Regulatory nucleic acid sequences

Abstract

The present invention relates to regulatory nucleic acid sequences, particularly CNS-specific promoters and elements thereof. The invention also relates to expression constructs, vectors, virions, pharmaceutical compositions and cells comprising such promoters, and to methods of use thereof. The regulatory nucleic acid sequences are particularly useful for gene therapy applications.

Description

[Technical Field]

[0001] The present invention relates to regulatory nucleic acid sequences, particularly CNS-specific promoters and elements thereof. The invention also relates to expression constructs, vectors, virions, pharmaceutical compositions and cells comprising such promoters, and to methods of use thereof. The regulatory nucleic acid sequences are particularly useful for gene therapy applications. [Background technology]

[0002] The following discussion is provided to aid the reader in understanding the present disclosure and does not constitute any admission as to the content or relevance of prior art.

[0003] After extensive study of the inner mechanisms of gene regulation in the body, the focus of research has recently shifted to modulating gene expression by introducing exogenous nucleic acid sequences into cells.

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

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

[0006] In gene therapy, controlling the expression of exogenous nucleic acids introduced into cells is crucial to the health and safety of the patient. Not only must the level of expression product be within the therapeutic window, but expression must also be in the required tissue or in a specific region within the required tissue. Expression outside the therapeutic window (i.e., lower or higher) or outside the therapeutic window, or even outside a specific region within the required tissue, may not be therapeutically useful or may even be 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 loss-of-function mutations in a single gene, DAT1 / SLC6A3 (Kurian et al., 2009). DAT1 / SLC6A3 encodes the presynaptic dopamine transporter, which is involved in the transfer of extraneuronal dopamine to dopaminergic neurons. The dopamine transporter transports dopamine, two sodium ions, and one chloride ion into cells using the driving force of the sodium gradient across the cell membrane. Consequently, 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 (Kurian et al., 2009).

[0008] A particular challenge in introducing a replacement DAT1 / SLC6A3 gene is that, in the non-disease state, DAT1 / SLC6A3 is specifically expressed in the midbrain, as shown in Figure 1 A. To best mimic the natural expression of DAT1 / SLC6A3, it is desirable to ensure that the replacement DAT1 / SLC6A3 gene is expressed in the midbrain (this is the location of dopaminergic neurons), while also preferably with minimal expression in other parts of the brain.

[0009] Therefore, there is a need for promoters that drive expression in the midbrain, among other CNS regions, as well as promoters that drive expression specifically in dopaminergic neurons in the midbrain.

[0010] Angelman syndrome is also a candidate for gene therapy by introducing a replacement gene. 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 maternally inherited copy of the UBE3A gene is active; loss of the maternal UBE3A gene leads to Angelman syndrome.

[0011] A particular challenge in introducing a replacement UBE3A gene is that, in non-disease states, UBE3A is broadly expressed in the brain, as shown in Figure 1B. To best mimic the natural expression of the UBE3A gene, it is preferable that the replacement UBE3A gene be broadly expressed in the brain.

[0012] Therefore, there is a need for promoters that drive expression in many or all regions of the brain (eg, pan-CNS).

[0013] Other diseases of the CNS are suitable targets for gene therapy; in some such diseases, targeted expression of therapeutic genes in specific CNS tissues may be desirable, while in others, which are more generalized, non-specific expression in the CNS may be suitable. [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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(1987) Nature 327:70 [Non-Patent Document 45] Lim et al., 2003, Genes & Development, 17, pp. 991-1008 [Non-Patent Document 46] Lim et al., 2003, Science, 299, 1540 [Non-Patent Document 47] Lee and Ambrose, 2001, Science, 294, 862 [Non-Patent Document 48] Lau et al., 2001, Science 294, pp. 858-861 [Non-Patent Document 49] Lagos-Quintana et al., 2002, Current Biology, 12, 735-739 [Non-Patent Document 50] Lagos-Quintana et al., 2001, Science, 294, 853-857 [Non-Patent Document 51] Lagos-Quintana et al., 2003, RNA, 9, 175-179 [Non-Patent Document 52] mousebrain.org / genesearch.html Summary of the Invention [Problem to be solved by the invention]

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

[0017] In a first aspect of the present invention, there is provided a synthetic central nervous system (CNS)-specific promoter comprising or consisting of a sequence according to any one of SEQ ID NOs: 1 to 8, 21 to 26, or a functional variant thereof.

[0018] In some embodiments, the synthetic CNS-specific promoter comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-8, 21-26.

[0019] The present invention therefore provides various synthetic CNS-specific promoters and functional variants thereof. It is generally preferred that promoters according to the invention that are variants of any one of SEQ ID NOS: 1-8, 21-26 retain at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of the reference promoter. Where appropriate, the activity is assessed using examples such as those described herein, although other methods may also be used.

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

[0021] In another aspect of the invention, there is provided a CNS-specific cis-regulatory element (CRE) comprising or consisting of a sequence according to any one of SEQ ID NOs: 9-11, 28-31, or a functional variant of any of them. In some embodiments, the CNS-specific CRE comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 9-11, 28-31.

[0022] It is generally preferred that a CNS-specific CRE according to the present invention that 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. Retention of activity can be assessed by comparing expression of a suitable reporter under the control of the reference promoter with an otherwise identical promoter containing the substituted CRE under equivalent conditions. Where appropriate, the activity is assessed using examples such as those described herein, although other methods can also be used.

[0023] Optionally, a CRE according to the present invention can be combined with an additional CRE to form a cis-regulatory module (CRM). Optionally, the additional CRE can be a CRE according to SEQ ID NOs: 9-11, 28-31 or a functional variant thereof, or they can be other CREs. Optionally, the additional CRE is CNS-specific.

[0024] In another aspect of the present invention, there is provided a synthetic CNS-specific promoter comprising or consisting of a CRE according to any one of SEQ ID NOs: 9-11, 28-31 or a functional variant thereof. In some embodiments, the CRE may be operably linked 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 invention, there is provided a minimal or proximal promoter comprising or consisting of a sequence according to any one of SEQ ID NOs: 12-13 or a functional variant thereof. In another aspect of the invention, there is provided a synthetic promoter comprising said minimal or proximal promoter, suitably a synthetic CNS-specific promoter comprising said minimal or proximal promoter. Optionally, the functional variant of the minimal or proximal promoter comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NOs: 12-13.

[0026] Optionally, any one of CNS-4, CNS-5_v2, CNS-6_v2, CNS-7_v2, CNS-8_v2 (SEQ ID NOs: 4-8) can function as a minimal or proximal promoter. Thus, synthetic CNS-specific promoters are provided comprising a minimal or proximal promoter according to any one of SEQ ID NOs: 12-13 or SEQ ID NOs: 4-8. Optionally, the minimal or proximal promoter can be operably linked to a CRE or CRM. The CRE can be a CRE according to the present invention or any other CRE. The CRM can comprise a CRE according to the present invention. Optionally, the CRE or CRM is CNS-specific.

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

[0028] The CRE, minimal / proximal promoter, or promoter of the present invention may be active in one or more of various parts of the CNS. The CNS is primarily composed of the brain and spinal cord. The retina, optic nerve, olfactory nerve, and olfactory epithelium are sometimes considered parts of the CNS along with the brain and spinal cord. This is because they are directly connected to brain tissue without intermediate nerve fibers. Optionally, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in the brain and spinal cord. Optionally, 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. Optionally, 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. Optionally, the CRE, minimal / proximal promoter, or promoter of the present invention may be active in one or more of various regions in the brain.

[0029] Non-limiting examples of brain regions include 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). Non-limiting examples of spinal cord regions include the cervical, thoracic, lumbar, sacral, and caudal 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 a majority of regions in the brain, i.e., at least 5, at least 6, at least 7, at least 8, or all 9 of the 9 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, such as 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 above regions of the brain and spinal cord. 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, such as 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 a majority of regions in the spinal cord, i.e., at least three, at least four or all five of the five regions of the spinal cord listed above.

[0030] In some embodiments, it may be desirable for the CRE, minimal / proximal promoter, or promoter of the present invention to exhibit predominant activity in one region of the CNS, and optionally in one region of the brain. Optionally, it may be desirable for the CRE, minimal / proximal promoter, or promoter of the present invention to exhibit activity in one region of the brain, but no activity or only minimal activity in the remainder 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, 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 exhibits no activity or only minimal activity in other regions of the brain.

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

[0032] In some embodiments, it may be desirable for a promoter to be active in a limited number of CNS cell types, or in one or fewer CNS cell types. In some embodiments, a CRE, minimal / proximal promoter, or promoter of the present invention is active in no more than four, three, two, or one CNS cell type from the CNS cell types listed above. In some embodiments, a CRE, minimal / proximal promoter, or promoter of the present invention is active in no more than two CNS cell types from the CNS cell types listed above, e.g., neurons and oligodendrocytes. In some embodiments, a 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, e.g., neurons.

[0033] In some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is active in a particular subtype of CNS cell, such as dopaminergic neurons. In certain 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 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 a particular type of CNS cell or a particular subtype of CNS cell, or in a particular region 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. Non-limiting examples of tissues outside the CNS include heart, liver, kidney, skeletal muscle, and spleen. Optionally, in some embodiments, the CRE, minimal / proximal promoter, or promoter of the present invention is not active, or only minimally active, in tissues or cells outside the CNS. Optionally, the CRE, minimal / proximal promoter, or promoter of the present invention is active in 1, 2, 3, 4, or fewer of the above tissues outside the CNS upon ICV delivery. Optionally, the CRE, minimal / proximal promoter, or promoter of the present invention is active in 1, 2, 3, 4, or fewer of the above tissues outside the CNS upon IV delivery.

[0035] Optionally, in some embodiments, it may be desirable for a CRE, minimal / proximal promoter, or promoter of the present invention to be active in the CNS, but also active in other tissues outside the CNS. Optionally, a 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 tissues outside the CNS upon ICV delivery. Optionally, a 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 tissues outside the CNS upon IV delivery.

[0036] In some embodiments, the CRE, minimal / proximal promoter, or synthetic promoter of the present invention may be active in both the CNS and the peripheral nervous system (PNS). When the CRE, minimal / proximal promoter, or synthetic promoter of the present invention is active in both the CNS and the PNS, the CRE, minimal / proximal promoter, or synthetic promoter of the present invention may be referred to as nervous system-specific (NS-specific). PNS refers to the portion of the nervous system that is outside the brain and spinal cord. Non-limiting examples of the peripheral nervous system include the cranial nerves, brachial plexus, thoracic and abdominal nerves, lumbar and sacral plexuses, and the 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 one, two, three, four, five, or six of the regions of the PNS listed above. In some embodiments, the CRE, CRM, minimal / proximal promoter or synthetic promoter of the present invention is active in a majority of regions in the PNS, i.e., at least four, at least five or all six of the six regions of the PNS listed above.

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

[0038] A CNS-specific promoter can be expressed in other non-CNS cells, but has a higher degree of expression in CNS cells, such as neurons in the brain and spinal cord, as well as non-neuronal or neural support cells located in the brain and spinal cord. For example, a CNS-specific promoter expresses a gene at least 25%, or at least 35%, or at least 45%, or at least 55%, or at least 65%, or at least 75%, or at least 80%, or at least 90%, or at least 95%, or any integer percentage between 25% and 95%, higher in cells located in the CNS, including neurons and non-neuronal cells located in the brain and spinal cord, compared to cells located outside the CNS.

[0039] Expression driven by a promoter of the invention in a desired tissue or cell is maintained 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, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 8 The period may 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 longer than 10 years. Expression can be 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 invention, there is provided an expression cassette comprising the synthetic CNS-specific promoter of any aspect of the invention operably linked to a sequence encoding an expression product. Optionally, the expression product is a gene, e.g., a transgene. In some embodiments, the expression product is a therapeutic expression product.

[0041] In a further aspect, a vector is provided comprising a synthetic CNS-specific promoter or expression cassette according to the present invention. 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 adenoviral vector, a retroviral vector, a herpes simplex vector, or a lentiviral vector. Lentiviral vectors have been 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 advantageous because they have a relatively large cloning capacity and do not express viral genes. A particularly preferred lentiviral vector system is based on HIV-1 (Jakobsson and Lundberg, 2006). Herpes simplex viral vectors and adenoviral vectors also show potential for use as gene transfer tools in the CNS, as they have shown 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 of particular interest because they typically do not integrate 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 to achieve efficient transduction in the CNS. Thus, AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAVrh10, AAVDJ8, 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 other embodiments, a particularly preferred AAV vector is AAVDJ8. In some embodiments, AAVrhlO is a particularly preferred AAV vector. Optionally, the AAV vector comprises a viral genome comprising the nucleic acid sequence of the present invention located between two inverted terminal repeats (ITRs). WO2019 / 028306 discloses various wild-type and modified AAV vectors that can be used, for example, in the CNS. In one embodiment, the AAV vector is capable of penetrating the blood-brain barrier after delivery of the AAV vector. In one embodiment, the AAV vector of the present invention is a replication-deficient recombinant AAV viral vector that lacks sequences encoding functional Rep and Cap proteins within its viral genome. These defective AAV vectors may lack most or all of the parent coding sequence and essentially retain only one or two AAV ITR sequences and the nucleic acid of interest for delivery to a cell, tissue, organ, or organism. Optionally, the AAV vector for use herein includes a virus that has been reduced to the minimum components necessary for transduction of the nucleic acid payload or cargo of interest. In this method, AAV vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and / or integration mechanisms found in wild-type viruses. In one embodiment, the AAV particles of the present invention are scAAV. In another embodiment, the AAV particles of the present invention are ssAAV.Methods for producing and / or modifying AAV particles have been widely disclosed in the art (see, e.g., 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 blood-brain barrier penetration following intravascular (e.g., intravenous or intra-arterial) administration (see, e.g., WO2014 / 144229, which discusses capsids engineered for efficient crossing of the blood-brain barrier, e.g., capsids or peptide inserts comprising VOY101, VOY201, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, and variants thereof).

