucleic acid is a variant of a reference sequence that retains the ability to function in the same manner as, for example, a CNS-specific CRE, a CNS-specific CRM, or a CNS-specific promoter. Alternative terms for such a functional variant include “biological equivalent” or “equivalent.”

[0191] It will be acknowledged that the ability of a given CRE, CRM, promoter, or other regulatory sequence to function as a CNS-specific enhancer is significantly determined by the ability of the sequence to bind to the same CNS-specific TF that binds to the reference sequence. Therefore, in most cases, a functional variant of a CRE or CRM contains most or all of the TFBS of the same TF as the reference CRE, CRM, or promoter. It is preferable, but not required, that the TFBS of the functional variant be in the same relative position (i.e., order and overall position) as the reference CRE, CRM, or promoter. It is also preferable, but not required, that the TFBS of the functional variant be in the same orientation as the reference sequence (note that in some cases, for example, they may exist in the opposite orientation as the reverse complement relative to the sequence in the reference sequence). It is also preferable, but not required, that the TFBS of the functional variant be co-stranded with the reference sequence. Therefore, in a preferred embodiment, the functional variant contains TFBS of the same TF as the reference sequence in the same order, same position, same orientation, and co-stranded. It will be understood that the sequences between TFBSs (in some cases referred to as spacer sequences or similar) are of little importance to the function of the CRE or CRM. Such sequences can usually vary considerably, and their lengths may change. However, in preferred embodiments, the spacing (i.e., the distance between adjacent TFBSs) is substantially identical in the functional variant to that in the reference sequence (e.g., differing by more than 20%, preferably by more than 10%, more preferably nearly identical). It will be apparent that in some cases, the functional variant of the CRE may exist in reverse orientation, for example, it may be the inverse complement of the CRE or a variant thereof, as described above.

[0192] The level of sequence identity between a functional variant and a reference sequence can also be an indicator or a retained functionality. High levels of sequence identity in CRE, CRM, or promoter TFBSs are generally of greater importance than sequence identity in spacer sequences (where there is little or no need for any sequence preservation). However, considering that the sequences in functional TFBSs do not need to correspond precisely to the consensus sequence, it can be acknowledged that a considerable degree of sequence variation may be accommodated even within TFBSs.

[0193] The ability of one or more TFs to bind to TFBS in a given functional variant can be determined by any relevant means known in the art, including, but not limited to, electrical mobility shift assays (EMSA), binding assays, chromatin immunoprecipitation (ChIP), and ChIP sequencing (ChIP-seq). In a preferred embodiment, the ability of one or more TFs to bind to a given functional variant is determined by EMSA. Methods for performing EMSA are well known in the art. A suitable approach is described by Sambrook et al., cited above. Numerous relevant publications describing this procedure are available, e.g., Hellman and Fried, Nat Protoc. 2007; 2(8): pp. 1849–1861.

[0194] "CNS-specific" or "CNS-specific expression" refers to the ability of a cis-regulatory element, cis-regulatory module, or promoter to enhance or drive gene expression in CNS cells (or CNS-derived cells) in a preferential or dominant manner compared to other tissues (e.g., liver, kidney, spleen, heart, muscle, and lung). Gene expression may be in the form of mRNA or protein. In preferred embodiments, CNS-specific expression is such that there is negligible expression in other (i.e., non-CNS) tissues or cells; that is, the expression is highly CNS-specific.

[0195] The ability of a CRE, CRM, or promoter to function as a CNS-specific CRE, CRM, or promoter can be readily assessed by those skilled in the art. Therefore, it is readily possible to determine whether any variant of the specific CRE, CRM, or promoter listed above remains functional (i.e., is a functional variant as defined above). For example, any given CRM to be evaluated can be operably linked to a minimal promoter (e.g., located upstream of CMV-MP or upstream of SEQ ID NO: 12 or 13) to measure the ability of the cis-regulatory element to drive CNS-specific expression of a gene (usually a reporter gene). Alternatively, a variant of a CRE or CRM can be substituted in place of a reference CRE or CRM in a synthetic CNS-specific promoter to determine its effect on CNS-specific expression driven by the modified promoter and compare it to the unmodified form. Similarly, the ability of a promoter to drive CNS-specific expression can be readily assessed by those skilled in the art (e.g., as described in the following examples). The expression level of a gene driven by a variant of a reference promoter can be compared to the expression level driven by the reference promoter. In some embodiments, a variant may be said to remain functional if the CNS-specific expression level driven by the variant promoter is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the expression level driven by the reference promoter. Suitable nucleic acid constructs and reporter assays for evaluating CNS-specific enhancement can be readily constructed, and suitable methodologies are provided by the examples described below.

[0196] CNS specificity can be identified, where gene expression (e.g., therapeutic or reporter genes) occurs preferentially or predominantly in CNS-derived cells. For example, preferential or dominant expression can be defined as when the level of expression is significantly higher in CNS-derived cells than in other types of cells (i.e., non-CNS-derived cells). For example, expression in CNS-derived cells may be at least 5 times higher than in non-CNS cells, preferably at least 10 times higher, and in some cases 50 times higher. For convenience, CNS-specific expression can be demonstrated, as appropriate, by comparing expression levels in various non-CNS cell lines, for example, primary CNS cells or CNS-derived cell lines, such as SH-Sy5y, Neuro2A, and U87-MG, with expression levels in liver-derived cell lines (e.g., Huh7 or HepG2), kidney-derived cell lines (e.g., HEK-293), cervical tissue-derived cell lines (e.g., HeLa), and / or lung-derived cell lines (e.g., A549), in muscle-derived cell lines, e.g., C2C12 or H2K cells (skeletal muscle) or H9C2 cells (heart).

[0197] The synthetic CNS-specific promoter of the present invention preferably exhibits reduced expression in non-CNS-derived cells, as appropriate, in C2C12, H9C2, Huh7, HEK-293, HeLa, and / or A549 cells, compared to a non-tissue-specific promoter, such as CMV-IE. The synthetic CNS-specific promoter of the present invention preferably has an activity of 50% or less, as appropriate, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less in non-CNS-derived cells (as appropriate, in C2C12, H9C2, Huh7, HEK-293, HeLa, and / or A549) compared to the CMV-IE promoter. Generally, it is preferable to minimize expression in non-CNS-derived cells, but in some cases this may not be necessary. Even if the synthetic CNS-specific promoter of the present invention exhibits higher expression in, for example, one or two non-CNS cells, it can still be considered a CNS-specific promoter as long as it exhibits overall higher expression in a variety of CNS cells compared to non-CNS cells.

[0198] The synthetic CNS-specific promoters of the present invention are preferably suitable for promoting expression in the target CNS, for example, for driving the CNS-specific expression of a transgene, preferably a therapeutic transgene. Preferred synthetic CNS-specific promoters of the present invention are suitable for promoting CNS-specific transexpression and have an activity of at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, or 400% of the activity of the synapsin-1 promoter in CNS cells. In some embodiments, the synthetic CNS-specific promoters of the present invention are suitable for promoting CNS-specific transgene expression at a level of at least 100% of the activity of the synapsin-1 promoter, preferably at a level of 150%, 200%, 300%, or 500% of the activity of the synapsin-1 promoter. Such CNS-specific expression is appropriately determined in CNS-derived cells, such as SH-Sy5y, Neuro2A, U87-MG cell lines, or primary CNS cells (primary human neurons, astrocytes, oligodendrocytes, microglia, and / or ependymal cells, as appropriate).

[0199] The synthetic CNS-specific promoter of the present invention may also be able to promote CNS-specific gene expression at levels of at least 50%, 100%, 150%, or 200% compared to CMV-IE in CNS-derived cells, such as SH-Sy5y, Neuro2A, U87-MG cell lines, or primary CNS cells (primarily human neurons, astrocytes, oligodendrocytes, microglia, and / or ependymal cells, as appropriate).

