Insulin gene therapy

Abstract

To provide new therapeutic approaches to mitigate inflammation in the CNS and stimulate neurogenesis.SOLUTION: Described herein is a gene construct comprising a nucleotide sequence encoding insulin, for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith.SELECTED DRAWING: None

Description

[Technical field]

[0001] Aspects of the present disclosure relate to a method for treating neuroinflammation, neurodegeneration and / or cognition disorders in mammals, particularly humans. It relates to the medical field involving insulin gene therapy for use in the treatment of cognitive decline. [Background technology]

[0002] Alzheimer's disease (AD), diabetes and obesity are growing epidemics worldwide. This leads to reduced life expectancy and poor quality of life (IDF Atlas 2015, www w.idf.org;Mayeux,R.et al.2012,Cold Sprin g Harb.Perspect.Med.2012,2:a006239). Data show that central nervous system (CNS) inflammation and insulin resistance are not only related to diabetes and obesity, but also to rather, it is important to understand the underlying mechanisms of AD and other neuropathological processes that underlie cognitive aging and dementia. (De Felice, FG, 201 3,J.Clin.Invest.23:531-539;Kullmann,S.et al.2016,Physiol.Rev 96:1169-1209;Guille mot-Legris,O.et al.,2017,Trends Neurosci .40:237-253;Dutheil S.et al.2016,Neurops ychopharmacology.41:1874-1887).

[0003] Administering recombinant insulin using the intranasal route to reach the central nervous system (CNS) Several studies have shown that memory function improves in both cognitively impaired individuals and normal adults. reported in (Craft, S. et al., 2012, Arch. Neur ol. 69:29 - 38; Reger, M. A. et al., 2006, Neurob iology of aging, 27:451 - 458). Long - term intranasal insulin infusion in a rat model of AD also improves cognition, reduces tau hyperphosphorylation, attenuates microglial activation, and promotes neurogenesis (Guo, Z. et al., 2017, Sci. R ep. 7:1 - 12).

[0004] However, the pharmacokinetics of the transnasal human insulin spray are inadequate, with a peak of insulin in the cerebrospinal fluid (CSF) after intranasal insulin administration, which rapidly declines 60 minutes later (Born, J., et al., 2002, Nat. Neurosci . 5(6):514 - 516). Therefore, this approach requires multiple administrations and has some local side effects of long - term exposure of the nasal mucosa to insulin (Schmid, V . et al., 2018, Diabetes Obes Metab. 20:1563 - 1577).

[0005] Considering the importance of neuroinflammation and neurogenesis in cognitive decline, a new therapeutic approach that reduces CNS inflammation and stimulates neurogenesis without all the drawbacks of existing treatments could be extremely important.

Prior Art Documents

Non - Patent Documents

[0006]

Non - Patent Document 1

[0007] In a first aspect, neuroinflammation, neurodegeneration and / or cognitive decline, or related thereto A gene construct comprising a nucleotide sequence encoding insulin for use in the treatment and / or prevention of associated diseases or conditions is provided.

[0008] In a preferred embodiment, the nucleotide sequence encoding insulin is operably linked to a ubiquitous promoter.

[0009] In another preferred embodiment, the ubiquitous promoter is selected from the group consisting of the CAG promoter and the CMV promoter, preferably the ubiquitous promoter is the CAG promoter.

[0010] In another preferred embodiment, the gene construct contains at least one target sequence for a microRNA expressed in the tissue where insulin expression is desired to be prevented, preferably at least one target sequence for a microRNA is selected from the target sequences that bind to microRNAs expressed in the mammalian heart and / or liver.

[0011] In another preferred embodiment, the gene construct contains at least one target sequence for a microRNA expressed in the liver and at least one target sequence for a microRNA expressed in the heart, preferably the target sequence for the microRNA expressed in the heart is selected from SEQ ID NO: 8 and 16 - 20, and the target sequence for the microRNA expressed in the liver is selected from SEQ ID NO: 7 and 9 - 15, more preferably the gene construct contains the target sequence for microRNA - 122a (SEQ ID NO: 7) and the target sequence for microRNA - 1 (SEQ ID NO: 8).

[0012] In another preferred embodiment, the nucleotide sequence encoding insulin is ​​​​​​​​​​​​(a) A nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2 or 3; ; (b) A nucleotide sequence having at least 60% sequence identity with the nucleotide sequence of SEQ ID NO: 4, 5 or 6; and ; and (c) A nucleotide sequence whose sequence is different from the sequence of the nucleotide sequence of (b) due to the degeneracy of the genetic code ; selected from the group consisting of.

[0013] In a second aspect, there is provided an expression vector comprising a gene construct according to the first aspect for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. ; ;

[0014] In a preferred embodiment, the expression vector is a viral vector, preferably the expression vector is a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, and a lentivirus vector, more preferably an adeno-associated virus vector. ;

[0015] In a preferred embodiment, the expression vector is an adeno-associated virus vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9 , rh10, rh8, Cb4, rh74, DJ, 2 / 5, 2 / 1, 1 / 2 or Anc8 0, preferably an adeno-associated virus vector of serotype 1, 2 or 9 , more preferably an adeno-associated virus vector of serotype 1 or 9.

[0016] In a third aspect, neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith For use in the treatment and / or prevention of associated diseases or conditions according to the first aspect A pharmaceutical composition comprising a gene construct according to the first aspect and / or an expression vector according to the second aspect together with one or more pharmaceutically acceptable ingredients is provided.

[0017] Diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive impairment are cognitive impairment, dementia, Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia (F TD), Parkinson's disease, Parkinsonian disorders, Parkinson's syndrome, Huntington's disease, traumatic brain injury, prion disease, dementia / neurocognitive problems due to HIV infection, age-related cognitive impairment / neurocognitive problems, tauopathy, multiple sclerosis and other neuroinflammatory / neurodegenerative diseases, preferably Alzheimer's disease, Parkinson's disease and / or Parkinsonian disorders more preferably selected from the group consisting of Alzheimer's disease or Parkinson's disease, A gene construct for use according to the first aspect and / or an expression vector for use according to the second aspect and / or a pharmaceutical composition for use according to the third aspect are also provided.

[0018] In some embodiments, the gene construct and / or the expression vector and / or the pharma ceutical composition are administered by intracerebrospinal fluid (CSF) administration.

[0019] In another aspect, a gene construct comprising a nucleotide sequence encoding insulin, wherein the nucleotide sequence encoding insulin is operably linked to a ubiquitous promoter and the gene construct comprises at least one target sequence for a micro RNA that is expressed in the tissue in which expression of insulin is desired, preferably at least one target sequence for a microRNA, and preferably at least one One target sequence binds to a microRNA expressed in the mammalian heart and / or liver A gene construct is provided that is selected from the target sequences that bind.

[0020] In a preferred embodiment, the gene construct has at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart and preferably the microRNA expressed in the heart is selected from SEQ ID NOs: 8 and 16-20 and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 7 and 9-15 and more preferably the gene construct comprises the target sequence of microRNA-122a (SEQ ID NO: 7) and the target sequence of microRNA-1 (SEQ ID NO: 8).

[0021] In another aspect, preferably the expression vector is a viral vector, more preferably the expression vector is a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, and a lentivirus vector and most preferably the expression vector is an adeno-associated virus vector, and an expression vector comprising the gene construct defined in the previous aspect is provided.

[0022] Description The inventors have developed an improved gene therapy strategy based on insulin gene therapy directed to the central nervous system (CNS) to counter neuroinflammation, neurodegeneration and / or cognitive decline. The long-term and effective expression of insulin provided by single administration of the vectors of the present invention into the CSF is an important advantage over other therapies. In particular, as detailed in the experimental section, the inventors have found the following unexpected advantages of brain-directed insulin gene therapy ​ : · The gene constructs and vectors described in this specification can achieve robust and widespread overexpression in the brain including the hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb (Examples 1 , 2, 3, 5).

[0023] · In an aging mouse model with age-related brain lesions such as widely used neuroinflammation, insulin expression in the brain using the gene constructs and vectors according to the present invention resulted in a significant reduction in neuroinflammation, an increase in neurogenesis and an increase in the number of astrocytes (Example 1) as well as short-term memory, improvement in long-term memory and learning ability (Example 5).

[0024] · In a mouse model of obesity and diabetes associated with widely used neuroinflammation and cognitive function decline, insulin expression in the brain using the gene constructs and vectors according to the present invention resulted in a significant reduction in neuroinflammation and an increase in the number of astrocytes (Example 2) .

[0025] Accordingly, the aspects and embodiments of the present invention described in this specification solve at least some of the problems and needs discussed in this specification.

[0026] Gene construct In a first aspect, a gene construct is provided that includes a nucleotide sequence encoding insulin. Preferably, the gene constructs described in this specification are for use as a medicament. More preferably, the gene constructs described in this specification are for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive function decline, or diseases or conditions associated therewith.

[0027] The "gene construct" described in this specification has the meaning that is understood by those skilled in the art in view of the present disclosure and is their customary and ordinary meaning. The "gene construct", also referred to as an "expression cassette" or an "expression construct", refers to a gene or a group of genes that contains a gene encoding a protein of interest and is operably linked to a promoter that controls its expression. The part of this application entitled "General Information" contains further details regarding the "gene construct". The term "operably linked" as used in this specification is further explained in the part of this application entitled "General Information". In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell. In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain may refer to expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain The part of this application entitled "General Information" contains further details regarding the "gene construct". The term "operably linked" as used in this specification is further explained in the part of this application entitled "General Information". The "operably linked" used in this specification is further explained in the part of this application entitled "General Information". is further explained in the part of this application entitled "General Information".