[0043] Methods for producing AAV vectors are well known in the art and are described, for example, in 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 University Press (1994); Samulski et al., J. Fir. 63: 3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66: 6922-30 (1992); Kimbauer et al., Vir. 219: 37-44 (1996); Zhao et al., Vir. 272: 382-93 (2000), the disclosures of each of which are incorporated herein by reference. Commonly used viral replication cells for the production of recombinant AAV viral 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, e.g., using cationic polymers or cationic lipids as known in the art. Various non-viral vectors are discussed in Selene Ingusci et al. (Gene Therapy Tools for Brain Diseases. Front. Pharmacol. 10:724. doi: 10.3389).

[0045] In a further aspect, there is provided a virion (viral particle) comprising a vector, optionally a viral vector, according to the invention. In some embodiments, the virion is an AAV virion.

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

[0047] For example, the AAV vector particles can be formulated as pharmaceutical compositions, which will be understood to necessarily include one or more active ingredients, most often pharmaceutically acceptable excipients.

[0048] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the amount of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dose, e.g., one-half or one-third of such a dose, etc.

[0049] In a further aspect, there is provided a synthetic CNS-specific promoter, expression cassette, vector, virion or pharmaceutical composition according to the invention for use as a medicament.

[0050] In a further aspect, there is provided a synthetic CNS-specific promoter, expression cassette, vector, virion or pharmaceutical composition according to the invention for use in therapy, i.e., the prevention or treatment of a medical condition or disease.

[0051] Optionally, the medical condition or disease is associated with aberrant gene expression, optionally in CNS tissue or cells. Optionally, the use is for gene therapy, preferably for use in treating a disease involving aberrant gene expression. Optionally, the medical condition or disease involving aberrant gene expression may be a disease of the CNS. Optionally, the medical condition or disease may be a monogenic disorder of the CNS. Optionally, the gene therapy involves 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 a further aspect, a cell is provided comprising a synthetic CNS-specific promoter, expression cassette, vector, or virion of the invention. In some embodiments, the cell is a mammalian cell, optionally a human cell. Optionally, the cell is a CNS cell. Optionally, the cell can be a neuron, astrocyte, oligodendrocyte, ependymal cell, or microglial cell. Optionally, the cell can be a human neuron, astrocyte, oligodendrocyte, ependymal cell, or microglial cell. The synthetic CNS-specific promoter can be episomal or in the genome of the cell.

[0053] In a further aspect, there is provided a synthetic CNS-specific CRE, synthetic CNS-specific promoter, expression cassette, vector, virion, or pharmaceutical composition as described herein for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease. Exemplary medical conditions or diseases relevant to this aspect are discussed below.

[0054] In a further aspect, there is provided a method of producing an expression product comprising providing a synthetic CNS-specific expression cassette, vector or virion of the invention in a CNS cell or tissue and expressing a gene of interest 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 aspect, a method of expressing a therapeutic transgene in a CNS cell is provided, comprising introducing into a CNS cell a synthetic CNS-specific expression cassette, vector, or virion as described herein and expressing an expression product (e.g., a gene of interest) present in the synthetic CNS-specific expression cassette, vector, or virion. The CNS cell can be, for example, a neuron, astrocyte, oligodendrocyte, ependymal cell, or microglial cell.

[0056] In a further aspect, there is provided a method of therapy of a subject, preferably a human, in need thereof, comprising: - administering to a subject an expression cassette, vector, virion or pharmaceutical composition as described herein comprising a sequence encoding a therapeutic product operably linked to a promoter according to the invention; and - expressing a therapeutic amount of a therapeutic product in the CNS of said subject. A method is provided that includes:

[0057] Optionally, the method is for the treatment, prevention, alleviation or amelioration of a neurological disease and / or disorder. Exemplary medical conditions or diseases relevant to this embodiment are discussed below.

[0058] Suitable methods of administration can be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection), including intravenous, intraarterial, intracranial, intramuscular, subcutaneous, intraarticular, intrathecal, and intradermal injection. Preferred methods of administration are intravenous, intraarterial, intracranial, and intrathecal injection.

[0059] In some embodiments, the method includes introducing into the CNS of a subject an expression cassette, vector, virion, or pharmaceutical composition as described herein, comprising a gene encoding a therapeutic product. A particular challenge with 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 animal's brain, e.g., intracranial injection, optionally intracerebroventricular (ICV) injection (see, e.g., Keiser et al., Curr Protoc Mouse Biol. 2018 Dec;8(4):e57). This administration method can be difficult to perform and potentially dangerous for the subject, making it a disadvantage for human gene therapy.

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

[0061] Optionally, the medical condition or disorder is a medical condition or disorder of the CNS, such as a neurological disease and / or disorder. Optionally, the medical condition or disease 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 chorea, 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 diseases, 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).

[0062] Optionally, 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] Additionally or alternatively, the expression product may be an antibody, antibody fragment or antibody-like scaffold protein.

[0064] Additionally or alternatively, the expression product can be a gene editing system (e.g., CRISPR-Cas9 system, TALEN, ZFN, etc.) directed to a disease allele.

[0065] Additionally or alternatively, the expression product may be one or more regulatory polynucleotides, such as therapeutic RNA or DNA molecules. For example, the regulatory polynucleotide may be miRNA or siRNA. The target gene may be any gene associated with any neurological disease, including, but not limited to, those listed herein. For example, siRNA duplexes or encoded dsRNAs can reduce or silence target gene expression in CNS cells, thereby ameliorating the symptoms of the neurological disease. In one non-limiting example, the target gene is huntingtin (HTT). In another non-limiting example, the target gene is microtubule-associated protein tau (MAPT).

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

[0067] In a further aspect, a method of expressing an expression product in the CNS is provided, comprising introducing into a CNS cell an expression cassette comprising a synthetic CNS-specific promoter comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 21 operably linked to the expression product. Optionally, the expression cassette is introduced into the CNS by ICV injection, and the expression product is widespread in the brain. Optionally, expression of the expression product in the brain is in at least six regions of the brain. Optionally, the synthetic CNS-specific promoter is capable of promoting CNS-specific expression of the expression product at a level at least 100%, 150%, or 200% compared to synapsin-1 (SEQ ID NO: 14) in the brain. Optionally, the expression cassette is introduced into the CNS by ICV injection, and the expression product is expressed in the cortex and hippocampus.

[0068] In a further aspect, a synthetic CNS-specific promoter is provided that comprises or consists of SEQ ID NO:2, SEQ ID NO:25, or SEQ ID NO:7, or a functional variant thereof, as discussed above. Optionally, such a synthetic CNS-specific promoter is capable of promoting widespread expression in the brain 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 regions of the brain. Optionally, a synthetic CNS-specific promoter comprising or consisting of SEQ ID NO:2 or a functional variant thereof is capable of promoting widespread intracranial expression of an expression product operably linked to the CNS-specific promoter when administered by IV injection. Optionally, a synthetic CNS-specific promoter comprising or consisting of SEQ ID NO:2 or a functional variant thereof does not promote expression in the midbrain. Optionally, a synthetic CNS-specific promoter comprising or consisting of SEQ ID NO:7 or SEQ ID NO:25, or a functional variant thereof, is capable of promoting expression in the cortex, hippocampus, and midbrain when administered by IV injection of an expression product operably linked to the CNS-specific promoter.

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

[0070] In a further aspect, a synthetic CNS-specific promoter is provided that comprises or consists of SEQ ID NO:3, SEQ ID NO:22, or SEQ ID NO:4, or a functional variant thereof, as discussed above. Optionally, the synthetic CNS-specific promoter is capable of promoting expression in the cortex and hippocampus when administered by ICV injection. Optionally, the synthetic CNS-specific promoter is not active, or only minimally active, in other regions of the brain. Optionally, a synthetic CNS-specific promoter that comprises or consists 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, is capable of promoting expression in the cortex, striatum, and hippocampus when administered by IV injection. Optionally, a synthetic CNS-specific promoter that comprises or consists of SEQ ID NO:4, SEQ ID NO:22, or a functional variant thereof, is further capable of promoting expression in the midbrain.

[0071] In a further aspect, a method of expressing an expression product in the CNS is provided, comprising introducing into a CNS cell an expression 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. Optionally, the expression cassette is introduced into the CNS by ICV injection, and the expression product is expressed in the cortex and hippocampus. Optionally, expression of the expression cassette is minimal in other regions of the brain. Optionally, the expression cassette is introduced into the CNS by ICV injection, and the expression product is expressed in the cortex and hippocampus. Optionally, the expression cassette is introduced into the CNS by IV injection, and the expression product is expressed in the cortex, striatum, and hippocampus.

[0072] In a further aspect, a synthetic CNS-specific promoter is provided that comprises or consists of SEQ ID NO: 5 or SEQ ID NO: 23, or a functional variant thereof, as discussed above. Optionally, the synthetic CNS-specific promoter is capable of promoting expression in the cortex, striatum, hippocampus, and midbrain. Optionally, the synthetic CNS-specific promoter is not active, or only minimally active, in other regions of the brain. Optionally, the synthetic CNS-specific promoter is administered by ICV injection.

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

[0074] In a further aspect, a synthetic CNS-specific promoter is provided that comprises or consists of SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:8, or a functional variant thereof, as discussed above. Optionally, the synthetic CNS-specific promoter is capable of promoting expression in the hippocampus, cortex, and midbrain when administered by ICV injection. Optionally, the synthetic CNS-specific promoter comprising or consisting of SEQ ID NO:6 or SEQ ID NO:24, or a functional variant thereof, is capable of promoting expression in the hippocampus, midbrain, and cerebellum when administered by IV injection. Optionally, the synthetic CNS-specific promoter comprising or consisting of SEQ ID NO:8 or SEQ ID NO:26, or a functional variant thereof, is capable of promoting expression in the hippocampus and midbrain when administered by IV injection. Optionally, the synthetic CNS-specific promoter is not active, or is only minimally active, in other regions of the brain. Optionally, the synthetic CNS-specific promoter comprising or consisting of SEQ ID NO:6, or a functional variant thereof, SEQ ID NO:24, or a functional variant thereof, SEQ ID NO:26, or a functional variant thereof, or SEQ ID NO:8, or a functional variant thereof, is primarily active in neurons. Optionally, the synthetic CNS-specific promoter comprising or consisting of SEQ ID NO: 8 or SEQ ID NO: 26 or a functional variant thereof is active primarily in dopaminergic neurons.

[0075] In a further aspect, a method of expressing an expression product in the CNS is provided, comprising introducing into a CNS cell an expression cassette comprising a synthetic CNS-specific promoter comprising or consisting of 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. Optionally, the expression cassette is introduced into the CNS by ICV injection, and the expression product is expressed in the hippocampus, cortex, and midbrain. Optionally, an expression cassette comprising or consisting of SEQ ID NO:6 or SEQ ID NO:24, or a functional variant thereof, is introduced into the CNS by IV injection, and the expression product is expressed in the hippocampus, midbrain, and cerebellum. Optionally, an expression cassette comprising or consisting of SEQ ID NO:8 or SEQ ID NO:26, or a functional variant thereof, is introduced into the CNS by IV injection, and the expression product is expressed in the hippocampus and midbrain. Optionally, expression is minimal in other regions of the brain.

[0076] In a further aspect, there is provided a method of expressing an expression product in a dopaminergic neuron, the method comprising introducing a synthetic CNS-specific expression cassette into the dopaminergic neuron 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 drawings]

[0077] [Figure 1A] Figure 1 shows the expression pattern of the DAT1 / SLC6A3 gene in coronal sections from the adult mouse brain (taken from the Alan Mouse Brain Atlas; mouse.brain-map.org). DAT1 / SLC6A3 is highly expressed in the midbrain. [Figure 1B] Figure 1 shows the expression pattern of the UBE3A gene in coronal sections from adult mouse brain (taken from the Alan mouse brain atlas; mouse.brain-map.org). UBE3A is widely expressed in the brain. [Figure 2A]1 shows the intracranial biodistribution in sagittal sections 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, and the transgene GFP under the control of the control promoter hSyn1. Scale bar is 1 mm. [Figure 2B] 1 shows the intracranial biodistribution of transgene GFP 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 ICV and IV in sagittal sections. Scale bar is 1 mm. [Figure 3A] 1 shows the intracranial biodistribution 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 coronal sections. Scale bar 1 mm. [Figure 3B] 1 shows the intracranial biodistribution in coronal sections 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. Scale bar is 1 mm. [Figure 4A] 1 shows the intracranial biodistribution of the 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 IV in coronal sections. Scale bar is 1 mm. [Figure 4B] 1 shows the intracranial biodistribution in coronal sections of the transgene GFP 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. Scale bar is 1 mm. [Figure 5A]1 shows the intracranial biodistribution in different parts of the brain at higher magnification of ICV-delivered 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) and the transgene GFP under the control of the control promoter hSyn1. Scale bar is 100 μm. [Figure 5B] 1 shows at higher magnification the intracranial biodistribution in various parts of the brain 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 hSynl. Scale bar is 100 μm. [Figure 6A] 1 shows, at higher magnification, the intracranial biodistribution of transgene GFP in various parts of the brain 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 an uninjected control. Scale bar is 100 μm. [Figure 6B] 1 shows, at higher magnification, the intracranial biodistribution of transgene GFP in various parts of the brain 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 an uninjected control. Scale bar is 100 μm. [Figure 7A]