[0200] As used herein, the term “nucleic acid” typically refers to oligomers or polymers of any length (preferably linear polymers) consisting essentially of nucleotides. A nucleotide unit generally comprises a heterocyclic base, a sugar group, and a phosphate group containing at least one, e.g., one, two, or three modified or substituted phosphate groups. Heterocyclic bases may include, among others, purine and pyrimidine bases, e.g., the widely naturally occurring nucleic acids adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated), unnatural, or derivatized bases. The sugar groups may include, in particular, pentose (pentofuranose) groups, for example, preferably ribose and / or 2-deoxyribose or arabinose, 2-deoxyarabinose, threose or hexose sugar groups common to naturally occurring nucleic acids, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modifications to phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or several other useful properties. The term “nucleic acid” more preferably encompasses DNA, RNA, and DNA-RNA hybrid molecules, specifically including hnRNA, premRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA-RNA hybrids. Nucleic acids may be naturally occurring, for example, found in nature or isolated from nature; or they may not be naturally occurring, for example, produced by recombinant DNA technology, and / or partially or entirely chemically or biochemically synthesized. “Nucleic acids” may be double-stranded, partially double-stranded, or single-stranded. In the case of single-stranded nucleic acids, the nucleic acid may be either a sense strand or an antisense strand.Furthermore, the nucleic acid can be either circular or linear.

[0201] As used herein, an "isolated" nucleic acid molecule or nucleic acid sequence, when referring to a nucleic acid, is one that lacks all or a portion of the sequences that are normally associated with it in nature, or one that is as found in nature but has heterologous sequences associated with it, or a molecule that has been dissociated from a chromosome.

[0202] The terms "identity" and "identical" refer to sequence similarity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules. Determination of sequence alignment and sequence identity can be performed, for example, using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), or the "BLAST2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).

[0203] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms can be found, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482, Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:pp. 237-44; Higgins and Sharp (1989) CABIOS 5:pp. 151-153; Corpet et al. (1988) Nucleic Acids Res. 16:pp. 10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:pp. 155-65; Pearson et al. (1994) Methods Mol. Biol. 24:pp. 307-31. This is described in Tatiana et al. (1999) FEMS Microbiol. Lett. 174: pp. 247-245. A detailed discussion of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215: pp. 403-410.

[0204] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, Maryland), for use in conjunction with several sequence analysis programs and is available on the Internet. Instructions on how to use this program to determine sequence identity are available on the Internet under the "Help" section of BLAST™. For comparison of nucleic acid sequences, the "BLAST2 sequences" function (Blastn) program of BLAST™ can be utilized using the default parameters. Nucleic acid sequences having greater similarity to a reference sequence will show an increase in the percentage of identity when evaluated by this method. Usually, the percentage of sequence identity is calculated over the entire length of the sequence.

[0205] For example, a global optimal alignment is found by the Needleman-Wunsch algorithm using the following scoring parameters as appropriate: match score: +2, mismatch score: -3, gap penalty: gap open 5, gap extension 2. The percentage of identity of the resulting optimal global alignment is calculated as appropriate by the ratio of the number of aligned bases to the overall length of the alignment, where the length of the alignment includes both matches and mismatches and is multiplied by 100.

[0206] The term “transcription factor binding site” (TFBS) is well known in the art. It will be apparent to those skilled in the art that TFBS sequences may be modified insofar as they are bound to the intended transcription factor (TF). The consensus sequences of the various TFBS disclosed herein are known in the art, and those skilled in the art can readily use this information to determine alternative TFBS. Furthermore, the ability to bind to a given putative sequence can readily be determined experimentally by those skilled in the art (e.g., by EMSA and other approaches well known in the art and discussed herein).

[0207] The meaning of "consensus sequence" is well known in the art. In this application, unless otherwise specified in the context, the following notation is used for consensus sequences. Consider the following exemplary DNA sequence: A[CT]N{A}YR

[0208] A means that A is always found at that position; [CT] represents either C or T at that position; N represents any base at that position; {A} means that any base other than A is found at that position. Y represents any pyrimidine, and R represents any purine.

[0209] In this application, "synthetic" means a nucleic acid molecule that does not occur in nature. The synthetic nucleic acids of the present invention are produced artificially, usually by recombinant techniques or de novo synthesis. Such synthetic nucleic acids may contain naturally occurring sequences (e.g., promoters, enhancers, introns, and other such regulatory sequences), but these may be present in circumstances not found in nature. For example, synthetic genes (or parts of genes) usually contain one or more nucleic acid sequences that are not contiguous in nature (chimeric sequences), and / or may include substitutions, insertions, deletions, and combinations thereof.

[0210] As used herein, "complementary" or "complementarity" refers to the Watson-Crick base pairing of two nucleic acid sequences. For example, for the sequence 5'-AGT-3', the complementary sequence 3'-TCA-5' binds. Complementarity between two nucleic acid sequences can be "partial," where only some of the bases bind to their complement, or it can be complete, where all the bases in the sequence bind to their complementary bases.

[0211] As used herein, the term "administration" refers to the introduction of a xenobiotic substance into a human or animal body. Administration may be, for example, intravenous, intra-arterial, or intracranial.

[0212] In this application, “transfection” broadly refers to any process of systematically introducing nucleic acids into cells, including the introduction of viruses and non-viral vectors, and includes or is equivalent to transformation, transduction, and similar terms and processes. Examples, though not limited to these, include transfection using viral vectors, transformation using plasmid vectors, electroporation (Fromm et al. (1986) Nature 319:791-73), lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7), microinjection (Mueller et al. (1978) Cell 15:579-85), Agrobacterium-mediated transduction (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7), direct DNA uptake, whisker-mediated transformation, and particulate guns (Klein et al. (1987) Nature 327:70).

[0213] As used herein, the term “transgene” refers to an exogenous nucleic acid sequence. In one example, the transgene is a gene that codes for an industrially or pharmaceutically useful compound or a gene that codes for a desirable trait. In yet another example, the transgene codes for a useful nucleic acid, such as an antisense nucleic acid sequence, and the expression of the antisense nucleic acid sequence inhibits the expression of the target nucleic acid sequence. The transgene preferably codes for a therapeutic product, such as a protein.

[0214] The term “vector” is well known in the art and, as used herein, refers to a nucleic acid molecule, such as double-stranded DNA, which may contain an inserted nucleic acid sequence according to the present invention. Vectors are used as appropriate to transport the inserted nucleic acid molecule into a suitable host cell. Vectors typically contain all the necessary elements to enable the transcription of the inserted nucleic acid molecule, preferably the translation of the transcript into a polypeptide. Vectors typically contain all the essential elements so that, once the vector is in a host cell, the vector can replicate independently of or simultaneously with the host chromosomal DNA, producing several copies of the vector and its inserted nucleic acid molecule. Vectors of the present invention may be episomal vectors (i.e., not integrated into the host cell genome) or vectors integrated into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, bluescript vectors (pBS), and their derivatives lacking pBR322 or bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors, such as artificial chromosomes (bacterial (BAC), yeast (YAC), or human (HAC)), may be used to accommodate larger insertion regions. Viral vectors are derived from, but are not limited to, viruses, and include retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpesviruses, hepatitis virus vectors, etc. Viral vectors are usually, though not always, replication-deficient because the viral genes essential for replication have been removed from the viral vector, thus losing their ability to proliferate in a given cell. However, some viral vectors can also be adapted to specifically replicate in a given cell, such as cancer cells, and are typically used to induce (cancer) cell-specific (tumor) lysis.Virosoms are an unspecified example of vectors containing both viral and nonviral elements, particularly those combining liposomes with inactivated HIV or influenza virus (Yamada et al., 2003). Another example involves viral vectors mixed with cationic lipids.