[0028] In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell. In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell. In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell. In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell. In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell. In some embodiments, the gene constructs described in this specification are suitable for expression in mammals. As used herein, "suitable for expression in mammals" means that the gene construct contains one or more regulatory sequences selected based on the mammalian host cell used for expression and that are operably linked to the nucleotide sequence to be expressed. Preferably, the mammalian host cell used for expression is a human, mouse or Chinese hamster ovary cell.

[0029] In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain In some embodiments, the gene constructs described in this specification contain a nucleotide sequence encoding insulin that is expressed in the CNS, preferably the brain, optionally in the CNS and / or brain of a mammal. In some embodiments, the gene constructs described in this specification are suitable for expression in the CNS, preferably the brain. In some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of the gene construct in the brain Expression in the building can be at least one or at least two or at least three or all gene constructs in the brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb. Expression can be meant. Expression can be evaluated using techniques such as qPCR, Western blot analysis or ELISA, which are described in the section entitled "General Information".

[0030] In the context of embodiments of the present invention, insulin expressed in the CNS and / or the brain; and gene constructs suitable for expression in the CNS and / or the brain are preferentially or dominantly (at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher , at least 90% higher, at least 100% higher, at least 150% higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher or more) expression compared to other organs or tissues. Other organs or tissues can be the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, other organs are the liver and / or the heart. Other organs can also be skeletal muscle. In one embodiment, the expression is undetectable in the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach and / or testis. In a preferred embodiment, the expression is undetectable in the liver and / or the heart. In another preferred embodiment, the expression is ​​​​​is undetectable in skeletal muscle. In some embodiments, the expression is in the liver, pancreas, adipose tissue , at least one, at least two, at least three, at least four, or selected from the group consisting of skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, and testis is undetectable in all organs. Expression can be evaluated using techniques such as qPCR , Western blot analysis, or ELISA, as described in the section entitled "General Information".

[0031] In some embodiments, the nucleotide sequence encoding insulin is operably linked to a ubiquitous promoter .

[0032] In some embodiments, the ubiquitous promoter described herein is a CAG promoter , CMV promoter, mini-CMV promoter, β-actin promoter , Rous sarcoma virus (RSV) promoter, elongation factor 1 alpha (EF1α) promoter , early growth response factor 1 (Egr-1) promoter, eukaryotic translation initiation factor 4A ( elF4A) promoter, ferritin heavy chain coding gene (FerH) promoter, ferritin heavy and light chain coding gene (FerL) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, GRP78 promoter, GRP94 promoter, heat shock protein 70 (hsp70) promoter, ubiquitin B promoter , SV40 promoter, beta-catenin promoter, ROSA26 promoter and PGK-1 promoter, selected from the group consisting of . In a preferred embodiment, the ubiquitous promoter is a CAG promoter and CMV

[0033] promoter It may be selected from the group consisting of promoters. In a preferred embodiment, the ubiquitous promoter is the CAG promoter. The CAG promoter has been demonstrated in the examples to be suitable for use in the gene constructs according to the present invention.

[0034] In some embodiments, the CAG promoter comprises, consists essentially of, or consists of a nucleotide sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69% , at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78 %, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 22. In some embodiments, the identity can be evaluated against a portion of SEQ ID NO: 22, such as at least 50%, 60%, 70%, 8 0%, 90%, 95% or 100% of SEQ ID NO: 22.

[0035] Another preferred ubiquitous promoter is the cytomegalovirus (CMV) promoter. In some embodiments, the CMV promoter has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73% , at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % nucleotide sequence having sequence identity, comprising, consisting essentially of, or consisting of. In some embodiments, the identity is evaluated against a portion of SEQ ID NO: 23, such as at least 50%, 60%, 7 0%, 80%, 90%, 95% or 100% of SEQ ID NO: 23.

[0036] Preferably, the CMV promoter is used together with an intron sequence. In some embodiments, the intron sequence has at least 60%, at least 6 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least​​ also 79%, at least 80%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, or consists essentially of, or consists of. In some embodiments the identity can be evaluated against a portion of SEQ ID NO: 21, such as at least 50%, 60%, 70%, 80%, 90% , 95% or 100% of SEQ ID NO: 21. Another preferred ubiquitous promoter is the miniCMV promoter. In some

[0037] embodiments, the miniCMV promoter has at least 60%, at least 61% , at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74 %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 25. comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 97%, at least 98%, at least 99% or 100% sequence identity. In some embodiments, the identity can be evaluated against a portion of SEQ ID NO: 25, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 25. Another preferred ubiquitous promoter is the EF1α promoter. In some embodiments, the EF1α promoter has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 26. comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 26. In some embodiments, the identity can be evaluated against a portion of SEQ ID NO: 26, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 26.

[0038]

[0039] Another preferred ubiquitous promoter is the RSV promoter. In some embodiments the RSV promoter comprises, consists essentially of, or consists of a nucleotide sequence having at least 60%, at least 61% , at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70 %, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 7 9%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 27. In some embodiments the identity can be evaluated against a portion of SEQ ID NO: 27, such as at least 50%, 60%, 70%, 80%, 90%, 9 5% or 100% of SEQ ID NO: 27.

[0040] In some embodiments, the gene construct comprises at least one target sequence of a microRNA that is expressed in the tissue in which insulin expression is to be prevented. In some embodiments, the nucleotide sequence encoding insulin is operably linked to a ubiquitous promoter and the gene construct comprises at least one target sequence of a microRNA that is expressed in the tissue in which insulin expression is to be prevented.

[0041] The descriptions of "ubiquitous promoter", "operably linked" and "microRNA" are provided in the section entitled "General Information". As used herein, "target sequence of a microRNA expressed in a tissue" or "target sequence that binds to a microRNA expressed in a tissue" or "binding site of a microRNA expressed in a tissue" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the said tissue. Expression can be evaluated using techniques such as qPCR, Western blot analysis or ELISA, as described in the section entitled "General Information". In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. As used herein, "target sequence of a microRNA expressed in the liver" or "target sequence that binds to a microRNA expressed in the liver" or "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the liver. Similarly, as used herein, "target sequence of a microRNA expressed in the heart" or "target sequence that binds to a microRNA expressed in the heart" or "binding site of a microRNA expressed in the heart" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the heart. In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. As used herein, "target sequence of a microRNA expressed in the liver" or "target sequence that binds to a microRNA expressed in the liver" or "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the liver. Similarly, as used herein, "target sequence of a microRNA expressed in the heart" or "target sequence that binds to a microRNA expressed in the heart" or "binding site of a microRNA expressed in the heart" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the heart. In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart.

[0042] In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. In some embodiments, at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver. Preferably, in some embodiments, the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart.

[0043] As used herein, "target sequence of a microRNA expressed in the liver" or "target sequence that binds to a microRNA expressed in the liver" or "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the liver. Similarly, as used herein, "target sequence of a microRNA expressed in the heart" or "target sequence that binds to a microRNA expressed in the heart" or "binding site of a microRNA expressed in the heart" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the heart. As used herein, "target sequence of a microRNA expressed in the liver" or "target sequence that binds to a microRNA expressed in the liver" or "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the liver. Similarly, as used herein, "target sequence of a microRNA expressed in the heart" or "target sequence that binds to a microRNA expressed in the heart" or "binding site of a microRNA expressed in the heart" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the heart. As used herein, "target sequence of a microRNA expressed in the liver" or "target sequence that binds to a microRNA expressed in the liver" or "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed in the liver. Similarly, as used herein, "target sequence of a microRNA expressed in the heart" or "target sequence that binds to a microRNA expressed in the heart" The "target sequence" or "binding site of microRNA expressed in the heart" is expressed in the heart Refers to a nucleotide sequence that is complementary or partially complementary to at least a part of the microRNA expressed.