[0033] Figure 1 shows the biodistribution 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 and 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 an overlay of the two together with the nuclear dye DAPI. The scale bar is 25 μm. [Figure 7B]

[0023] Figure 1 shows the biodistribution in the midbrain of the transgene GFP 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 ICV. The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows an overlay of the two together with the nuclear dye DAPI. Scale bar is 25 μm. [Figure 8A] Figure 1 shows the biodistribution of the transgene GFP in the midbrain under the control of IV-delivered 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), as well as the uninjected control. The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows an overlay of the two together with the nuclear dye DAPI. Scale bar is 25 μm. [Figure 8B]

[0023] Figure 1 shows the biodistribution in the midbrain of the transgene GFP 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. The left column shows GFP expression, the middle column shows TH+ positive cells (dopaminergic neurons), and the right column shows an overlay of the two together with the nuclear dye DAPI. Scale bar is 25 μm. [Figure 9] Figure 1 shows the biodistribution in various tissues of CNS-1-8 (SEQ ID NOS: 1-4, 23-26) and the transgene GFP under the control of the control promoter synapsin 1 (SEQ ID NOS: 14) delivered by ICV or IV. For this data, RNA extracted from organs throughout the body was converted to RNA and quantified by qPCR. The 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 CNS1-8 (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 taken at ×40 magnification across distinct brain regions while maintaining constant settings. Foreground immunostaining was defined by averaging the highest and lowest signals. Data are 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. CNS1-8 (SEQ ID NOs: 1-4, 23-26) show higher expression in the hippocampus than the 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] Figure 1 shows the expression of GFP under the control of CNS-1 (SEQ ID NO: 1) upon ICV delivery. Magnification: ×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 neuronal. [Figure 12] 1 shows the intracranial biodistribution in sagittal sections of CNS-8 (SEQ ID NO: 26) and the transgene GFP under the control of the control promoter hSynl delivered by ICV and IV. Scale bar is 1 mm. [Figure 13A] Figure 1 shows intracranial biodistribution in coronal sections of CNS-8 (SEQ ID NO: 26) delivered by ICV and the transgene GFP under the control of the control promoter hSynl. On the left, the scale bar is 1 mm. On the right, the brain region is shown at higher magnification. The scale bar is 100 μm. [Figure 13B]

[0023] Figure 1 shows the intracranial biodistribution in coronal sections of CNS-8 (SEQ ID NO: 26) delivered by IV and the transgene GFP under the control of the control promoter hSynl. On the left, the scale bar is 1 mm. On the right, the brain region is shown at higher magnification. The scale bar is 100 μm. [Figure 14A]

[0023] Figure 1 shows the biodistribution in the midbrain of the transgene GFP under the control of CNS-8 (SEQ ID NO: 26) delivered by ICV (top) and IV (bottom). The left column shows TH+ positive cells (dopaminergic neurons), the middle column shows GFP expression, and the right column shows an overlay of the two together with the nuclear dye DAPI. Scale bar is 25 μm. [Figure 14B] 1 shows a quantification of the percentage of dopaminergic neurons showing GFP expression (TH+GFP+ cells) among all dopaminergic neurons. The left portion of the graph shows the percentage of dopaminergic neurons showing GFP under the control of the control promoter Syn-1 in ICV and IV delivery. The center portion of the graph shows the quantification of the percentage of dopaminergic neurons showing GFP expression under the control of CNS-8 (SEQ ID NO: 26) when a low dose is administered in ICV and IV delivery (Example 1). The right portion of the graph shows the percentage of dopaminergic neurons showing GFP expression under the control of CNS-8 (SEQ ID NO: 26) when a high dose is administered in ICV and IV delivery (Example 2). [Figure 15] Figure 1 shows a comparison of the biodistribution in various tissues of the transgene GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at low or high doses. The left side shows the biodistribution of GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at low doses, and the right side shows the biodistribution of GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at high doses. The data for the biodistribution of GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at low doses are identical to the data shown in Figure 9. For this data, RNA extracted from organs throughout the body was converted to RNA and quantified by qPCR. [Figure 16A]Figure 1 shows the expression pattern of the faf1 gene in mouse PNS neurons derived from single-cell transcriptome data (Zeisel et al., 2018). Dark gray represents high expression, white represents no expression, and light gray represents low expression. faf1 is expressed in numerous PNS neurons. [Figure 16B] Figure 1 shows the expression pattern of the pitx3 gene in PNS neurons derived from single-cell transcriptome data (Zeisel et al., 2018). Dark gray represents high expression, white represents no expression, and light gray represents low expression. Pixt3 is expressed in sympathetic PNS neurons. DETAILED DESCRIPTION OF THE INVENTION

[0078] CRE and its functional variants Disclosed herein are various CREs that can be used in constructing CNS-specific promoters. Optionally, the CREs are CNS-specific. These CREs are generally derived from genomic promoter and enhancer sequences, but they are used herein in contexts entirely different from their native genomic environment. Generally, CREs constitute a small portion of a much larger genomic regulatory domain that controls the expression of the gene with which they are normally associated. Surprisingly, it has been found that many of these CREs are extremely small and can be isolated from their normal environment and retain their CNS-specific regulatory activity. This is surprising because recovery of regulatory sequences from the complex, "three-dimensional" native state of the genome often results in a significant loss of activity, so there is no reason to expect a given CRE to retain its observed level of activity once recovered from its native environment. It is even more surprising when a CRE retains its CNS-specific activity in an AAV vector. This is particularly true since AAV vectors contain inverted terminal repeats (ITRs) and 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 the CRE of the present invention can be altered without causing a substantial loss of activity. Functional variants of CRE can be prepared by altering the sequence of CRE, provided that alterations that are significantly detrimental to the activity of CRE are avoided. In view of the information provided in this disclosure, it is easy to modify CRE to provide functional variants. 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, and more particularly the promoter containing it, to be provided in a vector while occupying a minimal amount of the vector's payload, which is particularly important when the CRE is used in a volume-limited vector, such as an AAV-based vector.

[0081] The CRE of the present invention contains certain CNS-specific TFBSs. Generally, in functional variants of CREs, it is desirable that these CNS-specific TFBSs retain functionality. Those skilled in the art are well aware that TFBS sequences can be altered while retaining functionality. With this in mind, the sequences of TFBSs are usually exemplified by consensus sequences, in which some degree of variation usually exists. Further information about the variations that occur in TFBSs can be illustrated using a position weight matrix (PWM), which represents the frequency with which a given nucleotide is usually 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 sequence of any given TFBS of a CRE in a way that retains, or in some cases even enhances, CRE functionality. With this in mind, those skilled in the art have ample guidance as to how the TFBSs of any given TF can be modified while maintaining the ability to bind to the desired TF; for example, the Jaspar system scores putative TFBSs based on their similarity to a given PWM. Furthermore, CREs can be scanned against all PWMs from the JASPAR database to identify / analyze all TFBSs. Those skilled in the art can, of course, find additional guidance in the literature and can further use routine experimentation to confirm TF binding to putative TFBSs in any variant CRE. It will be apparent that significant sequence modifications can be made in CREs, even within the TFBSs in the CRE, while retaining function.

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

[0083] A functional variant of a CRE comprises a sequence that differs from a reference CRE element but substantially retains activity as a CNS-specific CRE. It will be apparent to those skilled in the art that the sequence of a CRE can be altered while retaining its ability to recruit appropriate CNS-specific transcription factors (TFs) and thereby enhance expression. A functional variant of a CRE may contain substitutions, deletions, and / or insertions compared to a reference CRE, provided that the substitutions do not render the CRE substantially non-functional.

[0084] In some embodiments, a functional variant of a CRE can be considered a CRE that substantially retains its activity when substituted for a reference CRE in a promoter. For example, a CNS-specific promoter comprising a functional variant of a given 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 comprising an unmodified CRE).

[0085] Optionally, a functional variant of a CRE retains a significant level of sequence identity to a reference CRE. Optionally, a functional variant comprises a sequence that is at least 70% identical to the reference CRE, more preferably at least 80%, 90%, 95% or 99% identical to the reference CRE.

[0086] Retention of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an otherwise identical promoter containing the replaced CRE under equivalent conditions. Suitable assays for assessing 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-regulatory module (CRM). The additional CREs can be provided upstream of the CRE according to the present invention or downstream of the CRE according to the present invention. The additional CREs can be the CREs disclosed herein, or they can be other CREs. Optionally, the additional CREs are CNS-specific.

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

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

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

[0091] In many instances, particularly for use in situations where the vector (e.g., a viral vector such as AAV) has limited capacity, shorter promoter sequences are preferred. Thus, in some embodiments, a synthetic CNS-specific CRM comprising at least one CRE according to SEQ ID NOs: 9-11, 28-31, or a functional variant thereof, has a length of 1000 or fewer nucleotides, e.g., 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. Those skilled in the art will understand that the sequence of a synthetic CNS-specific promoter can be varied while retaining its ability to recruit appropriate CNS-specific transcription factors (TFs) and recruit RNA polymerase II to provide CNS-specific expression of an operably linked sequence (e.g., an open reading frame). A functional variant of a synthetic CNS-specific promoter may contain 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] Thus, in some embodiments, a functional variant of a synthetic CNS-specific promoter can be considered a variant that substantially retains the CNS-specific promoter activity of the reference promoter. For example, a functional variant of a synthetic CNS-specific promoter preferably retains at least 70% of the activity of the reference promoter, more preferably at least 80% of that activity, more preferably at least 90% of that activity, more preferably at least 95% of that activity, and even more preferably 100% of that activity.

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

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

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

[0097] A functional variant of a given synthetic CNS-specific promoter may contain one or more additional CREs relative to those present in the reference synthetic CNS-specific promoter. The additional CREs may be provided, for example, upstream of the CRE present in the reference synthetic CNS-specific promoter or downstream of the CRE present in the reference synthetic CNS-specific promoter. The additional CREs may be CREs disclosed herein, or they may be other CREs.

[0098] A functional variant of a given synthetic CNS-specific promoter may contain 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, said one or more spacers may be longer or shorter than in the reference synthetic CNS-specific promoter.

[0099] It will be apparent that the synthetic CNS-specific promoters of the invention may comprise a CRE of the invention or a CRM comprising a CRE of the 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, segments of terminal repeats, response sites, stabilizing elements, destabilizing elements, splicing elements, etc., provided they do not render the promoter substantially non-functional.

[0100] In some embodiments, such a CNS-specific promoter is operably linked to one or more additional regulatory sequences. The additional regulatory sequences can, for example, enhance expression compared to a CNS-specific promoter that is not operably linked to the additional regulatory sequences. In general, it is preferred that the additional regulatory sequences do not substantially reduce the specificity of the CNS-specific promoter.

[0101] For example, a CNS-specific promoter in accordance with the present invention can be operably linked to sequences encoding UTRs (eg, 5' and / or 3' UTRs) and / or introns or the like.

[0102] In some embodiments, the CNS-specific promoter is operably linked to a sequence encoding a UTR, for example, a 5'UTR. The 5'UTR may contain various elements capable of regulating gene expression. The 5'UTR in a natural gene 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 as referred to herein may be the entire naturally occurring 5'UTR or a portion of a 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 in some cases may be significantly longer. Regulatory sequences that can be found in the 5'UTR include, but are not limited to: - binding sites for proteins that may affect mRNA stability or translation, - riboswitches, - sequences that promote or inhibit translation initiation, and - Introns within the 5'UTR involved in the regulation of gene expression and mRNA export Examples include:

[0103] When a regulatory sequence comprises both a 5'UTR and an intron, it may be referred to as a 5'UTR and an intron.

[0104] In some embodiments, such a synthetic CNS-specific promoter is operably linked to a sequence encoding a 5' UTR and an intron. In some embodiments, the 5' UTR and intron are derived from a CMV major immediate-term gene (CMV-IE gene). For example, the 5' UTR and intron from the CMV-IE gene optionally 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 allow for linkage to a 5'UTR, for example, sequences downstream of the transcription start site (TSS) in the promoter element can be removed (e.g., replaced with a 5'UTR).

[0106] The CMV-IE 5'UTR and intron are described in Simari et al., Molecular Medicine 4: 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 in Simari et al. are also described in WO2002 / 031137, which is incorporated herein by reference, and the regulatory sequences disclosed therein can also be used.

[0107] Other regulatory elements, e.g., other UTRs, that can be used in combination with promoters are known in the art, e.g., 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, 158-174 (2018), which is incorporated herein by reference.

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

[0109] In some embodiments, the sequence encoding the 5'UTR and intron comprises SEQ ID NO: 27 or a functional variant thereof. In some embodiments, a 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 linked to the CMV-IE 5'UTR and 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 linked 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] Preferred synthetic CNS-specific promoters of the present invention exhibit CNS-specific promoter activity in CNS cells 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 the synapsin-1, Camk2a, or NSE promoter. While higher levels of promoter activity are often preferred, this is not always the case, and thus, in some cases, more moderate levels of expression may be preferred. In some cases, it is desirable to have available a variety of promoters with different activity levels so that the level of expression can be tailored to requirements, and the present disclosure provides promoters with such a variety of activities. The activity of a given synthetic CNS-specific promoter of the invention compared to Syn-1 can be assessed 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 under the control of the Syn-1 promoter when the two promoters are provided in otherwise equivalent expression constructs and under equivalent conditions.