[0215] The terms “operably linked,” “operably connected,” or equivalent expressions, as used herein, refer to the arrangement of various nucleic acid elements relative to each other such that the elements are functionally connected and can interact with each other in the intended manner. Such elements include, but are not limited to, promoters, CREs (e.g., enhancers or other regulatory elements), promoter elements, polyadenylated sequences, one or more introns and / or exons, and coding sequences of the gene of interest to be expressed. When appropriately oriented or operationally linked, nucleic acid sequence elements may act together to modulate the activity of each other, ultimately affecting the level of expression of the expression product. Modulation means increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements can be expressed in terms of its 5' and 3' ends or its position upstream or downstream of another element or part (e.g., TSS or promoter element), and the distance between any particular elements can be referred to by the number of interposing nucleotides or base pairs between the elements. As those skilled in the art will understand, "operably linked" implies functional activity and is not necessarily related to innate positional linkage. In fact, when used in nucleic acid expression cassettes, CREs are usually located immediately upstream of the promoter element (this is a general case, but should not be explicitly interpreted as a restriction or exclusion of position within the nucleic acid expression cassette), but this is not necessary in vivo. For example, a naturally occurring regulatory element sequence downstream of the gene whose transcription is affected can function in the same way as if it were located upstream of the promoter. Thus, according to certain specific examples, the regulatory or enhancing effect of a regulatory element may be position-independent.

[0216] "Spacer sequence" or "spacer," as used herein, is a nucleic acid sequence that separates two functional nucleic acid sequences (e.g., TFBS, CRE, CRM, promoter element, etc.). It can be essentially any sequence, as long as it does not interfere with the functional nucleic acid sequence (e.g., a cis-regulatory element) functioning as desired (for example, this may occur if a silencer sequence is included, which would interfere with the binding of a desired transcription factor or similar). It is usually non-functional, as it exists only to position adjacent functional nucleic acid sequences at a distance from each other. In some embodiments, the spacer may have a length of 75, 50, 40, 30, 30, or 10 nucleotides or shorter.

[0217] Where used herein, the term "pharmaceutically acceptable" means consistent with the art, compatible with other components of a pharmaceutical composition, and not harmful to its recipient.

[0218] "Therapeutic dose" and similar phrases mean, for example, a dose or plasma concentration in a subject that provides a desired specific pharmacological effect, such as expressing a therapeutic gene in the CNS. While a therapeutic dose may not always be effective in treating the conditions described herein, such a dose will be considered therapeutically effective by those skilled in the art. Therapeutic doses may vary depending on the route and form of administration, the age and weight of the subject, and / or the disease or condition being treated.

[0219] The term “AAV vector,” as used herein, refers to an AAV vector nucleic acid sequence that is well known in the art and generally includes various nucleic acid sequences. As used herein, an AAV vector typically includes a heterogeneous nucleic acid sequence that is not of AAV origin as part of the vector. This heterogeneous nucleic acid sequence typically includes a promoter and other sequences intended for the genetic transformation of cells, as disclosed herein. Generally, the heterogeneous nucleic acid sequence is flanked by at least one, generally two, AAV reverse-terminal repeat sequences (ITRs). “AAV virion,” “AAV virus,” “AAV virus particle,” or “AAV vector particle” refers to a viral particle consisting of at least one AAV capsid polypeptide (including both variant AAV capsid polypeptides and non-variant parental capsid polypeptides) and a capsid-formed polynucleotide AAV vector. If the particle contains heterogeneous nucleic acid (i.e., polynucleotides other than the wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell), it may also be called an “AAV vector particle” or simply an “AAV vector.” Therefore, the production of an AAV virion or AAV particle necessarily involves the production of an AAV vector, since such a vector is contained within the AAV virion or AAV particle. The ITR may be derived from the same serotype as the capsid, or from a different serotype, selected from any of the serotypes listed in Table 1. An AAV vector typically has more than one ITR. In an example not limited to this, an AAV vector has a viral genome containing two ITRs. In one embodiment, the ITRs are of the same serotype as each other. In another embodiment, the ITRs are of different serotypes. Examples not limited to this include zero, one, or both ITRs that are of the same serotype as the capsid. Independently, each ITR may be about 100 to about 150 nucleotides in length.The ITR may be approximately 100–105 nucleotides long, 106–110 nucleotides long, 111–115 nucleotides long, 116–120 nucleotides long, 121–125 nucleotides long, 126–130 nucleotides long, 131–135 nucleotides long, 136–140 nucleotides long, 141–145 nucleotides long, or 146–150 nucleotides long. In one embodiment, the ITR is 140–142 nucleotides long. Examples of non-limiting ITR lengths include 102, 105, 130, 140, 141, 142, and 145 nucleotides long.

[0220] As used herein, the term “microRNA” refers to any type of interfering RNA, including but not limited to endogenous microRNAs and artificial microRNAs (e.g., synthetic miRNAs). Endogenous microRNAs are small RNAs naturally encoded in the genome that can modulate the generative utilization of mRNA. Artificial microRNAs can be any type of RNA sequence other than endogenous microRNAs that can modulate mRNA activity. MicroRNA sequences can be RNA molecules composed of any one or more of these sequences. MicroRNA (or "miRNA") sequences are described in publications such as Lim et al., 2003, Genes & Development, 17, pp. 991-1008; Lim et al., 2003, Science, 299, pp. 1540; Lee and Ambrose, 2001, Science, 294, pp. 862; Lau et al., 2001, Science, 294, pp. 858-861; Lagos-Quintana et al., 2002, Current Biology, 12, pp. 735-739; Lagos-Quintana et al., 2001, Science, 294, pp. 853-857; and Lagos-Quintana et al., 2003, RNA, 9, pp. 175-179. Examples of microRNAs include any RNA fragment of a large RNA, or miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNAs. See, for example, U.S. Patent Applications 20050272923, 20050266552, 20050142581, and 20050075492. A "microRNA precursor" (or "pre-miRNA") refers to a nucleic acid having a stem-loop structure into which a microRNA sequence is incorporated. "Mature microRNA" (or "mature miRNA") includes microRNAs that have been cleaved from microRNA precursors ("pre-miRNA") or synthesized (e.g., synthesized in the laboratory by cell-inclusive synthesis), and have a length of approximately 19 to 27 nucleotides. For example, mature microRNAs may have lengths of 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, or 27nt.Mature microRNA can bind to target mRNA and inhibit the translation of target mRNA.

[0221] The term "treatment" or "treating" refers to reducing, alleviating or eliminating one or more symptoms, signs or effects of a disease or condition. "Treatment", as used herein, thus includes any treatment of a disease in a mammal, particularly in a human, including (a) preventing a disease from occurring in a subject who is predisposed to having or at risk of acquiring the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., arresting its development, and (c) alleviating the disease, i.e., causing regression of the disease.

[0222] "Administration" of an agent to a subject includes any route of introduction or delivery of the agent to the subject for the purpose of exerting its intended function. Administration can be effected by any suitable route, including orally, intranasally, intraocularly, by eye drops, parenterally (intravascularly, intramuscularly, intraperitoneally or subcutaneously) or topically. Administration includes self-administration and administration by another person. Intravenous or intraarterial administration is of particular interest in the present invention.

[0223] The terms "individual", "subject" and "patient" are used synonymously and refer to any individual subject having a disease or condition for which treatment is needed. For the purposes of the present disclosure, the subject can be a primate, preferably a human or another mammal, such as a dog, cat, horse, pig, goat or cow, etc.