[0044] As described herein, a part of the microRNA expressed in the liver or a part of the microRNA expressed in the heart means a nucleotide sequence of at least 4, at least 5, at least 6, or at least 7 consecutive nucleotides of the microRNA. The binding site sequence can have complete complementarity with at least a part of the expressed microRNA (meaning that the sequences match exactly without mismatches occurring). Alternatively, the binding site sequence can be partially complementary to at least a part of the expressed microRNA (meaning that one mismatch can occur in 4, 5, 6, or 7 consecutive nucleotides). A partially complementary binding site preferably includes complete or nearly complete complementarity with the seed region of the microRNA (meaning that no mismatch occurs (complete complementarity) or one mismatch can occur (nearly complete complementarity) per 4, 5, 6, or 7 consecutive nucleotides between the seed region of the microRNA and its binding site). The seed region of the microRNA consists of the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA (meaning that the sequences match exactly without mismatches occurring). Alternatively, the binding site sequence can be partially complementary to at least a part of the expressed microRNA (meaning that one mismatch can occur in 4, 5, 6, or 7 consecutive nucleotides). A partially complementary binding site preferably includes complete or nearly complete complementarity with the seed region of the microRNA (meaning that no mismatch occurs (complete complementarity) or one mismatch can occur (nearly complete complementarity) per 4, 5, 6, or 7 consecutive nucleotides between the seed region of the microRNA and its binding site). The seed region of the microRNA consists of the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA Preferably, it includes complete or nearly complete complementarity with the seed region of the microRNA (meaning that no mismatch occurs (complete complementarity) or one mismatch can occur (nearly complete complementarity) per 4, 5, 6, or 7 consecutive nucleotides between the seed region of the microRNA and its binding site). The seed region of the microRNA consists of the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA NA's seed region and its binding site, no mismatch occurs (complete complementarity) or one mismatch can occur (nearly complete complementarity) per 4, 5, 6, or 7 consecutive nucleotides). The seed region of the microRNA consists of the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA No mismatch occurs (complete complementarity) or one mismatch can occur (nearly complete complementarity) per 4, 5, 6, or 7 consecutive nucleotides between the seed region of the microRNA and its binding site). The seed region of the microRNA consists of the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA The seed region of the microRNA consists of the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA from about nucleotide 2 to about nucleotide 8 of the microRNA. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA from the 5' region of the microRNA from about nucleotide 2 to about nucleotide 8. The part described herein is preferably in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA Preferably, it is in the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of the microRNA expressed in the liver or the microRNA expressed in the heart is through the RNA interference pathway or mRNA sequence is through the RNA interference pathway or mRNA can be mediated through direct translation control (inhibition). The present invention is in no way limited by the pathway ultimately utilized by miRNAs when inhibiting the expression of transgenes or encoded proteins and is not limited by the pathway ultimately utilized by miRNAs when inhibiting the expression of transgenes or encoded proteins .

[0045] In the context of the present invention, a target sequence that binds to a microRNA expressed in the liver has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67% with nucleotide sequences having a sequence identity of at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76 %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% and may be replaced by a nucleotide sequence comprising a nucleotide sequence having the same sequence identity. In some embodiments, the target sequence that binds to a microRNA expressed in the liver is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive stretches of nucleotides of SEQ ID NO: 7 or 9-15 and at least 60%, at least 6 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67% with a sequence identity of at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% and may be replaced by a nucleotide sequence comprising a nucleotide sequence having the same sequence identity. In some embodiments, the target sequence that binds to a microRNA expressed in the liver is 4, 5, 6, 7, 8, 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least At least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity may be replaced by a nucleotide sequence comprising a nucleotide sequence having.

[0046] In a preferred embodiment, the target sequence of the microRNA expressed in the liver is at least 60% identical to SEQ ID NO: 7 and at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68% , at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77 %, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or replaced by a nucleotide sequence comprising a nucleotide sequence having 100% sequence identity. In some embodiments, the target sequence that binds to the microRNA expressed in the liver is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of SEQ ID NO: 7, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more nucleotides and at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67% , at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76 %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 8 5%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity and may be replaced by a nucleotide sequence comprising a nucleotide sequence. In a further embodiment, at least one copy of the target sequence of the microRNA expressed in the liver described herein is present in the genetic construct of the present invention. In a further embodiment, 2, 3, 4, 5, 6, 7 or 8 copies of the target sequence of the microRNA expressed in the liver described herein are the genes of the present invention constructs. In a further embodiment, the target sequence of the microRNA expressed in the liver described herein is present in the genetic construct of the present invention. In a further embodiment, 2, 3, 4, 5, 6, 7 or 8 copies of the target sequence of the microRNA expressed in the liver described herein are the genes of the present invention constructs. In a further embodiment, at least one copy of the target sequence of the microRNA expressed in the liver described herein is present in the genetic construct of the present invention. In a further embodiment, 2, 3, 4, 5, 6, 7 or 8 copies of the target sequence of the microRNA expressed in the liver described herein are the genes of the present invention constructs. is present in the gene construct. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7 or 8 copies of the sequence miRT-122a (SEQ ID NO: 7 ) are present in the gene construct of the present invention . The preferred copy number of the target sequence of the microRNA expressed in the liver as described herein is 4.

[0047] The target sequence of the microRNA expressed in the liver as used herein is at least a detectable level of the target sequence of the microRNA expressed in the liver, as known to those skilled in the art . Exerts the activity of the bell. The activity of the target sequence of the microRNA expressed in the liver binds to its cognate microRNA expressed in the liver and, when operably linked to the transgene, mediates the off-targeting of transgene expression in the liver . This activity can be evaluated by measuring the level of transgene expression in the liver at the mRNA or protein level by standard assays known to those skilled in the art, such as qPCR, Western blot analysis or ELISA . . . .

[0048] In the context of the present invention, the target sequence of the microRNA expressed in the heart is at least 60%, at least 61%, at least 62%, at least 63 % with SEQ ID NO: 8 or 16 - 20, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 7 2%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85% . A nucleotide sequence comprising a nucleotide sequence having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity may be replaced by a nucleotide sequence. In some embodiments, the target sequence of the microRNA expressed in the heart is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66 %, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 7 5%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% or 100% sequence identity and may be replaced by a nucleotide sequence comprising a nucleotide sequence.

[0049] In a preferred embodiment, the target sequence of the microRNA expressed in the heart is the same as SEQ ID NO: 8 and at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68% , at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77 %, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity nucleotide sequence may be replaced by a nucleotide sequence containing. In some embodiments, the target of the microRNA expressed in the heart sequence is SEQ ID NO: 8 of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more consecutive extension and at least 60%, at least 61%, at least 62%, at least 63% , at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72 %, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 8 least 77%, at least 78%, at least 79%, at least 80%, at least 8 1%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity nucleotide sequence may be replaced by.

[0050] In a further embodiment, at least one copy of the target sequence of the microRNA expressed in the heart described herein is present in the gene construct of the present invention. In a further embodiment two, three four, five, six, seven or eight copies of the target sequence of the microRNA expressed in the heart described herein are present in the gene construct of the present invention. In a preferred embodiment one, two, three, four five, six, seven or eight copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 8) are present in the gene construct of the present invention. The preferred copy number of the target sequence of the microRNA expressed in the heart described herein is 4. five, six, seven or eight copies are present in the gene construct of the present invention. The preferred copy number of the target sequence of the microRNA expressed in the heart described herein is 4. The target sequence of the microRNA expressed in the heart as used herein, as known to those skilled in the art, exhibits at least a detectable level of activity of the target sequence of the microRNA expressed in the heart. The activity of the target sequence of the microRNA expressed in the heart binds to its cognate microRNA expressed in the heart and, when operably linked to a transgene, mediates the off-targeting of transgene expression in the heart. This activity can be measured by qPCR, Western

[0051] The target sequence of the microRNA expressed in the heart as used herein, as known to those skilled in the art, exhibits at least a detectable level of activity of the target sequence of the microRNA expressed in the heart. The activity of the target sequence of the microRNA expressed in the heart binds to its cognate microRNA expressed in the heart and, when operably linked to a transgene, mediates the off-targeting of transgene expression in the heart. This activity can be measured by qPCR, Western level of the target sequence of the microRNA expressed in the heart. The activity of the target sequence of the microRNA expressed in the heart binds to its cognate microRNA expressed in the heart and, when operably linked to a transgene, mediates the off-targeting of transgene expression in the heart. This activity can be measured by qPCR, Western level of the target sequence of the microRNA expressed in the heart. The activity of the target sequence of the microRNA expressed in the heart binds to its cognate microRNA expressed in the heart and, when operably linked to a transgene, mediates the off-targeting of transgene expression in the heart. This activity can be measured by qPCR, Western level of the target sequence of the microRNA expressed in the heart. The activity of the target sequence of the microRNA expressed in the heart binds to its cognate microRNA expressed in the heart and, when operably linked to a transgene, mediates the off-targeting of transgene expression in the heart. This activity can be measured by qPCR, Western level of the target sequence of the microRNA expressed in the heart. The activity of the target sequence of the microRNA expressed in the heart binds to its cognate microRNA expressed in the heart and, when operably linked to a transgene, mediates the off-targeting of transgene expression in the heart. This activity can be measured by qPCR, Western By standard assays known to those skilled in the art, such as Northern blot analysis or ELISA to measure the level of transgene expression in the heart at the mRNA or protein level can be evaluated.

[0052] In some embodiments, at least one copy of the target sequence of the microRNA expressed in the liver as described herein, and the microRNA expressed in the heart as described herein at least one copy of the target sequence are present in the gene construct of the present invention. In some embodiments, 2, 3, 4, 5, 6, 7, or 8 copies of the target sequence of the microRNA expressed in the liver as described herein, and 2, 3, 4, 5, 6, 7, or 8 copies of the target sequence of the microRNA expressed in the heart as described herein are present in the gene construct of the present invention. In further embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT-122a (SEQ ID NO: 7) and 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 8) are combined in the gene construct of the present invention. In further embodiments 4 copies of the nucleotide sequence encoding miRT-122a (SEQ ID NO: 7) and 4 copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 8) are combined in the gene construct of the present invention. 6, 7, or 8 copies are combined in the gene construct of the present invention. In further embodiments are combined in the gene construct of the present invention.