[0115] In addition to different activity levels, it may be desirable in some cases to have available promoters that are active in different regions of the brain. Therefore, it may be desirable to have promoters with different activity levels across different regions of the brain to allow for tailoring of expression levels to requirements, and the present disclosure provides promoters with such different activities. 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, with little or no expression in the remainder of the brain. This may be the case, for example, in the treatment of diseases such as dopamine transporter deficiency syndrome, where expression in the midbrain is desired. In some preferred embodiments, the CNS-specific promoters of the present invention are active in the midbrain. In some preferred embodiments, the CNS-specific promoters of the present invention are active in the midbrain with little or no activity in other regions of the brain. In some preferred embodiments, the CNS-specific promoters of the present invention are active in dopaminergic neurons. In some embodiments, the CNS-specific promoters of the present invention are active in dopaminergic neurons with little or no expression in other CNS cell types or CNS subtypes. Preferred synthetic CNS-specific promoters of the 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 invention relative to tyrosine kinase can be assessed 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 gene under the control of the tyrosine hydroxylase promoter in dopaminergic neurons when the two promoters are provided in otherwise equivalent expression constructs and under equivalent conditions.In some embodiments, the synthetic CNS-specific promoters of the invention are capable of increasing expression of a gene (e.g., a therapeutic gene or gene of interest) in a subject's dopaminergic neurons 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 relative to a known dopaminergic neuron-specific promoter, optionally the tyrosine hydroxylase promoter.

[0116] Alternatively, it may be preferable to have widespread expression in all or almost all regions of the brain, which may be the case, for example, in the treatment of diseases such as Angelman syndrome, where widespread expression throughout the brain is required.

[0117] In some embodiments, the synthetic CNS-specific promoters of the invention are capable of increasing expression of a gene (e.g., a therapeutic gene or gene of interest) in the CNS, or in CNS cells of a subject, 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 relative to a known CNS-specific promoter, as appropriate, the Syn1, Camk2a, or NSE promoter.

[0118] Preferred synthetic CNS-specific promoters of the present invention exhibit activity in non-CNS cells (e.g., Huh7 and HEK293 cells) that is 50% or less compared to CMV-IE, preferably 25% or less, more preferably 10% or less, and in some cases 5% or less, or 1% or less of CMV-IE.

[0119] In many instances, shorter promoter sequences are preferred, particularly for use in situations where the vector (e.g., a viral vector such as AAV) has limited capacity. Thus, in some embodiments, the synthetic CNS-specific promoter has a length of 1000 or fewer nucleotides, e.g., 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 surprising that a CNS-specific promoter would retain CNS-specific activity in an AAV vector, because the ITRs and different DNA structure of the AAV vector compared to the genome are known to affect promoter activity, and often the ITRs and different DNA structure negatively affect promoter activity.

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

[0123] If the gene encodes a protein, it can be essentially any type of protein. By way of non-limiting example, the protein can be an enzyme, an antibody or antibody fragment (e.g., a monoclonal antibody), a viral protein (e.g., REP-CAP, REV, VSV-G, or RD114), a therapeutic protein, or a toxic protein (e.g., caspase 3, 8, or 9).

[0124] In some preferred embodiments of the invention, the gene optionally encodes a therapeutic expression product, preferably a therapeutic polypeptide, suitable for use in the treatment of a disease or condition associated with aberrant 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 generally understood by those skilled in the art. The term relates to diseases that are suitable for treatment and / or prevention by administration of an active compound to the CNS, particularly to CNS cells. In some embodiments, the CNS disease is a neurological disease and / or disorder.

[0126] By way of non-limiting example, CNS diseases include septum pellucidum defect, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupil, Addie syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Gouttiere syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, and alternating hemiplegia. , Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic nervous system disorder meridian disorders, back pain, Barth syndrome, Batten disease, Becker's 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-Sulzberger syndrome, brachial plexus injury during birth, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysms, brain injury, Brown-Séquard syndrome, bulbar Spinal muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cavernoma, cavernous hemangioma, cavernous vascular malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, craniopathy, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation Cavemous Malformation), cerebral gigantism, hypoxic encephalopathy, cerebral palsy, cerebro-oculofacial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disease, chorea, chorea acanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic dysregulation, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, coma, complex regional pain syndrome, congenital facial diplegia, congenital myasthenia gravis, congenital myopathy, congenital cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis,Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cytomegalovirus infection, dancing eyes, dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, dementia, multi-infarct dementia, semantic dementia, subcortical dementia, dementia with Lewy bodies, dentatoruclear cerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental apraxia, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, autonomic dysfunction, dysgraphia, dyslexia, dysphagia, dyspraxia, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalopathy (familial neonatal), trigeminal region angiomatosis, epilepsy Hemiplegia, Erb's palsy, Erb-Duchenne and Dejerine-Klumpke palsy, essential tremor, extrapontine myelinolysis, Fabry's disease, Fahr's syndrome, syncope, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile convulsions, fibromuscular dysplasia, Fisher's syndrome, hypotonic infantile syndrome, foot drop, Friedreich's ataxia, frontal Cranial dementia, Gaucher disease, systemic gangliosidosis, Gerstmann syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent hemiplegia, hemifacial spasm, alternating hemiplegia Alterans), hereditary neuropathy, hereditary spastic paraplegia, hereditary polyneuropathy ataxia, herpes zoster, herpes zoster oticus, Hirayama syndrome, Holmes-Addie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, hydroanencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelia, hypercortisolism, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, childhood hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease, infantile spasms, inflammatory myopathy, forencephaly prolapse, intestinal lipodystrophy, intracranial cyst, intracranial hypertension, Isaacs syndrome,Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbone syndrome, Klein-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, Kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral cord syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levin-Critchley syndrome, dementia with Lewy bodies, lipid storage disease, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus - neurological sequelae, Lyme disease Neurological complications include Machado-Joseph disease, megaencephalopathy, megalencephaly, Melkersson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease, dyssensory femoral neuralgia, metachromatic leukodystrophy, microcephaly, migraine, Miller-Fisher syndrome, minor stroke, mitochondrial myopathy, Moebius syndrome, unilateral muscular atrophy, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, and orthostatic hypotension. Multiple system atrophy, muscular dystrophy, congenital myasthenia, myasthenia gravis, diffuse myeloablative sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, congenital myopathy, thyrotoxic myopathy, myotonia, congenital myotonia, narcolepsy, neuroacanthocytosis, neurodegeneration with cerebral iron deposition, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological outcome of cytomegalovirus infection, neurological manifestations of Pompe disease, neurological outcomes of lupus Sequelae, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, hereditary neuropathy, neurosarcoidosis, neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenate kinase-related neurodegeneration, paraneoplastic syndrome, abnormal sensations, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemicrania,Parry-Romberg disease, Pelizaeus-Merzbacher disease, Pena-Shocker syndrome type II, radicular cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's disease, compressed nerve, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, porencephaly, post-polio syndrome, post-herpetic neuralgia, post-infectious encephalomyelitis, postural hypotension, postural orthostatic tachycardia syndrome, postural tachycardia syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive facial hemiatrophy, progressive gait ataxia, progressive Multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, pseudotorch syndrome, pseudotoxoplasmosis syndrome, pseudotumor cerebri, psychogenic movement disorder, Ramsay-Hunt syndrome type I, Ramsay-Hunt syndrome type II, Rasmussen's encephalitis, reflex sympathetic dystrophy syndrome, Refsum's disease, infantile Refsum's disease, repetitive movement disorder, repetitive strain injury, restless legs syndrome, retrovirus-associated myelopathy, Rett's syndrome, Reye's syndrome, rheumatoid encephalitis, Riley-Day syndrome, sacral radicular cyst, Saint Vitus's chorea, salivary gland disease, Sandhoff's disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorder, semantic dementia, septo-optic dysplasia, severe myoclonic epilepsy in infants (SMEI), shaken baby syndrome, shingles, Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, convulsions, spina bifida, spinal cord infarction, spinal cord injury, spinal tumor, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic cerebral artery disease syndrome, short-lasting unilateral neuralgiform (SUNCT) headache, dysphagia, Sydenham chorea, syncope, syphilitic spinal sclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, spinal fistula, tardive dyskinesia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen's myotonia, thoracic outlet syndrome, thyrotoxic 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 paraparesis, Troyer's syndrome, tuberous sclerosis, vascular erectile tumors,The disease may be selected from vasculitis syndromes of the central and peripheral nervous system, von Economo disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash injury, Whipple disease, Williams syndrome, Wilson disease, Wolman disease, and X-linked spinal-bulbar muscular atrophy.

[0127] In some embodiments, the CNS disease is selected from the list consisting of dopamine transporter deficiency syndrome, attention deficit / hyperactivity disorder (ADHD), bipolar disorder, epilepsy, multiple sclerosis, tauopathy, Alzheimer's disease, Huntington's chorea, 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 diseases 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).

[0128] Various expression products suitable for treating the above conditions have been described in the art. Optionally, the nucleic acid encoding the expression product operably linked to a CRE, minimal / proximal promoter, or promoter in accordance with the present 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. Additionally or alternatively, the expression product operably linked to a CRE, minimal / proximal promoter, or promoter in accordance with the present invention may be a miRNA / CRISPR Cas9 directed against a disease allele.

[0129] CYP46A1 is the rate-limiting enzyme in cholesterol degradation and has been shown 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 the amount of viral cholesterol, as described in (Djelti et al., 2015), which is incorporated herein by reference. CYP46A1 has also been shown to be neuroprotective in Huntington's disease, as described in (Boussicault et al., 2016), which is 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 linked to a CRE, minimal / proximal promoter, or promoter in accordance with the present invention. Optionally, the CYP46A1 gene is operably linked to a synthetic promoter active in all regions of the CNS (pan-CNS) or a promoter active in more than 5, 6, 7, 8, or 9 of the brain regions listed above. Expression of CYP46A1 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 expression of CYP46A1 by the ubiquitous promoters CMV or CAG was found to be beneficial in a mouse Huntington's disease model (Kacher et al., 2019). Optionally, the CYP46A1 gene is operably linked to a synthetic promoter consisting of or comprising 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, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, pol- These 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 conductance regulator (CFTR).

[0131] Still other useful expression products include enzymes useful in enzyme replacement therapy, which are useful in a variety of conditions resulting from a deficiency in enzyme activity, for example, enzymes containing mannose-6-phosphate can be used in the therapy of lysosomal storage diseases (e.g., suitable genes include those encoding β-glucuronidase (GUSB)).

[0132] In some embodiments, exemplary polypeptide expression products include neuroprotective and anti-angiogenic polypeptides. Suitable polypeptides include, but are not limited to, glial-derived 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 epithelium-derived factor (PEDF), Wnt polypeptides, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibodies, soluble VEGFR, factor VIII (FVIII), factor IX (FIX), and members of the hedgehog family (such as 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), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFα, activin, inhibin, or bone morphogenetic proteins (BMPs). These include, but are not limited to, hormones and growth and differentiation factors including any one of the transforming growth factor alpha superfamily, including any one of BMP1-15, any one of the heregulin / neuregulin / ARIA / Neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0134] In some embodiments, useful expression products include proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemoattractant proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor alpha and beta, interferons (alpha, beta, and gamma), stem cell factor, and flk-2 / flt3 ligand. Gene products produced 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. Useful gene products also include complement regulatory proteins, such as complement regulatory proteins, complement regulatory protein (MCP), decay accelerating factor (DAF), CR1, CF2, and CD59.

[0135] In some embodiments, useful expression products include any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. Useful heterologous nucleic acid sequences also include cholesterol-regulating and / or lipid-regulating receptors, including 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 and estrogen receptors, vitamin D receptors, and other nuclear receptors. Further useful gene products include transcription factors such as 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 regulatory factor (IRF-1), Wilms tumor protein, ETS-binding proteins, STATs, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.

[0136] In some embodiments, useful expression products include non-naturally occurring polypeptides, for example, chimeric or hybrid polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions, or amino acid substitutions.

[0137] Further suitable expression products include microRNA (miRNA), interfering RNA, antisense RNA, ribozymes and aptamers.

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

[0139] Optionally, the synthetic CNS-specific expression cassette comprises a sequence that provides or encodes one or more, and preferably all, of a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Optionally, the expression cassette comprises a nucleic acid encoding a post-transcriptional regulatory element. Optionally, the expression cassette comprises a nucleic acid encoding a polyA element.

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

[0141] In some embodiments of the invention, the vector is a plasmid. Such a plasmid may contain a variety of other functional nucleic acid sequences, such as one or more selectable markers, one or more origins of replication, multiple cloning sites, etc. In some embodiments of the 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 expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1, 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. Many other vectors are well known and commercially available. For mammalian cell adenoviral vectors, the pSV and pCMV series of vectors are particularly well-known, non-limiting examples. There are many well-known yeast expression vectors, including, but not limited to, Yeast Integrating Plasmids (YIp) and Yeast Replicating Plasmids (YRp). For plants, the Ti plasmid of Agrobacterium is an exemplary expression vector, and plant viruses also provide suitable expression vectors, for example, 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, including AAV vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors. When the vector is a gene therapy vector, the vector preferably comprises a nucleic acid sequence encoding a therapeutic product, optionally a therapeutic protein, operably linked to a synthetic CNS-specific promoter of the present invention. The therapeutic protein may be a secretable protein. Non-limiting examples of secretable proteins are discussed above, and exemplary secretable therapeutic proteins include clotting factors, e.g., factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, toxic proteins, and the like.