[0224] The term "specifically active in a region or in a tissue" refers to a promoter that is predominantly active in that region or tissue, i.e., more active in that region or tissue than in other regions or tissues.

Examples

[0225] (Example 1) CNS transduction and vector distribution in vivo of CNS1-8 (SEQ ID NOs. 1-4, 23-26) operably linked to GFP were studied using AAV9. AAV plasmid preparation: hSyn.GFP plasmid containing the ssAAV2 reverse terminal repeat sequence was obtained from Addgene and used to generate a control AAV vector (Synapsin-1). The CNS1-8 (SEQ ID NOs. 1-4, 23-26) promoters were cloned into the hSyn.GFP plasmid and replaced the hSyn promoter by GeneArt® (Thermo Fisher Scientific, Germany). All plasmid DNA was prepared using the PureLink® HiPure Plasmid Maxiprep kit (number K210007; Thermo Fisher Scientific, Germany) according to the manufacturer's instructions and quantified using an Omega FLUOstar spectrophotometer (BMG Labtech, UK).

[0226] AAV Vector Preparation: Recombinant AAV2 / 9 (collectively referred to as AAV9) vectors encoding GFP were generated by a standard triple plasmid transfection method. Briefly, virus-producing human fetal kidney (HEK) 293T cells were co-transfected with three plasmids, pGFP regulated by various promoters (SEQ ID NOs. 1-4, 23-26), pGD9 encoding the AAV9 capsid, and pHGTI containing helper functions, using polyethyleneimine (PEI) (No. 24765; Polysciences, UK) at a stock concentration of 1 mg / ml in a molar ratio of 1:3:1. After 72 hours, cells were collected and lysed. The cell lysates and supernatants were treated with nucleases, filtered, and purified by affinity chromatography using POROS™ CaptureSelect™ AAVX resin (Thermo Fisher Scientific, Germany) with Primeview 5.0 software on AKTAprime plus (GE Healthcare Ltd, UK).

[0227] AAV Vector Titration: All vector preparations were titrated by qPCR against the GFP transgene using a QuantStudio® 3 system real-time PCR (Thermo Fisher Scientific, UK) according to the instructions for use of Luna® Universal qPCR Master Mix (No. M3003; New England Biolabs, UK). Data were analyzed using QuantStudio design and analysis software V5. The number of vector genomes was determined using primers designed to amplify segments of the GFP transgene (Table 5). All vectors were 1 × 10⁶ 13 The titer was matched to the vector genome / mL (vg / mL).

[0228] Animal Procedures: All animal experiments were conducted in accordance with the UK Home Office's Regulation and Animals (Scientific Procedures) Act 1986, within the guidelines of the University College London Ethics Review Board. Non-inbred CD1 mice (Charles River, UK) were housed in individually ventilated incubator cages (IVCs) at the Central Biological Services Unit, UCL, under standard conditions including a 12-hour light-dark cycle, constant temperature (21–23°C), humidity (60% ± 5%), and free access to solid feed and water. Experimental breeding pairs were time-mated after 6 weeks of age, and newborn littermates were used in these promoter studies. The offspring were weaned at P21 and euthanized at P35 for histological analysis.

[0229] Animal injection: All offspring were injected on the day of birth (P0). Offspring were subjected to transient hypothermic anesthesia before each injection method. For each injection method, four mice were injected per vector type, along with four uninjected controls, each uniquely identified by foot tattooing. Offspring were warmed to normal temperature and then returned to their mothers.

[0230] Intracranial injection of viral vector in neonatal pups: Neonatal pups were injected with 5 μl of viral vector (5 × 10 10 viral genomes / pup) into the cerebral lateral ventricles using a 33-gauge Hamilton needle (Fisher Scientific, UK) with established coordinates (Kim, Ji-Yoen et al., 2013), which are incorporated herein by reference. Injection into the ventricles bypasses the blood-brain barrier.

[0231] Intravenous injection of viral vector in neonatal pups: Neonatal pups were injected with 20 μl of viral vector (2 × 10 11 vg / pup) into the superficial temporal vein. The vein was visualized using fiberoptic transillumination, and the injection was performed using a 33-gauge Hamilton needle and a stereotactic microscope (Zeiss, Germany).

[0232] Perfusion and tissue preparation: Animals were anesthetized with isoflurane (5% induction chamber via nose cone, 1.5% maintenance). Transcardiac perfusion was performed by cutting the right atrium and injecting 10 mL of autoclaved PBS (phosphate-buffered saline) into the left ventricle until liver whitening was achieved. The brain and visceral organs were divided into two parts to enable different processing techniques depending on the following experiments. Half of the samples used for immunohistochemistry were post-fixed in 4% paraformaldehyde (PFA) for 48 hours and transferred to a 30% sucrose solution for cryoprotection at 4 °C until sectioning. Half of the brain was mounted on a cryomicrotome (Thermo Fisher HM430) at 40 μm thickness in either the coronal or sagittal plane and stored in TBSAF (Tris-buffered saline (TBS), 30% ethylene glycol, 15% sucrose, 0.05% sodium azide) at 4 °C. The half of the brain and visceral organ tissues used for molecular biology evaluation experiments were snap-frozen in dry ice and stored at -80 °C. Vector copy number (VCN) and gene expression (cDNA) qPCR analyses were performed according to standard DNA and / or RNA extraction protocols, respectively.

[0233] Histological analysis of GFP expression: GFP expression in mouse brains was evaluated by immunohistochemistry (IHC) and immunofluorescence (IHF).

[0234] Free-floating intravascular coagulation (IHC) using diaminobenzidine (DAB) immunoperoxidase staining: Brain sections were selected for either whole-brain analysis or sections from various brain regions (olfactory bulb, prefrontal cortex, striatum, hippocampus, midbrain, and cerebellum). All washing steps were performed three times in 1×TBS at room temperature (RT).

[0235] All brain sections were washed and then treated with 30% H2O2 in 1x TBS (Sigma Aldrich, UK) for 30 minutes, followed by blocking at RT for 30 minutes with 15% normal goat serum in TBST (1x TBS, 0.3% Triton X-100) (Vector Laboratories, UK). Samples were incubated with primary antibody (rabbit or chicken anti-GFP antibody from Table 6) at 4°C on an orbital shaker with constant agitation for 12–14 hours. Sections were washed and incubated at RT for 2 hours on an orbital shaker with the corresponding biotinylated secondary antibody (anti-rabbit or anti-chicken biotinylated secondary antibody from Table 6). Sections were washed and incubated with vector stain avidin-biotin solution (ABC Vector Stain, Vector Laboratories, UK). Sections were washed, and the reaction was visualized using DAB (Sigma Aldrich, UK) (10 mg DAB in 20 mL TBS and 6 ml 30% H2O2). The reaction was stopped after up to 7 minutes using ice-cold 1x TBS, and then mounted on glass slides.

[0236] Free-floating immunofluorescence: The same protocol as for DAB immunoperoxidase staining was used. Sections were washed with 1x TBS and blocked in 15% normal goat serum for 30 minutes. Sections were incubated with optimal primary antibodies (transgene markers and cell type markers from Table 6, rabbit / chicken anti-GFP and rabbit / chicken anti-tyrosine hydroxylase) diluted in 10% normal goat serum TBST and incubated overnight at 4°C. Sections were washed with TBS and incubated for 2 hours in secondary fluorophores (anti-chicken / rabbit Alexa flour secondary antibody from Table 6) diluted in 10% normal goat serum covered with RT. Sections were washed, treated with DAPI (4',6-diamidino-2-phenylindole, Sigma Aldrich, UK) for 2 minutes, transferred to ice-cold 1x TBS, and then mounted on glass slides.