[0053] In some embodiments, provided are the above gene constructs, wherein the target sequence of the microRNA expressed in the liver and the target sequence of the microRNA expressed in the heart are selected from the group consisting of the sequences of SEQ ID NOs: 7-20 and / or combinations thereof. In some embodiments ​​​​​​​In a form, the target sequence of the microRNA expressed in the heart is selected from SEQ ID NO: 8 and SEQ ID NOs: 16 to 20, and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NO: 7 and SEQ ID NOs: 9 to 15, and the above gene construct is provided. In some embodiments, the gene construct including the target sequence of microRNA-122a (SEQ ID NO: 7) and the target sequence of microRNA-1 (SEQ ID NO: 8) is provided. In some embodiments, the target sequence of the microRNA expressed in the liver is selected from SEQ ID NO: 7 and SEQ ID NOs: 9 to 15, and the above gene construct is provided. The above gene construct is provided. In some embodiments, the target sequence of microRNA-122a (SEQ ID NO: 7) and the target sequence of microRNA-1 (SEQ ID NO: 8) are included, and the above gene construct is provided.

[0054] The target sequence of the microRNA expressed in the liver and / or the target sequence of the microRNA expressed in the heart described in this specification exhibits at least a detectable level of activity. The activity of the target sequence of the microRNA can be the degradation of the mRNA including the target sequence of the microRNA. This degradation can be evaluated, for example, by measuring the expression / presence of the mRNA using any technique known to those skilled in the art. The expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA described in the section titled "General Information". The target sequence of the microRNA expressed in the liver and / or the target sequence of the microRNA expressed in the heart described in this specification exhibits at least a detectable level of activity. The activity of the target sequence of the microRNA can be the degradation of the mRNA including the target sequence of the microRNA. This degradation can be evaluated, for example, by measuring the expression / presence of the mRNA using any technique known to those skilled in the art. The expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA described in the section titled "General Information". The expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA described in the section titled "General Information". ELISA and the like.

[0055] The nucleotide sequence encoding insulin present in the gene construct according to the present invention can be derived from any insulin gene or insulin-encoding sequence including a mutant insulin gene or insulin-encoding sequence, or a codon-optimized insulin gene or insulin-encoding sequence. In some embodiments, the nucleotide sequence encoding insulin is a mouse, canine, or human insulin gene or insulin-encoding sequence, a mouse, canine, or human mutant insulin gene or insulin-encoding sequence, or a mutant insulin gene or insulin-encoding sequence, or a codon-optimized insulin gene or insulin-encoding sequence. The nucleotide sequence encoding insulin present in the gene construct according to the present invention can be derived from any insulin gene or insulin-encoding sequence including a mutant insulin gene or insulin-encoding sequence, or a codon-optimized insulin gene or insulin-encoding sequence. In some embodiments, the nucleotide sequence encoding insulin is a mouse, canine, or human insulin gene or insulin-encoding sequence, a mouse, canine, or human mutant insulin gene or insulin-encoding sequence, or a mouse, canine, or human codon-optimized insulin gene or insulin-encoding sequence. a mouse, canine, or human mutant insulin gene or insulin-encoding sequence, or is a mouse, dog or human codon-optimized insulin gene or insulin-encoding sequence. In some embodiments, the nucleotide sequence encoding insulin is from a human, chimpanzee, mouse, rat or dog insulin gene or insulin-encoding sequence; or a mutant insulin gene or insulin-encoding sequence from a human, chimpanzee, mouse, rat or dog; or a codon-optimized insulin gene or insulin- encoding sequence from a human, chimpanzee, mouse, rat or dog. A human sequence is preferred.

[0056] In a preferred embodiment, the nucleotide sequence encoding insulin present in the gene construct according to the invention encodes engineered insulin having a furin cleavage site. Such engineered insulin having a furin cleavage site is known to be processed in a highly efficient manner to produce mature insulin in non-pancreatic tissues. In some embodiments, the nucleotide sequence encoding engineered insulin having a furin cleavage site is (a) at least 60%, at least 61% , at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70 %, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 7 9%, at least 80%, at least 81%, at least 82%, at least 83%, at least At least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity or similarity A nucleotide sequence encoding a polypeptide comprising an amino acid sequence having; (b) At least 60%, at least 6 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; and (c) Due to the degeneracy of the genetic code, a nucleotide sequence whose sequence is different from the sequence of the nucleotide sequence of (a) or (b) Selected from the group consisting of .

[0057] Thus, in some embodiments, a preferred nucleotide encoding insulin ​The array encodes a polypeptide comprising an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity or similarity to the amino acid sequences of SEQ ID NOs: 1-3 or 41-44. SEQ ID NO: 1 represents the amino acid sequence of human insulin. SEQ ID NO: 2 represents the amino acid sequence of mouse insulin. SEQ ID NO: 3 represents the amino acid sequence of canine insulin. SEQ ID NO: 41 represents the amino acid sequence of human insulin having a furin cleavage site. SEQ ID NO: 42 represents the amino acid sequence of the human insulin mutant His-B10-Asp having a furin cleavage site. SEQ ID NO: 43 represents the amino acid sequence of mouse insulin. SEQ ID NO: 44 represents the amino acid sequence of chimpanzee insulin. In some embodiments, the identity can be evaluated against a portion of SEQ ID NOs: 1-3 or 41-44, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100%. at least 62%, at least 63%, at least 64%, at least 65%, at least 6 6%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88% at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99% or 100% identity or similarity to the amino acid sequences of SEQ ID NOs: 1-3 or 41-44. SEQ ID NO: 1 represents the amino acid sequence of human insulin. SEQ ID NO: 2 represents the amino acid sequence of mouse insulin. SEQ ID NO: 3 represents the amino acid sequence of canine insulin. SEQ ID NO: 41 represents the amino acid sequence of human insulin having a furin cleavage site. SEQ ID NO: 42 represents the amino acid sequence of the human insulin mutant His-B10-Asp having a furin cleavage site. SEQ ID NO: 43 represents the amino acid sequence of mouse insulin. SEQ ID NO: 44 represents the amino acid sequence of chimpanzee insulin. In some embodiments, the identity can be evaluated against a portion of SEQ ID NOs: 1-3 or 41-44, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100%. acid sequence of chimpanzee insulin. In some embodiments, the identity can be evaluated against a portion of SEQ ID NOs: 1-3 or 41-44, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100%. at least 50%, 60%, 70%, 80%, 90%, 95% or 100% such as the portion of SEQ ID NOs: 1-3 or 41-44 can be evaluated.

[0058] In some embodiments, the nucleotide sequence encoding insulin present in the gene construct according to the present invention is selected from the group consisting of SEQ ID NOs: 4 to 6 or 45 to 48 and has at least 60%, at least 61%, at least 62%, at least 63 %, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 7 2%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity. SEQ ID NO: 4 represents the nucleotide sequence of human insulin. SEQ ID NO: 5 represents the nucleotide sequence of mouse insulin. SEQ ID NO: 6 represents the nucleotide sequence of canine insulin. SEQ ID NO: 45 represents the nucleotide sequence of human insulin having a furin cleavage site. SEQ ID NO: 46 represents the nucleotide sequence of the human insulin mutant His-B10-Asp having a furin cleavage site. SEQ ID NO: 47 represents the nucleotide sequence of mouse insulin. SEQ ID NO: 48 represents the nucleotide sequence of chimpanzee insulin. In some embodiments, the identity is SEQ ID NO s: In some embodiments, the nucleotide sequence encoding insulin present in the gene construct according to the present invention is selected from the group consisting of SEQ ID NOs: 4 to 6 or 45 to 48 and has at least 60%, at least 61%, at least 62%, at least 63 %, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 7 2%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least At least 50%, 60%, 70%, 80%, 90%, 95 %, or 100% etc., can be evaluated for a part of SEQ ID NO: 4-6 or 45-48 .

[0059] The descriptions of "identity" or "sequence identity" and "similarity" or "sequence similarity" are provided in the section entitled "General Information".

[0060] In some embodiments, the nucleotide sequence encoding human insulin present in the gene construct according to the present invention has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79% , at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 4. In some embodiments, the identity can be evaluated for a part of SEQ ID NO: 4, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 4 .

[0061] ​In some embodiments, the nucleotide sequence encoding mouse insulin present in the gene construct according to the present invention has at least 60%, at least also 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74 %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with . In some embodiments, the identity is at least 50%, 6 0%, 70%, 80%, 90%, 95% or 100% etc. of SEQ ID NO: 5 or 47, and can be evaluated with respect to a part of SEQ ID NO: 5 or 47.

[0062] In some embodiments, the nucleotide sequence encoding canine insulin present in the gene construct according to the present invention has at least 60%, at least 61%, at least also 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least also 75%, at least 76%, at least 77%, at least 78%, at least 79% , at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, at least 99% or 100% identity. In some embodiments, the identity can be evaluated against a portion of SEQ ID NO: 6, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 6.