[0144] In some embodiments of the present invention, the vector is a viral vector, such as a retroviral, lentiviral, adenoviral, herpes simplex, or adeno-associated viral (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, AAVrhlO, 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 chimeric, meaning it contains components derived from at least two AAV serotypes, e.g., the ITRs of AAV2 and the capsid protein of AAV5. AAV9 is known to efficiently transduce CNS cells and tissues, particularly efficiently, and therefore AAV9 and its derivatives are particularly interesting for targeting CNS cells and tissues. AAV2g9 is known to efficiently transduce CNS cells and tissues, particularly efficiently, and therefore AAV2g9 and its derivatives are particularly interesting for targeting CNS cells and tissues. AAVrhlO is known to efficiently transduce CNS cells and tissues, particularly efficiently, and therefore AAVrhlO and its derivatives are particularly interesting for targeting CNS cells and tissues. Systemic or intravenous delivery of AAVrhlO has been shown to provide high transgene expression in the central nervous system, as described in (Tanguy et al., 2015), which is incorporated herein by reference, and therefore AAVrhlO is particularly preferred. AAVDJ8 is known to efficiently transduce CNS cells and tissues, particularly efficiently, and therefore AAVDJ8 and its derivatives are particularly interesting for targeting CNS cells and tissues. AAVDJ8 is preferred because it has been shown to efficiently target multiple brain regions and astrocytes, as described in (Hammond et al., 2017), which is 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 also of particular interest for targeting CNS cells and tissues.

[0146] The present invention further provides a recombinant virion (virus particle) comprising the vector described above.

[0147] Pharmaceutical Composition The vectors or virions of the invention can be formulated into pharmaceutical compositions using a pharmaceutically acceptable excipient, i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc. The pharmaceutical composition may be provided in the form of a kit. Pharmaceutical compositions and delivery systems suitable for AAV vectors and methods and uses thereof are known in the art.

[0148] Thus, a further aspect of the present invention provides a pharmaceutical composition comprising a vector or virion as described herein.

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

[0150] Pharmaceutical compositions can be formulated using 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 biodistribution (e.g., target 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 tunable expression of the payload of the invention. In some embodiments, a pharmaceutically acceptable excipient can 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 excipient is approved for human and veterinary use. In some embodiments, the excipient may be approved by the U.S. Food and Drug Administration. In some embodiments, the excipient can be of pharmaceutical grade. In some embodiments, the excipient can meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia. As used herein, excipients include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, as appropriate for the particular dosage form desired. Techniques for preparing various excipients and compositions for formulating pharmaceutical compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006, incorporated herein by reference in its entirety). The use of conventional excipient vehicles may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient vehicle may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or by otherwise interacting with any other components of the pharmaceutical composition in a deleterious manner.

[0151] Therapeutic Agents and Other Methods and Uses The present invention also provides synthetic CNS-specific promoters, expression cassettes, vectors, virions or pharmaceutical compositions according to various aspects of the invention for use in the treatment of diseases, preferably, optionally, diseases associated with aberrant gene expression in the CNS (e.g., genetic CNS diseases). Associated conditions, diseases and therapeutic expression products are discussed above.

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

[0153] The present invention also provides synthetic CNS-specific promoters, expression cassettes, vectors, virions according to various aspects of the invention for use in the manufacture of a pharmaceutical composition for the treatment of any of the conditions or diseases described herein.

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

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

[0156] In a preferred embodiment, the cell is a CNS cell, which may be ex vivo or in vivo. The CNS cell may be a primary neuron, astrocyte, oligodendrocyte, microglia, or ependymal cell. Alternatively, the CNS cell may be a CNS-derived cell line, e.g., an immortalized cell line.

[0157] The cells may be present in a CNS tissue environment (e.g., within the CNS of an animal) or may be isolated from CNS tissue, e.g., in cell culture. Optionally, the primary cells or cell lines are human cells.

[0158] A synthetic CNS-specific promoter, expression cassette or vector according to the invention can be inserted into the genome of a cell or can be episomal (eg, present in an episomal vector).

[0159] In a further aspect, the invention provides a method of producing an expression product, the method comprising providing a synthetic CNS-specific expression cassette according to the invention (preferably in a vector as described above) in a cell, preferably a CNS cell, and expressing the gene present in the synthetic CNS-specific expression cassette. Optionally, the method comprises maintaining the CNS cell under conditions suitable for expression of the gene. In culture, this may involve incubating the cell or tissue containing the cell under suitable culture conditions. Expression may, of course, be in vivo, e.g., in one or more cells in the CNS of a subject.

[0160] Optionally, the method comprises 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, such as an AAV vector, that contains a synthetic CNS-specific expression cassette.

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

[0162] The invention therefore provides, in some embodiments, an expression cassette, vector or virion according to the invention for use in gene therapy in a subject, preferably gene therapy through CNS-specific expression of a therapeutic gene. The therapy may include treatment of a disease through secretion of a therapeutic product from CNS cells, optionally a disease involving aberrant gene expression in the CNS, as discussed above.

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

[0164] The present invention also provides a method of gene therapy in a subject, preferably a human, in need thereof, comprising: - administering to a subject (and, where appropriate, introducing into the CNS of the subject) a synthetic CNS-specific expression cassette, vector, virion, or pharmaceutical composition of the invention comprising a gene encoding a therapeutic product; The present invention provides a method comprising:

[0165] The method optionally includes expressing a therapeutic amount of a therapeutic product from a gene in the CNS of the subject. Various conditions and diseases that can be treated are discussed above. Genes encoding suitable therapeutic products are discussed above.

[0166] The method optionally comprises administering to a subject a vector or virion according to the invention. Optionally, the vector is a viral gene therapy vector, for example an AAV vector.

[0167] In some embodiments, the method comprises administering the gene therapy vector systemically. Systemic administration can be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection). Preferred injection routes include intravenous, intramuscular, subcutaneous, intraarterial, intraarticular, intrathecal, and intradermal injection. In one embodiment, the gene therapy vector can be delivered into the CSF pathway by injection. Non-limiting examples of delivery into the CSF pathway include intrathecal and intraventricular administration.

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

[0169] In some embodiments, the viral gene therapy vector can be administered simultaneously or sequentially with one or more additional therapeutic agents or one or more saturating agents designed to prevent clearance of the vector by the reticuloendothelial system.

[0170] When the vector is an AAV vector, the vector dose is 1 x 10 10 gc / kg~1×10 15 gc / kg or more, as appropriate, 1 x 10 12 gc / kg~1×10 14 gc / kg, as appropriate, 5×10 12 gc / kg~5×10 13 It can be gc / kg.

[0171] Generally, the subject in need thereof is a mammal, preferably a primate, more preferably a human. Typically, the subject in need thereof exhibits symptoms characteristic of the disease. The method typically involves ameliorating the symptoms exhibited by the subject in need thereof by therapeutically expressing a therapeutic product. In one embodiment, the therapeutic method of the present invention can be used to reduce the decline in functional ability and activities of daily living, as measured by a standard assessment system, such as, but not limited to, the Total Functional Capacity (TFC) scale. In one embodiment, the method of the present invention can be used to improve performance on any assessment used to measure symptoms of a neurological disease.Such assessments include, but are not limited to, ADAS-cog (Alzheimer's Disease Assessment Scale-cognition), MMSE (Mini-Mental State Examination), GDS (Geriatric Depression Scale), FAQ (Functional Activities 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, IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly) Elderly), Neuropsychiatric Symptom Assessment, The Cohen-Mansfield Agitation Inventory, BEHAVE-AD, EuroQol, Short Form-36 and / or MBR Caregiver Strain Instrument or any of the other tests as described in Sheehan B (Ther Adv Neurol Disord. 5(6):349-358 (2012)), which are incorporated herein by reference in their entirety.

[0172] Gene therapy protocols for expressing therapeutic genes 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-carotid artery, hepatic artery, hepatic vein), intracranial administration, intramuscular injection, interstitial injection, instillation in the airways, and application to 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 contact the target cells with a catheter or implantable material containing the relevant vector, while more complex approaches can use jet injection devices and the like. Gene transfer into mammalian CNS cells has been performed using both ex vivo and in vivo procedures. Ex vivo approaches typically require the collection of CNS cells, in vitro transduction with an appropriate expression vector, and subsequent reintroduction of the transduced CNS cells into the CNS. This approach is generally less preferred due to the difficulties and risks of collecting and reintroducing CNS cells in the brain. In vivo gene transfer has been achieved by injecting DNA or viral vectors directly into the CNS, for example, by intracranial injection of viral vectors or by intravenous or intra-arterial injection.

[0173] In one embodiment, a gene therapy vector can be administered to a subject (e.g., to the CNS of a subject) in a therapeutically effective amount to reduce symptoms of the subject's neurological disorder (e.g., as determined using known assessments). In some embodiments, gene therapy vectors and compositions comprising gene therapy vectors can be administered in a manner that allows them to cross the blood-brain barrier, vascular barriers, or other epithelial barriers.

[0174] Gene therapy vectors can be used in combination with one or more other therapeutic, prophylactic, research, or diagnostic agents. "In combination with" is not intended to imply that the agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of the present invention. Compositions can be administered simultaneously with, prior to, or following one or more other desired therapeutic agents or medical procedures.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 such as memantine, antipsychotics, antidepressants, anticonvulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation such as methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or These include paclitaxel, kinase or phosphatase inhibitors, such as those targeting GSK3 (lithium) or PP2A, immunization with β-peptides or tau phosphoepitopes, anti-tau or anti-amyloid antibodies, dopamine depleting 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), acetylcholine release at the neuromuscular junction causing muscle paralysis, and steroids. These 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 predominant negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, mirtrazapine, venlafaxine for depression, anxiety, obsessive-compulsive behavior, and / or irritability), hypnotics (e.g., zopiclone and / or zolpidem for alterations of 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 methods 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 making and use of various embodiments of the invention are discussed in detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0177] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art in the areas relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but include a general class for which a particular example may be used for illustration. While technical terms are used herein to describe particular embodiments of the present invention, their use does not delimit the invention except as outlined in the claims.

[0178] The discussion of the background of the invention herein is included to explain the context of the invention and should not be construed as an admission that any of the material referred to was published, known, or part of the common general knowledge 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 an identifying citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents specifically referenced herein are incorporated by reference.

[0180] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and Sons 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 (M.J. 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 Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); and the series, Methods in Enzymology (Abelson and Simon, editors, Academic Press, Inc., New York), in particular volumes 154 and 155 (Wu et al., eds.) and volume 185, "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cell (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, volumes I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0181] The term "central nervous system" or "CNS" is 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 promoters of the present invention may be active in the brain and / or spinal cord. Preferably, the CNS is that of a mammal, even more preferably a human subject.

[0182] "CNS cell(s)" refers to cells found in or derived from the CNS (CNS tissue). CNS cells can be primary cells or cell lines (e.g., SH-Sy5y, Neuro2A, U87-MG). CNS cells can be in vivo (e.g., in CNS tissue) or in vitro (e.g., in cell culture). CNS cells consist of neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells. Neurons, as found in CNS tissue, contain a cell body, a long axon, and synaptic terminals. Neurons transmit electrical signals received at the cell body via their long axons to other cells near their synaptic terminals. Oligodendrocytes are a type of glial cell in the CNS that produce the myelin sheath that surrounds neuronal axons 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, assisting and regulating the transmission of electrical impulses. Microglia are resident macrophage cells in the brain and are involved in immune defense. Ependymal cells form the epithelial lining of the ventricles of the brain. The term "CNS cell(s)" as used herein includes neurons, astrocytes, oligodendrocytes, microglia, and / or ependymal cells. The promoters of the present invention can be active in any CNS cell (e.g., neurons). The promoters of the present invention can be active in more than one type of CNS cell (e.g., neurons and astrocytes). The promoters of the present invention can be active in all types of CNS cells (neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells). Furthermore, the synthetic CNS-specific promoters of the present invention can be active in one subtype of CNS cell, such as dopaminergic neurons or mature oligodendrocytes. In some embodiments, the synthetic CNS-specific promoters of the invention may be active only in one subtype of CNS cells, for example, dopaminergic neurons or mature oligodendrocytes.The CREs, proximal / minimal promoters, and promoters 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 CREs, proximal / minimal promoters, and promoters of the present invention may be active in specific CNS cell types, such as neurons, within all regions of the CNS. In other embodiments, the CREs, proximal / minimal promoters, and promoters of the present invention may be active in specific CNS cell types, such as neurons, within one or less regions of the CNS, such as the midbrain. In some embodiments, the CREs, proximal / minimal promoters, and promoters of the present invention may be active in all CNS cells, in all regions of the CNS. In some embodiments, the CREs, proximal / minimal promoters, and promoters of the present invention may be active in all CNS cells, in one or less regions of the CNS, such as the midbrain.

[0183] The term "cis-regulatory element" or "CRE" is a term well known to those skilled in the art and refers to a nucleic acid sequence, such as an enhancer, promoter, insulator, or silencer, that can regulate or modulate the transcription of adjacent genes (i.e., in cis). CREs are found near the gene they regulate. CREs usually regulate gene transcription by binding to TFs, i.e., they contain TFBSs. A single TF may bind to multiple CREs and thus control the expression of multiple genes (pleiotropy). CREs are usually, but not always, located upstream of the transcription start site (TSS) of the gene they regulate. In this context, an "enhancer" is a CRE that is operably associated with the gene it regulates, and can be found upstream, downstream, or even within its intron, enhancing (i.e., up-regulating) the transcription of the gene. Multiple enhancers may act in a coordinated manner to regulate the transcription of a single gene. In this context, a "silencer" refers to a CRE that binds to a TF called a repressor, which acts to prevent or down-regulate gene transcription. The term "silencer" can also refer to a region in the 3' untranslated region of a messenger RNA that binds to a protein that represses translation of that mRNA molecule, although this usage differs from its use in describing a CRE. Generally, a CRE of the present invention is a CNS-specific enhancer element (often referred to as a CNS-specific CRE or a CNS-specific CRE enhancer, or the like). In this context, it is preferred that the CRE is 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. CREs of the present invention are preferably relatively short in length, preferably 1000 nucleotides or less in length, for example they may be 800, 700, 600, 500, 400, 300, 200, 175, 150, 90, 80, 70, 60 or 50 nucleotides or less in length.The CRE of the present invention is typically provided in combination with an operably linked promoter element, which may be a minimal promoter or a proximal promoter, and the CRE of the present invention may enhance the CNS-specific activity of the promoter element.