[0237] Microscopy: Optical microscopy and fluorescence imaging were performed using a Leica DM4000B. All images were captured using a Leica DFC420 camera and Leica Application Suite V3.7 software, while maintaining constant light intensity, exposure, microscope calibration, and camera settings (Leica Microsystems, UK).

[0238] Quantitative measurements of 10 non-overlapping RGB images at 40x magnification of GFP staining intensity were performed by threshold analysis in selected brain regions: cortex, hippocampus, striatum, midbrain, and cerebellum. Foreground immunostaining was defined by averaging the highest and lowest signals, and the mean area percentage of immunoreactivity per field in each region of interest was calculated using Image-Pro 10 software (Media Cybernetics, USA).

[0239] Midbrain dopaminergic (mDA) neurons were quantified by counting TH-positive neurons and vector-driven GFP-expressing cells, and the percentage of bipositive neurons was calculated.

[0240] qRT-PCR for vector expression analysis: RNA was extracted from the brain and organs using the TRIzol® Plus RNA purification kit (Thermo Fisher Scientific, Germany) or the RNeasy mini-kit (Qiagen, UK) and quantified using Omega FLUOstar (BMG Labtech, UK). Contaminating DNA was removed from the total RNA (1-2 μg) using the DNase I (DNAse I) purification kit (NEB, UK), and then reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Bioscience, Thermo Fisher Scientific, Germany). Using 10 ng of cDNA, qPCR was performed using the Quantstudio™ Real-Time PCR System (Applied Biosystems, UK) with the Luna Taqman master mix (NEB, UK) and 300 nM primers (Table 5).

[0241] For the quantification of GFP transcripts, standardization was achieved by comparison with standard curves created by amplification from plasmid constructs specific to GFP and mGAPDH transcripts. mGAPDH was used as an endogenous control, and relative change multipliers were calculated as described for vector genome copy number analysis.

[0242] [Table 1]

[0243] [Table 2]

[0244] CNS 1-8 Construct Design: The promoter in this invention was designed by a combination of bioinformatics analysis and literature review.

[0245] CNS-5_v2, CNS-6_v2, CNS-7_v2, and CNS-8_v2 (SEQ ID NOs. 5-8) are longer versions of the promoters CNS-5, CNS-6, CNS-7, and CNS-8 (SEQ ID NOs. 23-26). That is, CNS-5_v2, CNS-6_v2, CNS-7_v2, and CNS-8_v2 (SEQ ID NOs. 5-8) are shortened, and the minimal promoter SYNP_CRE151 (SEQ ID NO. 12) is appended to this shorter version, resulting in CNS-5, CNS-6, CNS-7, and CNS-8 (SEQ ID NOs. 23-26). Due to the high sequence similarity between the longer and shorter versions of these synthetic promoters (e.g., CNS-5_v2 and CNS-5), they can be predicted to have similar expression patterns.

[0246] Results: GFP expression from CNS-1 to CNS-8 promoters (SEQ ID NOs: 1-4, 23-26) and the control promoter Syn1 (SEQ ID NO: 14) was first evaluated in the sagittal section, and the results are shown in Figures 2A-B. All tested promoters showed CNS expression, with varying intensities and distributions across various brain regions.

[0247] In animals injected with ICV, CNS-1 (SEQ ID NO: 1) showed the strongest expression, CNS-3 (SEQ ID NO: 3) showed the weakest expression, and the remaining promoters were between the two extremes. In particular, the expression of CNS-1 (SEQ ID NO: 1) was stronger and more uniform in the brain than the expression from the control promoter Syn1 (SEQ ID NO: 14).

[0248] In animals injected intravenously, CNS-4 (SEQ ID NO: 4) showed the strongest expression, CNS-3 (SEQ ID NO: 3) showed the weakest expression, and the remaining promoters were between the two extremes. Promoters CNS-1 to CNS-8 (SEQ ID NOs: 1 to 4, 23 to 26) all showed weaker expression than the control promoter Syn1.

[0249] Therefore, the method of administration (ICV vs. IV) affects both the strength and distribution of the CNS promoter.

[0250] GFP expression from CNS-1 to CNS-8 (SEQ ID NOs. 1-4, 23-26) promoters delivered by ICV, and from the control promoter Syn1 (SEQ ID NOs. 14), was then evaluated in coronal sections, and the results are shown in Figures 3A-B. Again, all tested promoters showed CNS expression, with varying intensities and distributions across various brain regions. Promoters CNS-1, CNS-2 (SEQ ID NOs. 1-2), and CNS-7 (SEQ ID NOs. 25) showed the strongest expression, CNS-3 (SEQ ID NOs. 3) showed the weakest expression, and the remaining promoters were between the two extremes. Promoters CNS-1 (SEQ ID NOs. 1), CNS-2 (SEQ ID NOs. 2), and CNS-7 (SEQ ID NOs. 25) showed similar expression levels to the control promoter Syn1.

[0251] GFP expression from CNS-1 to CNS-8 (SEQ ID NOs. 1-4, 23-26) promoters delivered by IV was also evaluated in the coronal section, and the results are shown in Figures 4A-B. Again, all tested promoters showed CNS expression, with varying intensities and distributions across various brain regions. Promoter CNS-3 (SEQ ID NOs. 3) showed the strongest expression, CNS-8 (SEQ ID NOs. 26) showed the weakest expression, and the remaining promoters were between these two extremes.

[0252] GFP expression from CNS-1 to CNS-8 (SEQ ID NOs. 1-4, 23-26) promoters delivered by ICV, and from the control promoter Syn1 (SEQ ID NOs. 14), was then visualized at higher magnification in coronal sections, and the results are shown in Figures 5A-B. At this higher magnification, CNS-1 (SEQ ID NOs. 1), CNS-2 (SEQ ID NOs. 2), and CNS-7 (SEQ ID NOs. 25) showed widespread intracranial expression, with CNS-1 (SEQ ID NOs. 1) showing the strongest expression. This expression appeared to be primarily neuronal for CNS-1 (SEQ ID NOs. 1) and CNS-2 (SEQ ID NOs. 2). As shown in Figure 11, the primarily neuronal expression of GFP when driven by CNS-1 (SEQ ID NOs. 1) in ICV delivery was also confirmed by double staining for CNS cell type. GFP expression when driven by CNS-7 (SEQ ID NOs. 25) was neuronal and astrocytic. CNS-3 (SEQ ID NO: 3) and CNS-4 (SEQ ID NO: 4) showed weaker expression, localized to the cortex and hippocampus. Expression for both CNS-3 (SEQ ID NO: 3) and CNS-4 (SEQ ID NO: 4) appeared to be primarily neuronal and astrocellular. CNS-5 (SEQ ID NO: 23) was more potently active in the cortex, striatum, hippocampus, and midbrain, but less so in the cerebellum. CNS-6 (SEQ ID NO: 24) and CNS-8 (SEQ ID NO: 26) were potently active in the hippocampus, followed by the cortex and midbrain, with less expression in other parts of the brain tested. Expression for CNS-6 (SEQ ID NO: 24) and CNS-8 (SEQ ID NO: 26) appeared to be primarily neuronal.

[0253] GFP expression from CNS-1~CNS-8 (SEQ ID NOs. 1~4, 23~26) promoters delivered by IV was visualized at higher magnification in the coronal section, and the results are shown in Figures 6A~B. CNS-1 (SEQ ID NOs. 1) was highly active in the cortex and hippocampus. Low levels of CNS-2 (SEQ ID NOs. 2) expression were observed in most tested areas away from the midbrain. CNS-3 (SEQ ID NOs. 3) and CNS-4 (SEQ ID NOs. 4) were expressed in the cortex, striatum, and hippocampus, with CNS-4 (SEQ ID NOs. 4) being expressed in the midbrain, but not with CNS-3 (SEQ ID NOs. 3). CNS-5 (SEQ ID NOs. 23) had minimal expression in all tested brain regions. CNS-6 (SEQ ID NOs. 24) was expressed in the hippocampus, midbrain, and cerebellum. CNS-7 (SEQ ID NOs. 25) was expressed in the cortex, hippocampus, and midbrain. CNS-8 (SEQ ID NO: 26) is active in the hippocampus and midbrain.