[0063] In some embodiments, the nucleotide sequence encoding human insulin present in the gene construct according to the invention is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65% with SEQ ID NO: 45 or 46, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74 %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, having at least 97%, at least 98%, at least 99% or 100% identity In some embodiments, the identity is at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 45 or 4 6, and can be evaluated against a portion of SEQ ID NO: 45 or 4

[0064] In some embodiments, the nucleotide sequence encoding chimpanzee insulin present in the gene construct according to the invention is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74% , at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83 %, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 48. In some embodiments, the identity is at least 50%, 60%, 70 %, 80%, 90%, 95% or 100% of SEQ ID NO: 48, and can be evaluated against a portion of SEQ ID NO: 48 . In some embodiments, the nucleotide sequence encoding insulin is evaluated against a portion of SEQ ID NO: 48

[0065] In some embodiments, the nucleotide sequence encoding insulin (a) A nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 1-3 or 41-44 and having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74 %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity or similarity; (b) A nucleotide sequence of SEQ ID NO: 4-6 or 45-48 and having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99% or 100% sequence identity of the nucleotide sequence; and (c) a nucleotide sequence that differs from the sequence of the nucleotide sequence of (a) or (b) due to the degeneracy of the genetic code is provided, a gene construct described herein selected from the group consisting of . Insulin encoded by the nucleotide sequences described herein (in particular, where the insulin sequence is described as having a minimum percentage of identity with a given SEQ ID NO) exhibits at least a detectable level of insulin activity. The activity of insulin

[0066] can be the regulation of hyperglycemia. More appropriately, in the context of the present disclosure, the activity of insulin can be evaluated at the level of the insulin signaling cascade. For example, the phosphorylation status of various proteins in the insulin signaling cascade, such as the tyrosine phosphorylation of IRS- 1 / 2, the phosphorylation of AKT, etc., can be determined. The phosphorylation status can be evaluated by Western blot analysis using, for example, antibodies that recognize phosphorylated tyrosine residues and / or antibodies that specifically recognize the phosphorylated forms of proteins such as IRS-1 / 2 and AKT. The activity of insulin can also be to reduce neuroinflammation, increase neurogenesis, or increase astrocytes . This activity can be determined by methods known to those skilled in the art, for example, by measuring the expression levels of inflammatory molecules, astrocyte markers, and / or neurogenic markers described in the experimental section . ​​​​​​​It could be evaluated by.

[0067] The following table summarizes the sequence identities at the DNA and protein levels for a representative number of insulin sequences suitable for use in the gene constructs of the present invention. [Table 1]

[0068] In some embodiments, the nucleotide sequence encoding insulin is operably linked to a tissue-specific promoter. In a preferred embodiment, the tissue-specific promoter is a CNS-specific promoter, more preferably a brain-specific promoter. As used herein, the CNS brain-specific promoter and / or brain-specific promoter also includes a promoter that directs expression in specific regions or cell subsets of the CNS and / or brain. Thus, the CNS-specific promoter and / or brain-specific promoter can also be selected from a hippocampus-specific promoter, a cerebellum-specific promoter, a cortex-specific promoter, a hypothalamus-specific promoter and / or an olfactory bulb-specific promoter, or any combination thereof.

[0069] The description of "tissue-specific promoter" is provided in the section entitled "General Information".

[0070] In some embodiments, the CNS-specific promoter described herein is the synapsin 1 promoter, the neuron-specific enolase (NSE) promoter, the calcium Tyrosine hydroxylase (TH) promoter, Forkhead box A2 (FOXA2) promoter, alpha-internexin (INA) promoter, nestin (NES ) promoter, glial fibrillary acidic protein (GFAP) promoter, aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter, myelin associated oligodendrocyte basic protein (MOBP) promoter, homeobox protein 9 (HB9) promoter, gonadotropin-releasing hormone (GnRH) pro moter, and myelin basic protein (MBP) promoter selected from the group consisting of

[0071] In some embodiments, the brain-specific promoters described herein are synapsin 1 promoter, neuron-specific enolase (NSE) promoter, calcium / cal modulin-dependent protein kinase II (CaMKII) promoter, tyrosine hy droxylase (TH) promoter, Forkhead box A2 (FOXA2) promoter alpha-internexin (INA) promoter, nestin (NES) promoter glial fibrillary acidic protein (GFAP) promoter, aldehyde de hydrogenase 1 family member L1 (ALDH1L1) promoter, myelin-related oligodendrocyte basic protein (MOBP) promoter, gonadotropin-releasing hormone (GnRH) promoter, and myelin basic protein (MBP) promoter selected from the group consisting of

[0072] In preferred embodiments, a CNS brain-specific promoter and / or a brain-specific promoter The promoter is synapsin 1 promoter. In some embodiments, the synapsin 1 pro moter has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71% , at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80 %, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 8 9%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence comprising or consisting essentially of or consisting of. In some embodiments, the identity is at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 28 and can be evaluated against a portion of SEQ ID NO: 28.

[0073] Another preferred CNS-specific promoter and / or brain-specific promoter is the cal cium / calmodulin-dependent protein kinase II (CaMKII) promoter. In some embodiments, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter has at least 60%, at least 6 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83% 、at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, comprising, consisting essentially of, or consisting of. In some embodiments the identity can be evaluated against a portion of SEQ ID NO: 29, such as at least 50%, 60%, 70%, 80%, 90% , 95% or 100% of SEQ ID NO: 29.

[0074] Another preferred CNS-specific promoter and / or brain-specific promoter is the glial fibrillary acidic protein (GFAP) promoter. In some embodiments , the glial fibrillary acidic protein (GFAP) promoter has at least 60% with SEQ ID NO: 30, at least 61%, at least 62%, at least 63%, at least 64 %, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 7 3%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99% or 1 00% nucleotide sequence having sequence identity, or consisting essentially of, or consisting of. In some embodiments, the identity is at least 50%, 60% of SEQ ID NO: 30, 70%, 80%, 90%, 95% or 100%, etc., for a part of SEQ ID NO: 30 and can be evaluated.

[0075] Another preferred CNS-specific promoter and / or brain-specific promoter is the nest in promoter. In some embodiments, the nestin promoter has at least 60% with SEQ ID NO: 31, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81% , at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9 9% or a nucleotide sequence having 100% sequence identity, or consisting essentially of or consisting of. In some embodiments, the identity is at least 5 0%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 31, such as evaluated against a portion of SEQ ID NO: 31.

[0076] Another preferred CNS-specific promoter is the homeobox protein 9 (HB9) promoter. In some embodiments, the homeobox protein 9 (HB9) promoter has at least 60%, at least 61%, at least 62% , at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 8 0%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with a nucleotide sequence, or consisting essentially of or consisting of. In some embodiments, the identity is the sequence At least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 32 and the like can be evaluated for a part of SEQ ID NO: 32.

[0077] Another preferred CNS-specific promoter and / or brain-specific promoter is the tyro sine hydroxylase (TH) promoter. In some embodiments, the tyrosine hydroxylase (TH) promoter has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74 %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with a nucleotide sequence that comprises, consists essentially of, or consists of. In some embodiments, the identity can be evaluated for a part of SEQ ID NO: 33, such as at least 50%, 60%, 70%, 80%, 9 0%, 95% or 100% of SEQ ID NO: 33.

[0078] Another preferred CNS-specific promoter and / or brain-specific promoter is the myelin It is a myelin basic protein (MBP) promoter. In some embodiments, myelin basic protein (MBP) promoter has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99% or 100% sequence identity with, consisting essentially of, or consisting of a nucleotide sequence. In some embodiments, the identity can be evaluated against a portion of SEQ ID NO: 34, such as at least 50%, 60%, 70%, 80% , 90%, 95% or 100% of SEQ ID NO: 34.

[0079] In some embodiments, the CNS-specific promoter and / or brain-specific promoter described herein directs expression of the nucleotide sequence in at least one cell of the CNS and / or brain. Preferably, the promoter directs expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70% of the cells of the CNS and / or brain. , direct expression at 80%, 90%, or 100%. As used herein, CNS brain-specific promoters and / or brain-specific promoters also include promoters that direct expression in specific regions or cell subsets of the CNS and / or brain. Thus, the CNS-specific promoters and / or brain-specific promoters described herein can also direct expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90%, or 100% of the cells of the hippocampus, cerebellum, cortex, hypothalamus, and / or olfactory bulb. Expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA, as described in the section entitled "General Information". The promoters used herein (particularly when the promoter sequence is described as having a minimum percentage identity with a given SEQ ID NO.) should exhibit at least the activity of a promoter known to those of ordinary skill in the art. Preferably, a promoter described as having a minimum percentage identity with a given SEQ ID NO. should control the transcription of a nucleotide sequence to which it is operably linked (i.e., at least a nucleotide sequence encoding insulin) when evaluated in an assay known to those of ordinary skill in the art. For example, such an assay can involve measuring the expression of a transgene. Expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA, as described in the section entitled "General Information".

[0080] Additional sequences may be present in the gene constructs of the present invention. Suitable exemplary additional sequences are known to those of ordinary skill in the art. sequences are known to those of ordinary skill in the art. When evaluated in an assay known to those of ordinary skill in the art, a promoter described as having a minimum percentage identity with a given SEQ ID NO. should control the transcription of a nucleotide sequence to which it is operably linked (i.e., at least a nucleotide sequence encoding insulin). For example, such an assay can involve measuring the expression of a transgene. Expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA, as described in the section entitled "General Information". Such an assay can involve measuring the expression of a transgene. Expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA, as described in the section entitled "General Information". Expression can be evaluated using techniques such as qPCR, Western blot analysis, or ELISA, as described in the section entitled "General Information".