[0184] The term "cis-regulatory module" or "CRM" generally refers to a functional regulatory nucleic acid module comprising two or more CREs; in the present invention, the CREs are typically CNS-specific enhancers, and thus the CRM is a synthetic CNS-specific regulatory nucleic acid. A CRM may comprise multiple CNS-specific CREs. Optionally, at least one of the CREs comprised in a CRM is a CRE according to SEQ ID NOs: 9-11, 28-31, or a functional variant thereof. Typically, multiple CREs within a CRM act together (e.g., additively or synergistically) to enhance transcription of a gene with which a promoter comprising the CRM is operably associated. There is considerable room for shuffling (i.e., rearranging), inverting (i.e., reversing orientation), and altering the spacing of CREs within a CRM. Thus, functional variants of the CRMs of the present invention include, inter alia, variants of the referenced CRMs in which the CREs within them have been shuffled and / or inverted, and / or the spacing between the CREs has been 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 must occur, i.e., transcription is initiated. A promoter allows for the appropriate activation or repression of transcription of a coding sequence under its control. A promoter usually contains specific sequences that are recognized and bound by multiple TFs. TFs bind to the promoter sequence, resulting in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of a gene. Many different promoters are known in the art.

[0186] The term "synthetic promoter" as used herein relates to a promoter that does not occur in nature. In this context, it typically comprises a CRE and / or CRM of the present invention operably linked to a minimal (or core) promoter or a CNS-specific proximal promoter (promoter element). The CRE and / or CRM of the present invention acts to enhance CNS-specific transcription of a gene operably linked to the synthetic promoter. While a portion of a synthetic promoter may occur in nature (e.g., a minimal promoter or one or more CREs in a promoter), a synthetic promoter as an entity does not occur in nature. Alternatively, a synthetic promoter may be a short, truncated version of a naturally occurring promoter.

[0187] As used herein, a "minimal promoter" (also known as a "core promoter") refers to a short DNA segment that is typically inactive or nearly inactive by itself but can mediate transcription when combined with other transcriptional regulatory elements. Minimal promoter sequences can be derived 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) immediate-early gene minimal promoter (CMV-MP), and the herpes thymidine kinase minimal promoter (MinTK). A minimal promoter typically includes a transcription start site (TSS) and immediately upstream elements, an RNA polymerase II binding site, and a general transcription factor binding site (often a TATA box). A minimal promoter may also include some elements downstream of the TSS, but these typically have little functionality without additional regulatory elements.

[0188] As used herein, the term "proximal promoter" refers to the proximal sequence upstream of a gene, which tends to contain a minimal promoter and at least some additional regulatory sequences, usually major regulatory elements. It often extends approximately 250 base pairs upstream of the TSS and includes specific TFBSs. The proximal promoter may also include one or more regulatory elements downstream of the TSS, such as UTRs or introns. In this case, the proximal promoter may optionally be a short, truncated version of a naturally occurring CNS-specific proximal promoter. The proximal promoter 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 a 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 the reference sequence that retains the ability to function in the same manner as, for example, a CNS-specific CRE, CNS-specific CRM, or CNS-specific promoter. Alternative terms for such functional variants include "biological equivalents" or "equivalents."

[0191] It will be appreciated 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 sequence's ability to bind to the same CNS-specific TF that binds to the reference sequence. Thus, in most cases, a functional variant of a CRE or CRM will contain most or all of the TFBSs of the same TF as the reference CRE, CRM, or promoter. The TFBSs of the functional variant are preferably, but not necessarily, in the same relative position (i.e., order and overall position) as the reference CRE, CRM, or promoter. It is also preferred, but not necessarily, that the TFBSs of the functional variant be in the same orientation as the reference sequence (note that in some cases, they may be present in the reverse orientation, e.g., as the reverse complement of a sequence in the reference sequence). It is also preferred, but not necessarily, that the TFBSs of the functional variant be on the same strand as the reference sequence. Thus, in a preferred embodiment, the functional variant contains the TFBSs of the same TFs as the reference sequence in the same order, position, orientation, and strand. It will also be understood that the sequences between the TFBSs (sometimes referred to as spacer sequences or the like) are not critical to the function of the CRE or CRM. Such sequences can typically vary significantly, and their length may be altered. 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., not different by more than 20%, preferably not different by more than 10%, more preferably nearly identical). It will be apparent that in some cases, a functional variant of a CRE may exist in the reverse orientation, e.g., may be the reverse complement of a 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 of retained functionality. A high level of sequence identity in TFBSs of CREs, CRMs, or promoters is generally of greater importance than sequence identity in spacer sequences (where there is little or no need for any conservation of sequence). However, given that the sequences of functional TFBSs do not need to correspond exactly to consensus sequences, it will be appreciated that a considerable degree of sequence variation can be accommodated even within a TFBS.

[0193] The ability of one or more TFs to bind to TFBSs in a given functional variant can be determined by any relevant means known in the art, including, but not limited to, electromobility 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 in Sambrook et al., cited above. Numerous relevant publications describing this procedure are available, for example, Hellman and Fried, Nat Protoc. 2007; 2(8): 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 expression of a gene in CNS cells (or in CNS-derived cells) in a preferential or predominant manner compared to other tissues (e.g., liver, kidney, spleen, heart, muscle, and lung). The expression of a gene can 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, i.e., 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 easily evaluated by those skilled in the art. Therefore, those skilled in the art can easily 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), and the ability of the cis-regulatory element to drive CNS-specific expression of a gene (usually a reporter gene) can be measured. Alternatively, a CRE or CRM variant can be substituted into a synthetic CNS-specific promoter in place of a reference CRE or CRM, and the effect on CNS-specific expression driven by the modified promoter can be determined and compared with the unmodified form. Similarly, the ability of a promoter to drive CNS-specific expression can be easily evaluated by those skilled in the art (e.g., as described in the Examples below). 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 assessing enhanced CNS-specific expression are readily constructed, and suitable methodologies are provided by the examples described below.

[0196] CNS specificity can be identified, where expression of a gene (e.g., a therapeutic or reporter gene) occurs preferentially or predominantly in CNS-derived cells. For example, preferential or predominant expression can be defined when the level of expression is significantly greater in CNS-derived cells than in other types of cells (i.e., non-CNS-derived cells). For example, expression in CNS-derived cells is suitably at least 5-fold higher than in non-CNS cells, preferably at least 10-fold higher than in non-CNS cells, and in some cases may be 50-fold higher. Conveniently, CNS-specific expression may be demonstrated by comparison of expression levels in various non-CNS cell lines, as appropriate, for example, primary CNS cells or CNS-derived cell lines, e.g., SH-Sy5y, Neuro2A, U87-MG, in muscle-derived cell lines, e.g., C2C12 or H2K cells (skeletal muscle) or H9C2 cells (heart), compared 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).

[0197] The synthetic CNS-specific promoters of the invention preferably exhibit reduced expression in non-CNS-derived cells, as appropriate C2C12, H9C2, Huh7, HEK-293, HeLa, and / or A549 cells, when compared to a non-tissue-specific promoter, e.g., CMV-IE. The synthetic CNS-specific promoters of the invention preferably have no more than 50%, as appropriate, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, or no more than 1% of the activity of the CMV-IE promoter in non-CNS-derived cells (as appropriate C2C12, H9C2, Huh7, HEK-293, HeLa, and / or A549). Generally, it is preferred that expression in non-CNS-derived cells be minimized, although in some cases this may not be necessary. Even if a synthetic CNS-specific promoter of the invention has, for example, higher expression in one or two non-CNS cells, it can still be a CNS-specific promoter, as long as it has overall higher expression in a variety of CNS cells relative to non-CNS cells.

[0198] The synthetic CNS-specific promoters of the present invention are preferably suitable for promoting expression in the CNS of a subject, e.g., for driving 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 transgene expression and have activity in CNS cells that is 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 some embodiments, the synthetic CNS-specific promoters of the present invention are suitable for promoting CNS-specific transgene expression at a level that is at least 100% of the activity of the synapsin-1 promoter, preferably 150%, 200%, 300%, or 500% of the activity of the synapsin-1 promoter. Such CNS-specific expression is suitably determined in CNS-derived cells, for example SH-Sy5y, Neuro2A, U87-MG cell lines or primary CNS cells (suitably primary human neurons, astrocytes, oligodendrocytes, microglia and / or ependymal cells).

[0199] The synthetic CNS-specific promoters of the invention may also be capable of promoting CNS-specific expression of a gene at a level 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 (primary human neurons, astrocytes, oligodendrocytes, microglia and / or ependymal cells, as appropriate).

[0200] The term "nucleic acid," as used herein, generally refers to an oligomer or polymer (preferably a linear polymer) of any length essentially composed of nucleotides. A nucleotide unit generally comprises a heterocyclic base, a sugar group, and at least one phosphate group, including, for example, one, two, or three modified or substituted phosphate groups. Heterocyclic bases can include, among others, purine and pyrimidine bases, such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), which are widely found in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (e.g., methylated), non-natural, or derivatized bases. Sugar groups may include, inter alia, pentose (pentofuranosyl) groups, such as, 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 contemplated 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 the phosphate group or sugar may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably encompasses DNA, RNA, and DNA-RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA-RNA hybrids. A nucleic acid can be naturally occurring, e.g., occurring in or isolated from nature, or non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partially or wholly chemically or biochemically synthesized. A "nucleic acid" can be double-stranded, partially double-stranded, or single-stranded. If single-stranded, the nucleic acid can be the sense strand or the antisense strand.Furthermore, nucleic acids can be circular or linear.

[0201] "Isolated," when referring to a nucleic acid, means a nucleic acid molecule or sequence that lacks all or part of the sequences that are normally associated with it in nature, or a sequence as it occurs in nature but with heterologous sequences that associate it with, or a molecule that has been dissociated from a chromosome.

[0202] Terms such as "identity" and "identical" refer to the sequence similarity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules. Sequence alignment and sequence identity determination 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 sequence" 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 are described, 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 73:237-244; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65, Pearson et al. (1994) Methods Mol. Biol. 24:307-31; (1999) FEMS Microbiol. Lett. 174:247-50. A detailed discussion of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[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), and on the Internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is 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 used using default parameters. Nucleic acid sequences with greater similarity to a reference sequence will exhibit increased percentage identity when assessed by this method. Typically, percentage sequence identity is calculated over the entire length of the sequence.

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

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

[0207] The meaning of "consensus sequence" is well known in the art. In this application, the following notation is used for consensus sequences unless the context indicates otherwise. 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 except A can be found at that position, Y represents any pyrimidine, and R represents any purine.

[0209] As used herein, "synthetic" refers to a nucleic acid molecule that does not occur in nature. Synthetic nucleic acids of the invention are produced artificially, typically 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 are present in a context that does not occur in nature. For example, a synthetic gene (or portion of a gene) may contain one or more nucleic acid sequences that are not normally contiguous in nature (chimeric sequences), and / or may include substitutions, insertions, and deletions, and combinations thereof.

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

[0211] The term "administration," as used herein, refers to the introduction of a xenobiotic into the human or animal body. Administration can be, for example, intravenous, intraarterial, or intracranial.

[0212] As used herein, "transfection" refers broadly to any process of deliberately introducing nucleic acid into a cell, and covers the introduction of viral and non-viral vectors, and includes or is equivalent to transformation, transduction, and similar terms and processes. Examples include, but are not limited to, transfection with viral vectors, transformation with plasmid vectors, electroporation (Fromm et al. (1986) Nature 319:791-3), lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7), microinjection (Mueller et al. (1978) Cell 15:579-85), Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7), direct DNA uptake, whisker-mediated transformation, and particle bombardment (Klein et al. (1987) Nature 327:70).

[0213] As used herein, the phrase "transgene" refers to an exogenous nucleic acid sequence. In one example, a transgene is a gene that encodes an industrially or pharmaceutically useful compound or a gene that encodes a desirable trait. In yet another example, a transgene encodes a useful nucleic acid, such as an antisense nucleic acid sequence, expression of which inhibits expression of a target nucleic acid sequence. The transgene preferably encodes a therapeutic product, e.g., a protein.