[0254] Expression from CNS1-8 (SEQ ID NOs. 1-4, 23-26) promoters and the control promoter Syn1, delivered by ICV, was visualized at higher magnification in the midbrain, and the results are shown in Figures 7A-B. CNS1-4 (SEQ ID NOs. 1-4) and Syn1 (SEQ ID NOs. 14) showed some GFP expression in the midbrain. Double staining with a marker for dopaminergic neurons (TH+) showed that some GFP expression from the Syn1 (SEQ ID NOs. 14) promoter was localized to dopaminergic neurons, while only a small amount of GFP expression from CNS1-4 (SEQ ID NOs. 1-4) was localized to dopaminergic neurons. CNS-6 (SEQ ID NOs. 24) and CNS-7 (SEQ ID NOs. 25) showed minimal expression in the midbrain. CNS-5 (SEQ ID NOs. 23) showed expression in the midbrain, but its expression did not appear to be localized to dopaminergic neurons. The majority of GFP expression driven by the CNS-8 (SEQ ID NO: 26) promoter is localized to dopaminergic neurons.

[0255] Expression from CNS1-8 (SEQ ID NOs. 1-4, 23-26) promoters delivered by IV was also visualized in the midbrain, and the results are shown in Figures 7A-B. CNS-1-4 (SEQ ID NOs. 1-4) showed minimal GFP expression in the midbrain, and the majority of GFP-positive cells were not dopaminergic neurons. CNS-5 (SEQ ID NOs. 23), CNS-6 (SEQ ID NOs. 24), and CNS-7 (SEQ ID NOs. 25) showed no GFP expression in the midbrain after IV delivery. On the other hand, CNS-8 (SEQ ID NOs. 26) showed strong expression in the midbrain, and the majority of cells showing GFP expression were dopaminergic neurons.

[0256] Figure 9 shows the in vivo distribution of the transgene GFP in various tissues, under the control of CNS-1~8 (SEQ ID NOs. 1~4, 23~26) and the control promoter Syn-1 (SEQ ID NO. 14), delivered by ICV and IV.

[0257] In ICV delivery, CNS-1 to 4 (SEQ ID NOs: 1 to 4) showed activity in the heart, while the remaining tested promoters, CNS-5 to 8 (SEQ ID NOs: 23 to 26), were not active in the heart. In IV delivery, CNS-1 to 4 (SEQ ID NOs: 1 to 4) showed activity in the heart, while the remaining tested promoters, CNS-5 to 8 (SEQ ID NOs: 23 to 26), were not active in the heart. The control promoter Syn-1 also showed very low activity in the heart in both ICV and IV delivery.

[0258] In ICV delivery, CNS-1-4 (SEQ ID NOs: 1-4), CNS-6 (SEQ ID NO: 24), CNS-8 (SEQ ID NO: 26), and the control promoter Syn-1 (SEQ ID NO: 14) showed activity in the liver, while the remaining tested promoters did not. In IV delivery, CNS-1-4 (SEQ ID NOs: 1-4), CNS-6 (SEQ ID NO: 24), CNS-8 (SEQ ID NO: 26), and the control promoter Syn-1 (SEQ ID NO: 14) showed activity in the liver, while the remaining tested promoters did not.

[0259] In ICV delivery, CNS1-3 (SEQ ID NOs: 1-3) and CNS-8 (SEQ ID NO: 26) showed activity in the kidney, while CNS4-7 (SEQ ID NOs: 4, 23-25) and the control promoter Syn-1 (SEQ ID NO: 14) did not show activity in the kidney. In IV delivery, CNS2-3 (SEQ ID NOs: 2-3) and CNS-8 (SEQ ID NO: 26) showed activity in the kidney, while the remaining tested promoters and the control promoter Syn-1 (SEQ ID NO: 14) did not.

[0260] In ICV delivery, CNS-3 (SEQ ID NO: 3) shows activity in skeletal muscle, while CNS-1~2 (SEQ ID NOs: 1~2) and CNS-4~8 (SEQ ID NOs: 4, 23~26) do not show activity in skeletal muscle. In IV delivery, CNS-1~3 (SEQ ID NOs: 1~3) show activity in skeletal muscle, while CNS-4~8 (SEQ ID NOs: 4, 23~26) do not show activity in skeletal muscle. The control promoter Syn-1 (SEQ ID NO: 14) does not show activity in skeletal muscle.

[0261] In ICV delivery, CNS-1 (SEQ ID NO: 1) and CNS-7~8 (SEQ ID NOs: 25~26) show activity in the spleen, while CNS-2~6 (SEQ ID NOs: 2~4, 23~24) and the control promoter Syn-1 (SEQ ID NO: 14) do not show activity in the spleen. In IV delivery, CNS-2 (SEQ ID NO: 2) and CNS-7~8 (SEQ ID NOs: 25~26) show activity in the spleen, while CNS-1 (SEQ ID NO: 1), CNS-3~6 (SEQ ID NOs: 3~4, 23~24) and the control promoter Syn-1 (SEQ ID NO: 14) do not show activity in the spleen.

[0262] mm 2The GFP immunoreactivity percentage per unit area was measured in various brain regions, and the results are shown in Figure 10. As previously mentioned, GFP was controlled by CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), CNS-3 (SEQ ID NO: 3), CNS-4 (SEQ ID NO: 4), CNS-5 (SEQ ID NO: 23), CNS-6 (SEQ ID NO: 24), CNS-7 (SEQ ID NO: 25), CNS-8 (SEQ ID NO: 26), and control promoter Syn-1 (SEQ ID NO: 14), delivered by ICV and IV. With ICV delivery, control promoter Syn-1 (SEQ ID NO: 14), CNS-1 (SEQ ID NO: 1), and CNS-2 (SEQ ID NO: 2) showed extremely high GFP immunoreactivity percentages in the cortex, followed by CNS-7 (SEQ ID NO: 25) and CNS-4 (SEQ ID NO: 4), while the remaining promoters tested showed very little or no GFP immunoreactivity in the cortex. In IV delivery, the control promoter Syn-1 (SEQ ID NO: 14) exhibited a high percentage of GFP immunoreactivity in the cortex, while the remaining promoters tested showed very little or no GFP immunoreactivity in the cortex.

[0263] In ICV delivery, CNS-1 (SEQ ID NO: 1) and CNS-2 (SEQ ID NO: 2) showed extremely high GFP immunoreactivity percentages in the striatum, followed by CNS-4 (SEQ ID NO: 4), CNS-5 (SEQ ID NO: 23), and CNS-7 (SEQ ID NO: 25). The remaining promoters tested showed very little or no GFP immunoreactivity in the striatum. In IV delivery, CNS-2 (SEQ ID NO: 2) and CNS-3 (SEQ ID NO: 3) showed low GFP immunoreactivity percentages in the striatum, and the remaining promoters tested showed very little or no GFP immunoreactivity in the striatum. The control promoter Syn-1 (SEQ ID NO: 14) showed extremely high GFP immunoreactivity in the striatum in both ICV and IV delivery.