[0081] Additional sequences may be present in the gene constructs of the present invention. Suitable exemplary additional The plus array includes terminal inverted repeats (ITRs), the SV40 polyadenylation signal (SEQ ID NO: 3 7), the rabbit β-globin polyadenylation signal (SEQ ID NO: 38), and the CMV enhancer -sequence (SEQ ID NO: 24). In the context of the present invention, "ITR" is intended to include one 5' ITR and one 3' ITR each derived from the AAV genome. Preferred ITRs are those of AAV2 and are represented by SEQ ID NO: 35 (5' ITR) and SEQ ID NO: 36 (3' ITR). Within the context of the present invention, the CMV enhancer sequence (SEQ ID NO: 24) and the CMV promoter sequence (SEQ ID NO: 23) are included as two separate sequences or a single sequence (SEQ ID NO: 39). Each of these additional sequences may be present in the gene construct according to the present invention. In some embodiments, the nucleotide sequence encoding insulin as described in the present specification is included, and one 5' ITR and one 3' ITR, preferably an AAV2 ITR, more preferably an AAV2 ITR represented by SEQ ID NO: 30 (5 ' ITR) and SEQ ID NO: 31 (3' ITR) are further included. Gene constructs are provided. In some embodiments, a polyadenylation signal, preferably the SV40 polyadenylation signal (preferably represented by SEQ ID NO: 32 ) and / or the rabbit β-globin polyadenylation signal (preferably represented by SEQ ID NO: 33 ) are further included, and gene constructs containing the nucleotide sequence encoding insulin as described herein are provided. Additional nucleotide sequences such as those encoding signal sequences, nuclear localization signals, expression enhancers, etc. are present in the (one or more) nucleotides encoding insulin

[0082] sequences. ​​​​​ It may be operably linked to the chID sequence.

[0083] In some embodiments, a nucleotide sequence encoding insulin may be included in a genetic construct that does not include a target sequence of a microRNA expressed in a tissue in which insulin expression is desired to be prevented. A genetic construct is provided.

[0084] In some embodiments, the level of sequence identity or similarity used herein is preferably 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0085] Expression vector The genetic constructs described herein can be placed in an expression vector. Thus, in another aspect, an expression vector comprising the genetic constructs described herein is provided. Preferably, the expression vectors described herein are for use as a medicament. Preferably, the expression vectors described herein are for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or diseases or conditions associated therewith.

[0086] An explanation of "expression vector" is provided in the section entitled "General Information".

[0087] In some embodiments, the expression vector is a viral expression vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral vector. It can be a viral vector selected from the group consisting of a lentivirus, a retroviral vector, and a lentiviral vector. A preferred viral vector is an adeno-associated viral vector. The description of "viral expression vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information".

[0088] The description of "viral expression vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information". Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. The description of "adeno-associated viral vector" is provided in the section titled "General Information".

[0089] In some embodiments, the vector is an adeno-associated vector or an adeno-associated virus selected from the group consisting of AAV of serotype 1 (AAV1), AAV of serotype 2 (AAV2), AAV of serotype 3 (AAV3), AAV of serotype 4 (AAV4), AAV of serotype 5 (AAV5), AAV of serotype 6 (AAV6), AAV of serotype 7 (AAV7), AAV of serotype 8 (AAV8), AAV of serotype 9 (AAV9), AAV of serotype rh10 (AAVrh10), AAV of serotype rh8 (AAVrh8), AAV of serotype Cb4 (AAVCb4), AAV of serotype rh74 (AAVrh74), AAV of serotype DJ (AAVDJ), AAV of serotype 2 / 5 (AAV2 / 5), AAV of serotype 2 / 1 (AAV2 / 1), AAV of serotype 1 / 2 (AAV1 / 2), AAV of serotype Anc80 (AAVAnc80). It is a viral vector or an adeno-associated virus-derived vector (AAV). In a preferred embodiment form, the vector is AAV of serotype 1, 2 or 9 (AAV1, AAV2 or AA V9). These AAV serotypes have been demonstrated in the examples to be suitable for use as expression vectors according to the present invention. In a particularly preferred embodiment, the expression vector is an adeno-associated virus vector of serotype 9 or 1.

[0090] In a preferred embodiment, the expression vector is AAV1 or AAV9, preferably AAV 9, and contains a gene construct containing a nucleotide sequence encoding insulin and at least one target sequence of a microRNA expressed in a tissue where insulin expression is to be prevented.

[0091] In another preferred embodiment, the expression vector is AAV1 or AAV9, preferably A AV1, and contains a gene construct containing a nucleotide sequence encoding insulin, which may not contain the target sequence of a microRNA expressed in a tissue where insulin expression is to be prevented.

[0092] In a preferred embodiment, the expression vector is AAV9-CAG-hIns-dmiRT containing a gene construct encoding human insulin operably linked to the CAG promoter and the miRNA target sequences miRT-1 and miRT-122a. The gene construct may further contain a rabbit β-globin polyadenylation signal. In another preferred embodiment, the expression vector is AAV1-CAG-hIns containing a gene construct encoding human insulin operably linked to the CAG promoter. The gene construct The substance may further contain a rabbit β-globin polyadenylation signal.

[0093] Composition In a further aspect, the gene constructs described herein and / or the viral vectors described herein are optionally provided together with one or more pharmaceutically acceptable components. A composition is provided. Preferably, the compositions described herein are for use as a medicament. Preferably, the compositions described herein are for the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or diseases or conditions associated therewith. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions.

[0094] As used herein, "pharmaceutically acceptable components" may include pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients. Thus, one or more pharmaceutically acceptable components may be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, 22nd edition. Pharmaceutical Press (2013). / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions. / or prevention. Preferably, in some embodiments, the composition is a pharmaceutical composition. Compositions such as those described herein may also be referred to as gene therapy compositions.

[0095] Further compounds may be present in the compositions of the invention. Said compounds are for the delivery of the composition. can be useful. Suitable compounds in this context are complexes, nanoparticles, micelles and / or liposomes that complex or capture each of the components described herein and are able to cross the cell membrane and deliver them. Many of these compounds are known in the art. Suitable compounds include polyethyleneimine (PEI), or polypropyleneimine or polyethyleneimine copolymer (PEC) and similar cationic polymers including conductors; synthetic amphiphiles (SAINT-18); lipofectin (trademark); DOTAP. One skilled in the art will know which type of formulation is most suitable for the compositions described herein. And the like that complex or capture each of the components described herein and are able to form complexes, nanoparticles, micelles and / or liposomes that cross the cell membrane and deliver them. A compound that can form complexes, nanoparticles, micelles and / or liposomes that complex or capture each of the components described herein and are able to cross the cell membrane and deliver them. Many of these compounds are known in the art. Suitable compounds include polyethyleneimine (PEI), or polypropyleneimine or polyethyleneimine copolymer (PEC) and similar cationic polymers including conductors; synthetic amphiphiles (SAINT-18); lipofectin (trademark); DOTAP. Or polypropyleneimine or polyethyleneimine copolymer (PEC) and similar cationic polymers including conductors; synthetic amphiphiles (SAINT-18); lipofectin (trademark); DOTAP. Including conductors; synthetic amphiphiles (SAINT-18); lipofectin (trademark); DOTAP. (trademark); DOTAP are included. One skilled in the art will know which type of formulation is most suitable for the compositions described herein. One skilled in the art will know which type of formulation is most suitable for the compositions described herein.

[0096] Methods and Uses In a further aspect, there is provided a gene construct described herein for use as a medicament. There is further provided an expression vector described herein for use as a medicament. There is further provided a pharmaceutical composition described herein for use as a medicament. There is also provided a gene construct described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is further provided an expression vector described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is further provided a pharmaceutical composition described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is further provided a pharmaceutical composition described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. Neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is also provided a gene construct described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. Neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is further provided an expression vector described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is further provided an expression vector described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. Neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. There is further provided a pharmaceutical composition described herein for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith.

[0097] Thus, in some embodiments, the gene constructs and / or the expression vectors described herein and / or the pharmaceutical compositions described herein are for use in the treatment and / or prevention of neuroinflammation. In some embodiments the gene constructs and / or the expression vectors described herein and / or the pharmaceutical compositions described herein are for use in the treatment and / or prevention of neurodegeneration. In some embodiments the gene constructs and / or the expression vectors described herein and / or the pharmaceutical compositions described herein are for use in the treatment and / or prevention of cognitive decline. In the context of the present invention, "neuroinflammation", "neurodegeneration" and "cognitive decline" are each replaceable by "neuroinflammation or a disease or condition associated therewith", "neurodegeneration or a disease or condition associated therewith" and "cognitive decline or a disease or condition associated therewith" respectively. In some embodiments, the diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive decline are cognitive impairment, dementia, Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia (FTD), Parkinson's disease, Parkinsonian disorders, Parkinsonism, Huntington's disease, traumatic brain injury, prion disease, HIV infection-related dementia / neurocognitive problems, age-related dementia / neurocognitive problems, tauopathy, multiple sclerosis and other neuroinflammatory / neurodegenerative diseases. In preferred embodiments, the diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive decline are Alzheimer's disease, Parkinson's

[0098] In some embodiments, the diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive decline are cognitive impairment, dementia, Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia (FTD), Parkinson's disease, Parkinsonian disorders, Parkinsonism, Huntington's disease, traumatic brain injury, prion disease, HIV infection-related dementia / neurocognitive problems, age-related dementia / neurocognitive problems, tauopathy, multiple sclerosis and other neuroinflammatory / neurodegenerative diseases. In preferred embodiments, the diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive decline are Alzheimer's disease, Parkinson's disease, Parkinson's syndrome, Huntington's disease, traumatic brain injury, prion disease, HIV infection-related dementia / neurocognitive problems, age-related dementia / neurocognitive problems, tauopathy, multiple sclerosis and other neuroinflammatory / neurodegenerative diseases. In preferred embodiments, the diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive decline are Alzheimer's disease, Parkinson's disease, Parkinson's syndrome, Huntington's disease, traumatic brain injury, prion disease, HIV infection-related dementia / neurocognitive problems, age-related dementia / neurocognitive problems, tauopathy, multiple sclerosis and other neuroinflammatory / neurodegenerative diseases. In preferred embodiments, the diseases or conditions associated with neuroinflammation, neurodegeneration and / or cognitive decline are Alzheimer's disease, Parkinson's Parkinson's disease and / or Parkinson-like diseases, preferably Alzheimer's disease and / or Parkinson's disease.