[0214] The term "vector" is well known in the art and, as used herein, refers to a nucleic acid molecule, e.g., double-stranded DNA, into which a nucleic acid sequence according to the present invention may be inserted. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements to allow transcription of the inserted nucleic acid molecule, preferably translation of the transcript into a polypeptide. A vector typically contains all of the essential elements so that, once the vector is in a host cell, the vector can replicate independently of or co-operating with the host chromosomal DNA, resulting in the generation of several copies of the vector and its inserted nucleic acid molecule. The vectors of the present invention may be episomal vectors (i.e., not integrated into the genome of the host cell) or vectors that integrate 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 pBR322 or their derivatives lacking bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. To accommodate larger inserts, larger vectors such as artificial chromosomes (bacterial (BAC), yeast (YAC), or human (HAC)) can be used. Viral vectors are derived from viruses and include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, hepatitis virus vectors, etc. Viral vectors are usually, but not necessarily, replication-deficient because viral genes essential for replication have been removed from the viral vector, rendering them unable to propagate in a given cell. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as cancer cells, and are typically used to induce (cancer) cell-specific (tumor) lysis.Virosomes are a non-limiting example of vectors that contain both viral and non-viral elements; in particular, they combine liposomes with inactivated HIV or influenza viruses (Yamada et al., 2003). Another example includes 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 one another such that the elements are functionally connected and can interact with one another in the intended manner. Such elements include, but are not limited to, a promoter, a CRE (e.g., an enhancer or other regulatory element), a promoter element, a polyadenylation sequence, one or more introns and / or exons, and the coding sequence of a gene of interest to be expressed. When properly oriented or operably linked, nucleic acid sequence elements may act together to modulate each other's activity, ultimately affecting the level of expression of the expression product. By modulate, we mean to increase, decrease, or maintain the level of activity of a particular element. The position of each element relative to other elements can be expressed in terms of the 5' and 3' ends of each element or its position upstream or downstream of another element or moiety (e.g., a TSS or promoter element), and the distance between any particular elements can be referred to by the number of intervening nucleotides or base pairs between the elements. As will be understood by those skilled in the art, operably linked implies functional activity and does not necessarily relate to the natural positional linkage. Indeed, when used in a nucleic acid expression cassette, the CRE is usually located immediately upstream of the promoter element (this is generally the case, but should not be clearly interpreted as a limitation or exclusion of the position within the nucleic acid expression cassette), but this does not have to be the case in vivo; for example, a regulatory element sequence naturally occurring downstream of the gene whose transcription is affected can function in the same way as when located upstream of the promoter. Thus, according to certain embodiments, the regulatory or enhancing effect of a regulatory element may be position-independent.

[0216] As used herein, a "spacer sequence" or "spacer" refers to a nucleic acid sequence that separates two functional nucleic acid sequences (e.g., TFBS, CRE, CRM, promoter element, etc.). It can have essentially any sequence, as long as it does not prevent the functional nucleic acid sequence (e.g., a cis-regulatory element) from functioning as desired (e.g., this can occur when it contains a silencer sequence, preventing the binding of a desired transcription factor or the like). Typically, it is non-functional, as it exists solely to space adjacent functional nucleic acid sequences apart from one another. In some embodiments, the spacer can have a length of 75, 50, 40, 30, 30, or 10 nucleotides or less.

[0217] The term "pharmaceutically acceptable," as used herein, is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0218] "Therapeutically effective amount" and similar phrases refer to a dosage or plasma concentration in a subject that provides the specific desired pharmacological effect, e.g., expression of a therapeutic gene in the CNS. Although a therapeutically effective amount may not always be effective in treating the conditions described herein, such a dosage would be considered therapeutically effective by one of skill in the art. A therapeutically effective amount may vary based on the route and dosage 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, is well known in the art and generally refers to an AAV vector nucleic acid sequence that includes a variety of nucleic acid sequences. An AAV vector, as used herein, typically includes a heterologous nucleic acid sequence not of AAV origin as part of the vector. This heterologous nucleic acid sequence typically includes a promoter, as disclosed herein, and other sequences of interest for genetic transformation of a cell. Generally, the heterologous nucleic acid sequence is flanked by at least one, and typically two, AAV inverted terminal repeat (ITR) sequences. An "AAV virion" or "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid polypeptide (including both variant AAV capsid polypeptides and non-variant parent capsid polypeptides) and an encapsidated polynucleotide AAV vector. When a particle includes heterologous nucleic acid (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell), it may also be referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of AAV virions or particles necessarily includes production of AAV vectors, since such vectors are contained within AAV virions or particles. The ITRs may be derived from the same serotype as the capsid, selected from any of the serotypes listed in Table 1, or from a different serotype than the capsid. AAV vectors typically have more than one ITR. In a non-limiting example, 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. Non-limiting examples include none, one, or both ITRs having the same serotype as the capsid. Independently, each ITR can be about 100 to about 150 nucleotides in length.The ITRs can be approximately 100-105, 106-110, 111-115, 116-120, 121-125, 126-130, 131-135, 136-140, 141-145, or 146-150 nucleotides in length. In one embodiment, the ITRs are 140-142 nucleotides in length. Non-limiting examples of ITR lengths are 102, 105, 130, 140, 141, 142, and 145 nucleotides in length.

[0220] As used herein, the term "microRNA" refers to any type of interfering RNA, including, but not limited to, endogenous microRNA and artificial microRNA (e.g., synthetic miRNA). Endogenous microRNA is a small RNA naturally encoded in the genome that can regulate the productive utilization of mRNA. Artificial microRNA can be any type of RNA sequence other than endogenous microRNA that can regulate the activity of mRNA. A microRNA sequence can be an RNA molecule 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, 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 larger RNA, or include miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNAs. See, e.g., U.S. Patent Applications Nos. 20050272923, 20050266552, 20050142581, and 20050075492. "MicroRNA precursor" (or "pre-miRNA") refers to a nucleic acid having a stem-loop structure incorporating a microRNA sequence. "Mature microRNA" (or "mature miRNA") includes microRNAs that are cleaved from microRNA precursors ("pre-miRNA") or synthesized (e.g., synthesized in the laboratory by cell-free synthesis) and have a length of about 19 nucleotides to about 27 nucleotides; for example, a mature microRNA can have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, or 27 nt.Mature microRNAs can bind to target mRNAs and inhibit their translation.

[0221] The term "treatment" or "treating" refers to reducing, ameliorating, or eliminating one or more symptoms, symptoms, or effects of a disease or condition. "Treatment," as used herein, therefore, includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who is susceptible to or at risk of acquiring the disease, but who has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., halting its development, and (c) relieving 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 a subject to perform its intended function. Administration can be by any suitable route, including orally, intranasally, intraocularly, by eye drop, parenterally (intravascularly, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and administration by another. Intravenous or intra-arterial administration is of particular interest in the present invention.

[0223] The terms "individual," "subject," and "patient" are used interchangeably and refer to any individual subject having a disease or condition in need of treatment. For purposes of this disclosure, a subject may be a primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or cow.

[0224] The term "specifically active in a region or 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. [Example]

[0225] Example 1 CNS transduction and vector biodistribution of CNS1-8 (SEQ ID NOS: 1-4, 23-26) operably linked to GFP was studied using AAV9. AAV plasmid preparation: The hSyn.GFP plasmid, containing ssAAV2 inverted terminal repeats, was obtained from Addgene and used to generate a control AAV vector (synapsin-1). The CNS 1-8 (SEQ ID NOS: 1-4, 23-26) promoter was cloned into the hSyn.GFP plasmid to replace the hSyn promoter using GeneArt® (Thermo Fisher Scientific, Germany). All plasmid DNA was prepared using the PureLink™ HiPure Plasmid Maxiprep Kit (product 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 (referred to throughout as AAV9) vectors encoding GFP were generated by a standard triple-plasmid transfection method. Briefly, virus-producing human embryonic kidney (HEK) 293T cells were cotransfected with three plasmids: pGFP controlled by various promoters (SEQ ID NOS: 1-4, 23-26), pGD9 encoding the AAV9 capsid, and pHGTI containing helper functions, at a stock concentration of 1 mg / ml and a molar ratio of 1:3:1 using polyethyleneimine (PEI) (product no. 24765; Polysciences, UK). After 72 hours, cells were harvested and lysed. Cell lysates and supernatants were nuclease-treated, filtered, and purified by affinity chromatography on AKTAprime plus (GE Healthcare Ltd, UK) using POROS™ CaptureSelect™ AAVX resin (Thermo Fisher Scientific, Germany) and Primeview 5.0 software.

[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) with Luna® Universal qPCR Master Mix according to the manufacturer's instructions (number M3003; New England Biolabs, UK). Data were analyzed using QuantStudio Design and Analysis Software V5. Primers designed to amplify a segment of the GFP transgene (Table 5) were used to determine vector genome numbers. All vectors were titrated at 1 × 10 13 Titers were matched to vector genomes / mL (vg / mL).

[0228] Animal Procedures: All animal experiments were conducted within the guidelines of the University College London Ethics Committee, in accordance with UK Home Office regulations and the Animals (Scientific Procedures) Act 1986. Outbred CD1 mice (Charles River, UK) were housed in individually ventilated cages (IVC) at the Central Biological Services Unit, UCL, under standard conditions with a 12-h light-dark cycle, constant temperature (21–23°C), humidity (60% ± 5%), and free access to chow food and water. Experimental breeding pairs were time-mated after 6 weeks of age, and newborn litters were used for these promoter studies. Pups were weaned at P21 and euthanized for tissue analysis at P35.

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

[0230] Neonatal intracranial injection of viral vectors: Pups were inoculated with 5 μl of viral vector (5 × 10) using a 33-gauge Hamilton needle (Fisher Scientific, UK) using established coordinates (Kim, Ji-Yoen et al., 2013), which are incorporated herein by reference. 10 The viral genome / pup was injected into the lateral cerebral ventricle. Injection into the ventricle bypasses the blood-brain barrier.

[0231] Neonatal intravenous injection of viral vectors: Each pup received 20 μl of viral vector (2 × 10 11 vg / pup) was injected into the superficial temporal vein. The vein was visualized using fiber optic transillumination, and the injection was performed using a 33-gauge Hamilton needle under a stereoscopic operating microscope (Zeiss, Germany).

[0232] Perfusion and tissue preparation: Animals were anesthetized with isoflurane (5% induction chamber, 1.5% maintenance via nose cone). Transcardial perfusion was performed by cutting the right atrium and injecting 10 mL of autoclaved phosphate-buffered saline (PBS) into the left ventricle until liver blanching was achieved. The brain and visceral organs were divided into two halves to allow for different processing techniques depending on the subsequent experiment. The half used for immunohistochemistry was post-fixed in 4% paraformaldehyde (PFA) for 48 hours and transferred to a 30% sucrose solution for cryoprotection at 4°C until sectioning. The half brains were mounted on a freezing microtome (Thermo Fisher HM430) at 40 mm 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. Brain halves 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] Tissue analysis of GFP expression: GFP expression in mouse brain was assessed by immunohistochemistry (IHC) and immunofluorescence (IHF).

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

[0235] All brain sections were washed, then treated with 30% H2O2 (Sigma-Aldrich, UK) in 1x TBS for 30 minutes and blocked with 15% normal goat serum (Vector Laboratories, UK) in TBST (1x TBS, 0.3% Triton X-100) for 30 minutes at room temperature. Samples were incubated in primary antibodies (rabbit or chicken anti-GFP antibodies from Table 6) for 12–14 hours at 4°C on an orbital shaker with constant agitation. Sections were washed and incubated with the corresponding biotinylated secondary antibodies (anti-rabbit or anti-chicken biotinylated secondary antibodies from Table 6) for 2 hours at room temperature on an orbital shaker. Sections were washed and incubated with Vectastain avidin-biotin solution (ABC Vector Stain, Vector Laboratories, UK). Sections were washed and the reaction visualized with DAB (Sigma Aldrich, UK) (10 mg DAB in 20 mL TBS, 6 ml 30% H2O2). The reaction was stopped after a maximum of 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 antibodies from Table 6) diluted in 10% normal goat serum covered at 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] microscope: Light microscopy and fluorescence imaging were performed using a Leica DM4000B, and all images were captured using a Leica DFC420 camera and Leica Application Suite V3.7 software, keeping light intensity, exposure, microscope calibration, and photographic camera settings constant (Leica Microsystems, UK).

[0238] Quantitative measurements of GFP staining intensity were performed by threshold analysis on 10 non-overlapping RGB images at ×40 magnification 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] Quantification of midbrain dopaminergic (mDA) neurons was performed by counting TH-positive neurons and vector-driven GFP-expressing cells, and the percentage of double-positive neurons was calculated.

[0240] qRT-PCR for vector expression analysis: RNA was extracted from brains and organs using the TRIzol™ Plus RNA Purification Kit (Thermo Fisher Scientific, Germany) or the RNeasy Mini Kit (Qiagen, UK) and quantified using an Omega FLUOstar (BMG Labtech, UK). Contaminating DNA was removed from total RNA (1–2 μg) using a DNase I (DNAse I) Purification Kit (NEB, UK), followed by reverse transcription using a High-Capacity cDNA Reverse Transcription Kit (Applied Bioscience, Thermo Fisher Scientific, Germany). qPCR was performed using 10 ng of cDNA and Luna Taqman Master Mix (NEB, UK) on a Quantstudio™ Real-Time PCR System (Applied Biosystems, UK) with 300 nM primers (Table 5).

[0241] For quantification of GFP transcripts, normalization was achieved by comparison to standard curves generated by amplification from plasmid constructs specific for GFP and mGAPDH transcripts. mGAPDH was used as an endogenous control, and relative fold changes were calculated as described for vector genome copy number analysis.

[0242] [Table 1]

[0243] [Table 2]

[0244] CNS 1-8 construct design The promoters in this invention were 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 long versions of 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) were shortened, and the minimal promoter SYNP_CRE151 (SEQ ID NOS: 12) was added to the shortened versions to arrive at 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 profiles.

[0246] result GFP expression from the CNS-1 through CNS-8 promoters (SEQ ID NOs: 1-4, 23-26) and from the control promoter Syn1 (SEQ ID NO: 14) was first assessed in sagittal sections, and the results are shown in Figure 2A-B. All promoters tested exhibited CNS expression, with varying intensity and distribution across different brain regions.

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

[0248] In IV-injected animals, CNS-4 (SEQ ID NO: 4) showed the strongest expression, CNS-3 (SEQ ID NO: 3) showed the weakest, and the rest of the promoters fell 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] Thus, the method of administration (ICV vs. IV) affects both the strength and distribution of CNS promoters.