[0264] In ICV delivery, CNS-1 to 8 (SEQ ID NOs: 1 to 4, 23 to 26) showed intermediate or high GFP immunoreactivity percentages in the hippocampus, and higher than the control promoter Syn-1 (SEQ ID NO: 14). In IV delivery, CNS-1 to 8 (SEQ ID NOs: 1 to 4, 23 to 26) showed very little or no GFP immunoreactivity in the hippocampus, while the control promoter Syn-1 (SEQ ID NO: 14) showed very high GFP immunoreactivity in the hippocampus.

[0265] In ICV delivery, CNS-1 (SEQ ID NO: 1) and CNS-2 (SEQ ID NO: 2) showed extremely high GFP immunoreactivity percentages in the midbrain, followed by CNS-7 (SEQ ID NO: 25), CNS-5 (SEQ ID NO: 23), and CNS-4 (SEQ ID NO: 4). The remaining promoters tested showed very little or no GFP immunoreactivity in the cortex. In IV delivery, CNS-1 to 8 (SEQ ID NOs: 1 to 4, 23 to 26) showed very little or no GFP immunoreactivity in the midbrain, while the control promoter Syn-1 (SEQ ID NO: 14) showed extremely high GFP immunoreactivity in the midbrain. In particular, CNS-8 (SEQ ID NO: 26) in IV delivery showed very low GFP immunoreactivity in the midbrain, but showed activity in dopaminergic neurons in the midbrain, as shown in Figure 8B.

[0266] In ICV delivery, CNS-1 to 8 (SEQ ID NOs: 1 to 4, 23 to 26) showed intermediate or low GFP immunoreactivity percentages in the cerebellum. The GFP immunoreactivity percentages of CNS-1 (SEQ ID NO: 1) and CNS-5 to 8 (SEQ ID NOs: 23 to 26) were higher than that of the control promoter Syn 1 (SEQ ID NO: 14). In IV delivery, the control promoter Syn 1 (SEQ ID NO: 14) had a high GFP immunoreactivity percentage in the cerebellum, while the remaining promoters tested showed very little or no GFP immunoreactivity in the cerebellum.

[0267] In particular, CNS-1 (SEQ ID NO: 1) showed high or intermediate GFP immunoreactivity percentages per region in all brain regions tested during ICV delivery. Similarly, CNS-2 (SEQ ID NO: 2) showed high GFP immunoreactivity percentages per region in four of the five tested regions (away from the cerebellum) during ICV delivery. CNS-8 (SEQ ID NO: 26) showed extremely low or no GFP immunoreactivity percentages per region in all brain regions tested, but still exhibited expression in dopaminergic neurons.

[0268] (Example 2) The in vivo distribution of the transgene GFP under the control of CNS-8 (SEQ ID NO: 26) was further investigated at higher doses (referred to as high doses herein) via IV and ICV delivery. Intracranial and intravenous injections were performed as described in Example 1, with intracranial injection yielding 5 × 10⁶ 11 Inject the viral genome / offspring, 2 × 10 12 The dose was administered intravenously at a dose of vg / pig (10 times higher). The doses used in IV and ICV delivery in Example 1 are referred to as low doses in this specification.

[0269] As shown in Figures 2B and 12, the in vivo distribution of GFP under CNS-8 control when a low dose was administered (Example 1) was very similar to the in vivo distribution of GFP under CNS-8 control when a high dose was administered (Example 2) in the sagittal section for both ICV and IV delivery.

[0270] Similarly, as shown in Figures 3B, 5B, and 13A, the in vivo distribution of CNS-8-regulated GFP after low-dose administration was remarkably similar to the in vivo distribution of CNS-8-regulated GFP after high-dose administration, both at high and low magnifications, in the coronal section during ICV delivery.

[0271] As shown in Figures 4B, 6B, and 13B, the in vivo distribution of GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at low doses was remarkably similar to the in vivo distribution of GFP under the control of CNS-8 when administered at high doses in the coronal section during IV delivery, at both high and low magnification.

[0272] Similarly, as shown in Figures 7B, 8B, and 14A, GFP expression under the control of CNS-8 (SEQ ID NO: 26) in the midbrain after low-dose administration was remarkably similar to GFP expression under the control of CNS-8 (SEQ ID NO: 26) after high-dose administration in both ICV and IV delivery. This was supported by the quantification of GFP-positive dopaminergic neurons, as shown in Figure 14B, which indicated no difference in the percentage of GFP-positive dopaminergic neurons between low and high-dose administration in both ICV and IV delivery. Therefore, although there was no overall difference in GFP expression under the control of the CNS-8 promoter (SEQ ID NO: 26) between low and high-dose administration, this indicates that low doses are sufficient to show GFP expression in dopaminergic neurons and that increasing the dose does not result in higher GFP expression. In some cases, the lowest dose that shows the desired expression pattern may be preferred.

[0273] A comparison of the in vivo distribution of the CNS-8 (SEQ ID NO: 26)-regulated transgene GFP in various tissues after low and high dose administration demonstrated that dose affected GFP expression. In the liver, GFP expression was very similar across doses for ICV delivery, but low for IV delivery. In the heart, GFP expression was not detected at low doses for either ICV or IV delivery, but was detected at high doses for IV delivery. Similarly, in skeletal muscle, GFP expression was not detected at low doses for either ICV or IV delivery, but was detected at high doses for both ICV and IV delivery. However, in the spleen, higher GFP expression was detected at low doses compared to high doses for both IV and ICV delivery. Similarly, in the kidney, higher GFP expression was detected at low doses compared to high doses for both IV and ICV delivery. These data suggest that administration of different doses of the viral genome may result in different expression patterns and levels in tissues other than the CNS.

[0274] Therefore, changing the dose did not alter GFP expression and levels in the CNS, but it did alter the expression pattern and levels in tissues other than the CNS. Thus, it may be possible to find an optimal dose that maintains the expression pattern and levels in the CNS while depending on the requirements for expression patterns and levels in tissues other than the CNS. For example, if activity in the CNS as well as in the liver, spleen, and kidneys is required, a low dose may be administered via ICV or IV. Alternatively, if activity in the CNS as well as in at least the heart and skeletal muscle is required, a high dose may be administered via IV delivery.

[0275] (Example 3) The tissue expression patterns of the faf1 and pitx3 genes, for which CRE / proximal promoters were designed from CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4, were investigated in a single-cell transcriptome dataset (Zeisel et al., 2018). Due to the proximity of the CRE / proximal promoter to the gene, it is predicted that the CRE / proximal promoter assists in gene regulation (He et al., 2014). Assuming that the CRE / proximal promoter regulates the expression of its nearest gene, the gene expression pattern provides an indicator of a possible expression profile of the synthetic promoter, including the CRE / proximal promoter.

[0276] The single-cell transcriptome dataset (Zeisel et al., 2018) contains single-cell RNA sequencing of 500,000 cells of any type derived from the CNS and PNS of adult mice. The resource is publicly available at mousebrain.org / genesearch.html and provides a useful tool for determining the possible expression of synthetic promoters CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4 in the PNS. Genes with designed CRE / proximal promoters for CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4 have been added to the web tool, and the expression patterns of the faf1 and pitx3 genes are shown in Figures 16A and 16B. The gray gradient indicates the intensity of RNA expression detected in the database (Zeisel et al., 2018). faf1 is expressed in a large number of PNS neurons; therefore, synthetic promoters containing a CRE or proximal promoter designed from the faf1 gene, such as CNS-5 and CNS-5_v2, are predicted to have strong expression in the PNS. pitx3 is expressed in sympathetic PNS neurons; therefore, synthetic promoters containing a CRE designed from the pitx3 gene, such as CNS-2, CNS-3, or CNS-4, are predicted to have expression in PNS sympathetic neurons. Similar analyses of lmx1b and pitx2 showed no expression in the PNS above the analysis cutoff score (trinization score less than 0.95, data not shown); therefore, CNS-1, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are not predicted to be active in PNS neurons.