[0099] Accordingly, the gene constructs and / or the expression vectors and / or the pharmaceutical compositions described herein can be regarded as anti-neuroinflammatory agents, neurodegenerative agents and / or anti-cognitive decline agents. Accordingly, this can also be regarded as an anti-aging agent.

[0100] The embodiments disclosed herein can also be used to treat and / or prevent neuroinflammation, neurodegeneration, and / or cognitive decline associated with any

[0101] In some embodiments, the gene constructs and / or the expression vectors and / or the pharmaceutical compositions for use described herein are accompanied by the expression of the gene construct in the CNS, preferably the brain.

[0102] Preferably, according to some embodiments, the gene constructs and / or the expression vectors and / or the pharmaceutical compositions for use described herein are administered by intracerebrospinal fluid (CSF) administration.

[0103] In a further aspect, there is provided a treatment method comprising administering the gene constructs, expression vectors or pharmaceutical compositions described herein. Preferably, the treatment method is for treating and / or preventing neuroinflammation, neurodegeneration and / or cognitive decline, or diseases or Administering a vector or pharmaceutical composition means administering a therapeutically effective amount of a gene construct, expression vector or pharmaceutical composition to a subject in need thereof.

[0104] In a further aspect, there is provided the use of a gene construct, expression vector or pharmaceutical composition described herein for the manufacture of a medicament. Preferably, in some embodiments, said medicament is for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith.

[0105] In a further aspect, there is provided the use of a gene construct, expression vector described herein for a medical treatment. Preferably, in some embodiments, the said medical treatment is the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith.

[0106] In another aspect, there is provided a method of improving memory and / or learning in a subject, the method comprising the step of administering to the subject a gene construct described herein and / or an expression vector described herein and / or a composition described herein. In preferred embodiments, an effective amount of a gene construct, expression vector or composition is administered. As used herein, "effective amount" is an amount sufficient to exert a beneficial or desired result. In preferred embodiments, the subject being treated is an elderly subject and / or a subject diagnosed with a metabolic disorder or disease, preferably obesity and / or diabetes. In some embodiments, memory is recognition memory and / or recall memory, preferably recognition memory It may be memory. In some embodiments, the memory can be sensory memory; short-term and / or long-term memory , preferably short-term and / or long-term memory. In some embodiments, the memory can be implicit (or procedural) memory and / or explicit (or declarative) memory. In preferred embodiments, the memory can also be by spatial memory. In some embodiments , the learning can be spatial learning. Further descriptions of the various types of memory are included in the section entitled "General Information" .

[0107] In preferred embodiments of the gene construct for use according to the present invention, the expression vector for use, the composition for use, the method and the use, the subject to be treated is an elderly subject and / or a subject diagnosed with a metabolic disorder or disease. In other words, the use according to the present invention of the gene construct for use, the expression vector for use, the composition for use, the method and the use, in some embodiments , neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith, is associated with aging and / or a metabolic disorder or disease and / or caused by these. Complications of metabolic disorders or diseases may also be included.

[0108] As used herein, an elderly subject may preferably mean a subject 50 years or older, preferably 55 years or older, more preferably 60 years or older, and most preferably 65 years or older.

[0109] In other embodiments of the gene construct for use according to the present invention, the expression vector for use, the composition for use, the method and the use, the subject to be treated is not an elderly subject, ​​and / or 50 years old or younger, 45 years old or younger, 40 years old or younger, 35 years old or younger, 30 years old or younger, 25 years old are the following subjects.

[0110] In other embodiments of the gene construct for use according to the present invention, the expression vector for use, the composition for use, the method, and the use, the subject to be treated is a subject not diagnosed with a metabolic disorder or a disease. In other words, in some embodiments of the gene construct for use according to the present invention, the expression vector for use, the composition for use, the method, and the use, a central nervous system disorder or disease, or a condition related thereto, is not related to and / or not caused by aging and / or a metabolic disorder or disease. That is, the gene construct for use according to the present invention, the expression vector for use, the composition for use, the method, and several embodiments of the use, in a central nervous system disorder or disease, or a condition related thereto, is not related to and / or not caused by aging and / or a metabolic disorder or disease.

[0111] Metabolic disorders and diseases include metabolic syndrome, diabetes, obesity, obesity-related comorbidities, diabetes-related comorbidities, hyperglycemia, insulin resistance, glucose intolerance, fatty liver, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), coronary heart disease (CHD), hyperlipidemia, atherosclerosis, endocrine diseases, osteoporotic sarcopenic obesity syndrome (OSO), diabetic nephropathy, chronic kidney disease (CKD), cardiac hypertrophy, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, ocular angiogenesis, neurodegeneration, dementia, and may include depression, adenoma, and carcinoma. Diabetes includes prediabetes, hyperglycemia, type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the young (MODY), monogenic diabetes, neonatal diabetes, gestational diabetes, labile diabetes, idiopathic diabetes, drug- or chemical-induced diabetes, stiff-man syndrome, lipoatrophic diabetes, latent autoimmune diabetes in adults. ​​​​​​​​​​​​Latent autoimmune diabetes in adults (LADA) may be included. Obesity includes overweight, central / upper body obesity, peripheral / lower body obesity, morbid obesity, osteoporotic sarcopenic obesity syndrome (OSO), childhood obesity, Mendelian (monogenic) syndromic obesity, Mendelian non-syndromic obesity, polygenic obesity may be included. Preferred metabolic disorders or diseases are obesity and / or diabetes.

[0112] In some embodiments of the gene construct for use according to the present invention, the expression vector for use, the composition for use, the method and the use, the subject to be treated has a risk of developing neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith. In the context of the gene construct for use according to the present invention, the expression vector for use, the pharmaceutical composition for use, the method and the use, treatment and / or treatment and / or medicine may be accompanied by the expression of the gene construct in the CNS, preferably the brain. In some embodiments, there is no detectable expression in tissues other than the CNS and / or the brain. In some embodiments, the expression of the gene construct in the brain may mean the expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or

[0113] In the context of the gene construct for use according to the present invention, the expression vector for use, the pharmaceutical composition for use, the method and the use, the expression of the gene construct in the brain may mean the expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, the expression of the gene construct in the brain may mean the expression of the gene construct in at least one or at least two or at least three or all of the brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb. In some embodiments, the expression in the CNS and / or the brain may mean specific expression in the CNS and / or the brain. In one embodiment, the expression in the CNS and / or the brain may mean specific expression in the CNS and / or the brain. In one embodiment, the expression in the brain may mean the expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, the expression of the gene construct in the brain may mean the expression of the gene construct in at least one or at least two or at least three or all of the brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb. In some embodiments, the expression in the CNS and / or the brain may mean specific expression in the CNS and / or the brain. In one embodiment, the expression in the CNS and / or the brain may mean specific expression in the CNS and / or the brain. In one embodiment, the expression in the CNS and / or the brain may mean specific expression in the CNS and / or the brain. In some embodiments, the expression in the CNS and / or the brain may mean specific expression in the CNS and / or the brain. In one embodiment, the expression is undetectable in the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach and / or testis. In a preferred embodiment, the expression is undetectable in the liver and / or the heart. In another preferred embodiment, the expression is undetectable in skeletal muscle. In some embodiments, the expression is not associated with expression in at least 1, at least 2, at least 3, at least 4, or all of the organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, and testis. The description of CNS-specific expression and / or brain-specific expression is provided in the section entitled "General Information". The description of CNS-specific expression and / or brain-specific expression is provided in the section entitled "General Information".

[0114] Expression can be evaluated using techniques such as qPCR, Western blot analysis or ELISA, as described in the section entitled "General Information". The description of "CNS", "brain", "hypothalamus", "hippocampus", "cerebellum", "cortex", and "olfactory bulb" is provided in the section entitled "General Information".

[0115] In the context of the gene constructs for use according to the invention, expression vectors for use, pharmaceutical compositions for use, methods and uses, the gene constructs and / or expression vectors and / or pharmaceutical compositions and / or medicaments can be administered by administration into the CSF (cerebrospinal fluid) (via cisterna magna, intrathecal or intracerebroventricular delivery). The preferred mode of administration, and preferably the preferred mode of administration in humans, is intracerebroventricular.