[0250] GFP expression from the CNS-1 to CNS-8 (SEQ ID NOs: 1-4, 23-26) promoters delivered by ICV and from the control promoter Syn1 (SEQ ID NO: 14) was then assessed in coronal sections, and the results are shown in Figure 3A-B. Again, all tested promoters showed CNS expression, with varying intensity and distribution across various brain regions. Promoters CNS-1, CNS-2 (SEQ ID NOs: 1-2), and CNS-7 (SEQ ID NO: 25) showed the strongest expression, CNS-3 (SEQ ID NO: 3) showed the weakest expression, and the rest of the promoters fell between the two extremes. Promoters CNS-1 (SEQ ID NO: 1), CNS-2 (SEQ ID NO: 2), and CNS-7 (SEQ ID NO: 25) showed similar expression levels to the control promoter Syn1.

[0251] GFP expression from the CNS-1 through CNS-8 (SEQ ID NOs: 1-4, 23-26) promoters delivered by IV was also assessed in coronal sections, and the results are shown in Figure 4A-B. Again, all promoters tested showed CNS expression, with varying intensity and distribution across various brain regions. Promoter CNS-3 (SEQ ID NO: 3) showed the strongest expression, CNS-8 (SEQ ID NO: 26) showed the weakest expression, and the rest of the promoters fell between the two extremes.

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

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

[0254] Expression from the CNS1-8 (SEQ ID NOS: 1-4, 23-26) promoter 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 fraction 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 the CNS1-8 (SEQ ID NOS: 1-4, 23-26) promoter 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] The biodistribution in various tissues of CNS-1 to 8 (SEQ ID NOs: 1 to 4, 23 to 26) and the transgene GFP under the control of the control promoter Syn-1 (SEQ ID NO: 14) delivered by ICV and by IV is shown in Figure 9 .

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

[0258] With 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, whereas the remaining promoters tested did not. With 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, whereas the remaining promoters tested did not.

[0259] With ICV delivery, CNS1-3 (SEQ ID NOs: 1-3) and CNS-8 (SEQ ID NO: 26) showed activity in the kidney, whereas CNS-4-7 (SEQ ID NOs: 4, 23-25) and the control promoter Syn-1 (SEQ ID NO: 14) showed no activity in the kidney. With IV delivery, CNS-2-3 (SEQ ID NOs: 2-3) and CNS-8 (SEQ ID NO: 26) showed activity in the kidney, whereas the rest of the promoters tested and the control promoter Syn-1 (SEQ ID NO: 14) did not.

[0260] With ICV delivery, CNS-3 (SEQ ID NO: 3) is active in skeletal muscle, whereas CNS-1 to 2 (SEQ ID NOs: 1 to 2) and CNS-4 to 8 (SEQ ID NOs: 4, 23 to 26) are inactive in skeletal muscle. With IV delivery, CNS-1 to 3 (SEQ ID NOs: 1 to 3) are active in skeletal muscle, whereas CNS-4 to 8 (SEQ ID NOs: 4, 23 to 26) are inactive in skeletal muscle. The control promoter Syn-1 (SEQ ID NO: 14) is inactive in skeletal muscle.

[0261] In ICV delivery, CNS-1 (SEQ ID NO: 1), CNS-7 to CNS-8 (SEQ ID NO: 25 to CNS-8) are active in the spleen, whereas CNS-2 to CNS-6 (SEQ ID NO: 2 to CNS-4, 23 to CNS-4) and the control promoter Syn-1 (SEQ ID NO: 14) are inactive in the spleen. In IV delivery, CNS-2 (SEQ ID NO: 2), CNS-7 to CNS-8 (SEQ ID NO: 25 to CNS-8) are active in the spleen, whereas CNS-1 (SEQ ID NO: 1), CNS-3 to CNS-6 (SEQ ID NO: 3 to CNS-4, 23 to CNS-4) and the control promoter Syn-1 (SEQ ID NO: 14) are inactive in the spleen.

[0262] mm 2The percentage of GFP immunoreactivity per promoter was measured in various regions of the brain, and the results are shown in Figure 10. As previously described, GFP was placed under the control of 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 the control promoter Syn-1 (SEQ ID NO: 14), delivered by ICV and by IV. With ICV delivery, the control promoters Syn-1 (SEQ ID NO: 14), CNS-1 (SEQ ID NO: 1), and CNS-2 (SEQ ID NO: 2) had the highest percentage of GFP immunoreactivity in the cortex, followed by CNS-7 (SEQ ID NO: 25) and CNS-4 (SEQ ID NO: 4), while the rest of the promoters tested showed very little or no GFP immunoreactivity in the cortex. With IV delivery, the control promoter Syn-1 (SEQ ID NO: 14) had a high percentage of GFP immunoreactivity in the cortex, while the rest of the promoters tested had very little or no GFP immunoreactivity in the cortex.

[0263] With ICV delivery, CNS-1 (SEQ ID NO: 1) and CNS-2 (SEQ ID NO: 2) had very 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), with the remainder of the promoters tested showing very little or no GFP immunoreactivity in the striatum. With IV delivery, CNS-2 (SEQ ID NO: 2) and CNS-3 (SEQ ID NO: 3) had low GFP immunoreactivity percentages in the striatum, with the remainder of the promoters tested showing very little or no GFP immunoreactivity in the striatum. The control promoter Syn-1 (SEQ ID NO: 14) showed very high GFP immunoreactivity in the striatum with both ICV and IV delivery.

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

[0265] With ICV delivery, CNS-1 (SEQ ID NO: 1) and CNS-2 (SEQ ID NO: 2) had 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 rest of the promoters tested showed very little or no GFP immunoreactivity in the cortex. With IV delivery, CNS-1-8 (SEQ ID NOs: 1-4, 23-26) 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. Notably, CNS-8 (SEQ ID NO: 26) with 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] With ICV delivery, CNS-1 to CNS-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 CNS-8 (SEQ ID NOs: 23 to 26) were higher than that of the control promoter Syn-1 (SEQ ID NO: 14). With IV delivery, the control promoter Syn-1 (SEQ ID NO: 14) had a high GFP immunoreactivity percentage in the cerebellum, while the rest of the tested promoters had very little or no GFP immunoreactivity in the cerebellum.

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

[0268] Example 2 The biodistribution of the transgene GFP under the control of CNS-8 (SEQ ID NO: 26) was further investigated at higher doses (herein referred to as high doses) in IV and ICV delivery. Intracranial and intravenous injections were performed as described in Example 1, except that intracranial injections were performed at 5x10 11 Inject 2 x 10 viral genomes / pup 12 vg / pup injected intravenously (10-fold higher dose). The dose used in the IV and ICV delivery in Example 1 is referred to herein as the low dose.

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

[0270] Similarly, as shown in Figures 3B, 5B, and 13A, the biodistribution of GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at low doses was very similar to the biodistribution of GFP under the control of CNS-8 when administered at high doses in coronal sections for ICV delivery at high and low magnifications.

[0271] As shown in Figures 4B, 6B, and 13B, the biodistribution of GFP under the control of CNS-8 (SEQ ID NO: 26) when administered at low doses was very similar to the biodistribution of GFP under the control of CNS-8 when administered at high doses in coronal sections upon IV delivery at 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 when a low dose was administered was very similar to GFP expression under the control of CNS-8 (SEQ ID NO: 26) when a high dose was administered in both ICV and IV delivery. This was supported by quantification of GFP-positive dopaminergic neurons, which showed no difference in the percentage of GFP-positive dopaminergic neurons between low and high doses in both ICV and IV delivery, as shown in Figure 14B. Thus, there was no overall difference in GFP expression under the control of the CNS-8 promoter (SEQ ID NO: 26) between low and high doses, indicating that a low dose is sufficient to show GFP expression in dopaminergic neurons and that increasing the dose does not result in higher GFP expression. Where appropriate, the lowest dose that shows the desired expression pattern may be preferred.

[0273] Comparison of the biodistribution of the transgene GFP under the control of CNS-8 (SEQ ID NO: 26) in various tissues when administered at low or high doses showed that the dose affected GFP expression. In the liver, GFP expression was very similar between doses in ICV delivery but low at the low dose in IV delivery. In the heart, GFP expression was not detected at the low dose in either ICV or IV delivery, but was detected at the high dose in IV delivery. Similarly, in skeletal muscle, GFP expression was not detected at the low dose in either ICV or IV delivery, but was detected at the high dose in both ICV and IV delivery. However, in the spleen, higher GFP expression was detected at the low dose compared to the high dose in both IV and ICV delivery. Similarly, higher GFP expression was detected at the low dose compared to the high dose in both IV and ICV delivery. This data indicates that administration of different doses of viral genome can result in different expression patterns and levels in tissues other than the CNS.

[0274] Thus, changing the dose did not alter GFP expression and levels in the CNS, but did alter the expression pattern and expression level in tissues other than the CNS. Therefore, it may be possible to find an optimal dose depending on the expression pattern and level requirements in tissues other than the CNS, while maintaining the expression pattern and level in the CNS. For example, if activity in the CNS as well as in the liver, spleen, and kidney 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 heath 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 from CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4 were engineered, were investigated in single-cell transcriptome datasets (Zeisel et al., 2018). Due to the proximity of the CRE / proximal promoter to its gene, the CRE / proximal promoter is predicted to assist in regulating the gene (He et al., 2014). Assuming that the CRE / proximal promoter regulates the expression of its nearest gene, the expression pattern of the gene provides an indication of the potential expression profile of the synthetic promoter containing 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 from the CNS and PNS of adult mice. The resource is publicly available at mousebran.org / genesearch.html and provides a useful tool for determining the potential expression of synthetic promoters CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4 in the PNS. Genes with engineered CRE / proximal promoters for CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4 were 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 many PNS neurons, and therefore synthetic promoters containing a CRE or proximal promoter engineered from the faf1 gene, e.g., CNS-5 and CNS-5_v2, are predicted to have strong expression in the PNS. pitx3 is expressed in sympathetic PNS neurons, and therefore synthetic promoters containing a CRE engineered from the pitx3 gene, e.g., CNS-2, CNS-3, or CNS-4, are predicted to have expression in PNS sympathetic neurons. Similar analysis of lmx1b and pitx2 showed no expression in the PNS above the cutoff score for the analysis (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] References Boussicault, 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] Allocation 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 comprising a sequence according to SEQ ID NO: 1 or 21 or a sequence that is at least 90% identical to any one of SEQ ID NOs: 1 and 21.

2. 2. The synthetic CNS-specific promoter of claim 1, wherein a synthetic CNS-specific promoter having at least 90% sequence identity retains at least 25%, 50%, 75%, 80%, 85%, 90%, 95% or 100% of the activity of a synthetic CNS-specific promoter comprising a sequence according to SEQ ID NO: 1 or 21.

3. 10. The synthetic CNS-specific promoter of claim 1, which is broadly active in the brain when administered by intracerebroventricular (ICV) injection.

4. 4. The synthetic CNS-specific promoter of claim 3, which is active at a level that is at least 100%, 150%, or 200% of the activity of the synapsin-1 promoter (SEQ ID NO: 14) in the brain.

5. 10. An expression cassette comprising the synthetic CNS-specific promoter of any one of claims 1 to 4 operably linked to a nucleic acid sequence encoding an expression product.

6. A vector comprising the synthetic CNS-specific promoter of any one of claims 1 to 4 or the expression cassette of claim 5.

7. The vector of claim 6, which is a viral vector.

8. The vector of claim 7, which is a lentiviral vector or an AAV vector.

9. A virion comprising the vector of any one of claims 6 to 8.

10. 10. A pharmaceutical composition comprising a synthetic CNS-specific promoter according to any one of claims 1 to 4, an expression cassette according to claim 5, a vector according to any one of claims 6 to 8 or a virion according to claim 9.

11. 11. A synthetic CNS-specific promoter according to any one of claims 1 to 4, an expression cassette according to claim 5, a vector according to any one of claims 6 to 8, a virion according to claim 9 or a pharmaceutical composition according to claim 10 for use as a medicament or for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease.

12. 10. A cell comprising the synthetic CNS-specific promoter of any one of claims 1 to 4, the expression cassette of claim 5, the vector of any one of claims 6 to 8 or the virion of claim 9.

13. 10. A composition for use in a method for producing an expression product comprising the synthetic CNS-specific expression cassette of claim 5, the method comprising providing the synthetic CNS-specific expression cassette in a CNS cell and expressing the expression product present in the synthetic CNS-specific expression cassette.

14. 14. The composition of claim 13, wherein the expression product is broadly expressed in the brain when the expression cassette is provided by ICV injection.

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

16. 10. A composition for use in a method for expressing a therapeutic transgene in a CNS cell, comprising the synthetic CNS-specific expression cassette of claim 5, the vector of any one of claims 6 to 8, or the virion of claim 9, the method comprising the step of introducing the synthetic CNS-specific expression cassette, vector, or virion into a CNS cell.

17. 17. The composition of claim 16, wherein the expression cassette, vector or virion is introduced by intravenous injection.

18. 18. The composition of claim 17, wherein the injection is into one of the cephalic, median, or basilic veins.

19. 11. A synthetic CNS-specific expression cassette according to claim 5, a vector according to any one of claims 6 to 8, a virion according to claim 9 or a pharmaceutical composition according to claim 10 for use in a method of therapy of a subject, preferably a human, in need thereof, The method comprises: - administering to a subject the expression cassette, vector, virion or pharmaceutical composition comprising a sequence encoding a therapeutic product operably linked to a promoter according to any one of claims 1 to 4, and - expressing a therapeutic amount of a therapeutic product in the CNS of said subject. Including, An expression cassette, a vector, a virion or a pharmaceutical composition.

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