[0277] ReferencesBoussicault, L. et al. (2016) 'CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease', Brain, 139(3), pp. 953-970. doi: 10.1093 / brain / awv384.Djelti, F. et al. (2015) 'CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer's disease', Brain, 138(8), pp. 2383-2398. doi: 10.1093 / brain / awv166.Hammond, S. L. et al. (2017) 'Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection', PLoS ONE, 12(12), pp. 1-22. doi: 10.1371 / journal.pone.0188830.He, B. et al. (2014) 'Global view of enhancer-promoter interactome in human cells', Proceedings of the National Academy of Sciences of the United States of America, 111(21). doi: 10.1073 / pnas.1320308111.Jakobsson, J. and Lundberg, C. (2006) 'Lentiviral vectors for use in the central nervous system', Molecular Therapy. The American Society of Gene Therapy, 13(3), pp. 484-493. doi: 10.1016 / j.ymthe.2005.11.012.Kacher, R. et al. (2019) 'CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington's disease', Brain: a journal of neurology, 142(8), pp. 2432-2450. doi: 10.1093 / brain / awz174.Kim, Ji-Yoen; Ash, Ryan T.; CAballos-Diaz; CArolina, Levites, Yona; Golde, Todd E.;Smirnakis, Stelios M.; Jankowsky, J. L. (2013) 'Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo', European Journal of Neuroscience, 37(8), pp. 1203-1220. doi: 10.1111 / ejn.12126.Viral.Tanguy, Y. et al. (2015) 'Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice', Frontiers in Molecular Neuroscience, 8(JULY), pp. 1-10. doi: 10.3389 / fnmol.2015.00036.Zeisel, A. et al. (2018) 'Molecular Architecture of the Mouse Nervous System', Cell, 174(4), p. 999-1014.e22. doi: 10.1016 / j.cell.2018.06.021.

[0278] Sequence information

[0279] Table 3A

[0280] Table 3B

[0281]

Table 3C

[0282]

Table 3D

[0283] Table 3E

[0284] Table 4A

[0285] Table 4B

[0286] Table 5

[0287] Table 6

[0288] Synapsin-1 (SEQ ID NO: 14)

[0289] [ka]

Claims

1. A synthetic CNS-specific promoter containing a sequence or a functional variant thereof that follows one of sequence numbers 1-8 or 21-26.

2. The synthetic CNS-specific promoter according to claim 1, comprising a sequence that is at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 1-8 or 21-26.

3. A synthetic CNS-specific promoter according to claim 1 or 2, wherein a functional variant of the synthetic CNS-specific promoter retains at least 25%, 50%, 75%, 80%, 85%, 80%, 95%, or 100% of the activity of the reference promoter.

4. A CNS-specific cis-regulatory element (CRE) containing a sequence following any one of sequence numbers 9-11 or 28-31, or a functional variant thereof.

5. A CNS-specific cis-regulating element (CRE) according to claim 4, comprising a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 9-11 or 28-31.

6. A synthetic CNS-specific cis-regulating module (CRM) comprising the CRE described in claim 4 or 5.

7. A synthetic CNS-specific promoter comprising the CRE described in claim 4 or 5 or the CRM described in claim 6.

8. An isolated minimal or proximal promoter containing a sequence or a functional variant thereof that follows any one of sequence numbers 12-13.

9. An isolated minimal or proximal promoter according to claim 8, comprising a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 12 to 13.

10. A synthetic CNS-specific promoter comprising the minimal or proximal promoter described in claim 8 or 9.

11. A synthetic CNS-specific promoter according to any one of claims 1 to 3, comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 21 or a functional variant thereof, which is broadly active in the brain when administered by intraventricular (ICV) injection.

12. The synthetic CNS-specific promoter according to claim 11, which is active at a level of at least 100%, 150%, or 200% of the activity of synapsin-1 (SEQ ID NO: 14) in the brain.

13. A synthetic CNS-specific promoter according to any one of claims 1 to 3, comprising or consisting of SEQ ID NO: 8 or SEQ ID NO: 26, which is active in the midbrain when administered by intravenous (IV) injection.

14. The synthetic CNS-specific promoter according to claim 13, which is active in dopaminergic neurons.

15. An expression cassette comprising a synthetic CNS-specific promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, operably linked to a nucleic acid sequence encoding an expression product.

16. A vector comprising a synthetic CNS-specific promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, or an expression cassette according to claim 15.

17. The vector according to claim 16, which is a viral vector, for example, an AAV vector, an adenovirus vector, a retrovirus vector, or a lentivirus vector.

18. The vector according to claim 17, which is a lentiviral vector.

19. The vector according to claim 17, which is an AAV vector.

20. A vilion comprising the vector according to any one of claims 17, 18, or 19.

21. A pharmaceutical composition comprising a synthetic CNS-specific promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, an expression cassette according to claim 15, a vector according to claim 16, 17, 18, or 19, or a virion according to claim 20.

22. A synthetic CNS-specific promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, an expression cassette according to claim 15, a vector according to claim 16, 17, 18, or 19, a virion according to claim 20, or a pharmaceutical composition according to claim 21, for use as a pharmaceutical.

23. A cell comprising a synthetic CNS-specific promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, an expression cassette according to claim 15, a vector according to claim 16, 17, 18, or 19, or a virion according to claim 20.

24. A synthetic CNS-specific promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, an expression cassette according to claim 15, a vector according to claim 16, 17, 18, or 19, a virion according to claim 20, or a pharmaceutical composition according to claim 21, for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

25. A method for producing an expression product, comprising the steps of providing a synthetic CNS-specific expression cassette according to claim 15 in CNS cells and expressing an expression product present in the synthetic CNS-specific expression cassette.

26. The method according to claim 25, wherein the synthetic CNS-specific expression cassette comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 21 or functional variants thereof, and the expression product is widely expressed in the brain when the expression cassette is provided by ICV injection.

27. The method according to claim 26, wherein the CNS-specific expression cassette drives expression at a level of at least 100%, 150%, or 200% compared to the activity of synapsin-1 (SEQ ID NO: 14) in the brain.

28. The method according to claim 25, wherein the synthetic CNS-specific expression cassette comprises or consists of SEQ ID NO: 8 or SEQ ID NO: 26 or functional variants thereof, and the expression product is expressed in the midbrain when administered by intravascular injection.

29. The method according to claim 28, wherein the expression product is expressed in dopaminergic neurons.

30. A method for expressing a therapeutic transgene in CNS cells, comprising the step of introducing a synthetic CNS-specific expression cassette according to claim 15, a vector according to claim 16, 17, 18, or 19, or a virion according to claim 20 into CNS cells.

31. A method for expressing a therapeutic transgene in CNS cells according to claim 30, wherein an expression cassette, vector, or virion is introduced by intravenous injection.

32. The method according to claim 31, wherein the injection is made in one of the radial cephalic vein, median vein, or ulnar cephalic vein.

33. A therapeutic method for a target that requires it, preferably for a human being. - A step of administering to a target an expression cassette according to claim 15, a vector according to claim 16, 17, 18, or 19, a virion according to claim 20, or a pharmaceutical composition according to claim 21, which includes a sequence encoding a therapeutic product operably linked to a promoter according to any one of claims 1, 2, 3, 7, 10, 11, 12, 13, or 14, and - A method comprising the step of expressing a therapeutic amount of a therapeutic product in the target CNS.

34. A method for expressing an expression product in a dopaminergic neuron, comprising the step of introducing the synthetic CNS-specific expression cassette described in claim 15 into the dopaminergic neuron by intravascular injection, wherein the CNS-specific expression cassette comprises SEQ ID NO: 8 or SEQ ID NO: 26 or a functional variant thereof.

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