[0116] In the context of the gene constructs for use according to the invention, expression vectors for use, pharmaceutical compositions for use, methods and uses, the gene constructs and / or expression vectors and / or pharmaceutical compositions and / or medicaments can be administered by administration into the CSF (cerebrospinal fluid) (via cisterna magna, intrathecal or intracerebroventricular delivery). The preferred mode of administration, and preferably the preferred mode of administration in humans, is intracerebroventricular. ​​​​​and / or the pharmaceutical composition and / or medicament may be administered by intraparenchymal administration.

[0117] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, genetic constructs and / or expression vectors and and / or the pharmaceutical composition and / or the medicament may be administered by intranasal administration.

[0118] As used herein, "intra-CSF administration," "intranasal administration," "intracembrane administration," "intracisternal administration," The terms "administration," "intrathecal administration," and "intracerebroventricular administration" are included in the section of this application entitled "General Information." is explained in.

[0119] In a preferred embodiment, the treatment or therapy or use of the medicaments described herein is In some embodiments, the treatments or The treatment or use or administration of a drug occurs annually or at least once every 2, 3, 4, 5, 6, 7, 8, 9 or may be repeated every 10 years (including the interval between any two of the listed values).

[0120] The subjects to be treated include cats, rodents (preferably mice, rats, gerbils and monkeys). higher mammals, such as rats, dogs, or humans; It could be.

[0121] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, the genetic constructs and / or or an expression vector and / or a pharmaceutical composition and / or a medicament, preferably comprising Show at least one, at least two, at least three, at least four, or all : - Decrease in neuroinflammation; - Increase in neurogenesis; - Increase in the number of astrocytes; - Decrease in neurodegeneration; - Symptom alleviation (described later in this specification); and - Improvement of parameters (described later in this specification).

[0122] A decrease in neuroinflammation may mean that the inflammation of neural tissue decreases. This can be evaluated using techniques known to those skilled in the art, such as measurement of (neural) inflammation markers, as is done, for example, in experimental sections. Exemplary markers that may be used in this regard are Il-1b, Il-6, and NfkB. In this context, "decrease" (respectively "improvement") means a detectable decrease (respectively detectable improvement) using assays known to those skilled in the art, such as assays performed in the experimental section. The decrease can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% decrease. The decrease can be seen at least 1 week, 1 month, 6 months, 1 year, or more after treatment with the gene construct and / or expression vector and / or composition of the present invention. Preferably, the decrease is observed after a single administration . In some embodiments, the decrease is preferably observed for at least 1 week, 1 month , 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years, or more after a single administration. . In some embodiments, the decrease is preferably observed for at least 1 week, 1 month , 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years, or more after a single administration.

[0123] An increase in neurogenesis may mean that neurons are produced by neural stem cells. This This can be evaluated using techniques known to those skilled in the art, such as the measurement of neurogenesis markers, as performed, for example, in the laboratory. Exemplary markers that can be used in this regard are Dcx, Ncam, and Sox2. In this context, "increase" (respectively, "improvement") means at least a detectable increase (respectively, a detectable improvement) using an assay known to those skilled in the art, such as an assay performed in the laboratory. A decrease can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% decrease. An increase can be seen at least 1 week, 1 month, 6 months, 1 year, or more after treatment using the gene construct and / or expression vector and / or composition of the present invention. Preferably, the increase is observed after a single administration. In some embodiments, the increase is preferably observed for at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years, or more after a single administration. An increase in the number of astrocytes can mean that the number of astrocytes increases. This can be evaluated using techniques known to those skilled in the art, such as the measurement of astrocyte markers, as performed, for example, in the laboratory. Exemplary markers that can be used in this regard are Gfap and S100b. In this context, "increase" (respectively, "improvement") means at least a detectable increase using an assay known to those skilled in the art, such as an assay performed in the laboratory.

[0124] Means an increase (each a detectable improvement). A decrease can be at least 5%, at least 10% , at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% decrease. An increase can be seen at least 1 week, 1 month, 6 months, 1 year or more after treatment with the gene constructs and / or expression vectors and / or compositions of the invention. Preferably, the increase is observed after a single administration. In some embodiments , the increase is preferably observed after a single administration, at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years or more.

[0125] A decrease in neurodegeneration can mean a decrease in the loss of neuron structure or function, including neuron death . This can be evaluated by using techniques known to those skilled in the art such as immunocytochemistry, immunohistochemistry and by medical imaging techniques such as MRI to study neuron morphology and synaptic changes (by measuring the density of proteins located at synapses), or by analyzing the expression levels of some aging and neurodegeneration markers . In this context, "decrease" (each "improvement") means at least a detectable decrease (each a detectable improvement) using assays known to those skilled in the art, such as assays performed in the experimental section . A decrease can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least It can be a decrease of up to 100%. The increase can be seen at least 1 week, 1 month, 6 months, 1 year or more after treatment using the gene construct and / or expression ve ctor and / or composition of the present invention. Preferably, the increase is observed after a single administration. In some embodiments, the increase is preferably, after a single administration, at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 2 0 years or more.

[0126] The alleviation of symptoms means that the progression of typical symptoms (e.g., neuroinflammation, neurodegeneration, cognitive decline, memory loss , learning ability decline, synaptic loss, tau phosphorylation) has slowed down in the individual, the cells, tissues or organs of the individual, as evaluated by a physician. The decrease of typical symptoms can mean a slowdown in the progression of symptom onset or a complete disappearance of the symptoms. The symptoms, and thus the decrease of the symptoms, can be evaluated in substantially the same ways as those used for the diagnosis of neuroinflammation, neurodegeneration, cognitive decline, and related diseases, including various clinical examinations and routine laboratory tests. Clinical examinations can include behavioral tests and cognitive tests . Laboratory tests can include both macroscopic and microscopic methods, molecular methods, radiographic methods such as X-rays, biochemical methods, immunohistochemical methods, etc. Memory and learning can be evaluated in mice, for example, by, as described in the experimental section, for example, the novel object recognition test and / or the Morris water maze test . The alleviation of symptoms is at least 1 week, 1 month, 6 months after treatment using the gene construct and / or expression vector and / or composition of the present invention. , may be seen 1 year or more later. Preferably, the alleviation is observed after a single administration. In some embodiments, the alleviation is preferably observed for at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years or more after a single administration.

[0127] The improvement of the parameter may mean the improvement of the result after the behavioral test, the improvement of the expression of serum and CSF markers, the improvement of the expression of apoptosis / neurogenesis cell markers, etc. The improvement of the parameter may be seen at least 1 week, 1 month, 6 months, 1 year or more after the treatment using the gene construct and / or expression vector and / or composition of the present invention. Preferably, the improvement is observed after a single administration. In some embodiments, the improvement is preferably observed for at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years after a single administration, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years or more. .

[0128] In the context of the gene construct for use according to the present invention, the expression vector for use, the pharmaceutical composition for use, the method and the use, the gene construct and / or expression vector and / or pharmaceutical composition described herein preferably alleviate one or more symptoms of neuroinflammation, neurodegeneration and / or cognitive impairment, or a disease associated therewith, in an individual, cells, tissues or organs of said individual, or alleviate one or more characteristics or symptoms of cells, tissues or organs of said individual.

[0129] The gene construct and / or expression vector and / or pharmaceutical composition described herein The product preferably has, at least 1 week, 1 month, 6 months, 1 year or more after treatment using the gene construct and / or expression vector and / or composition, a symptom or characteristic of the patient or the cells, tissues or organs of said patient reduced as described herein such that (for example, it is no longer detectable or has slowed down), the symptom or characteristic can be alleviated.

[0130] The gene constructs and / or expression vectors and / or pharmaceutical compositions and / or medicaments described herein may be suitable for administration to cells, tissues and / or organs in vivo of an individual suffering from or at risk of developing neuroinflammation, neurodegeneration and / or cognitive impairment, or a disease associated therewith, and may be administered in vivo, ex vivo or in vitro. The gene constructs and / or expression vectors and / or pharmaceutical compositions and / or medicaments described herein may be administered directly or indirectly to cells, tissues and / or organs in vivo of an individual suffering from or at risk of developing neuroinflammation, neurodegeneration and / or cognitive impairment, or a disease associated therewith, and may be administered directly or indirectly in vivo, ex vivo or in vitro. The mode of administration can be intravenous, intramuscular, intrathecal, intraventricular, intraperitoneal, inhalation, intranasal, intraocular and / or

[0131] intraparenchymal administration. Preferred modes of administration are intranasal, intraparenchymal and administration into the CSF (via cisternal, intrathecal or intraventricular delivery). Administration into the CSF is most preferred. The most preferred mode of administration, and in some cases the preferred mode of administration in humans, is intraventricular.

[0132] The gene construct and / or expression vector and / or composition and / or pharmaceutical of the present invention can be administered directly or indirectly using appropriate means known in the art. Improvements in the means for providing the gene construct and / or expression vector and / or composition and / or pharmaceutical of the present invention to an individual or to the cells, tissues, organs of said individual are expected, considering the progress already achieved. Such future improvements can, of course, be incorporated to achieve the mentioned effects of the present invention. The gene construct and / or expression vector and / or composition and / or pharmaceutical can be delivered directly to an individual, the cells, tissues or organs of said individual. Depending on the disease or condition, the cells,

[0133] tissues or organs of said individual can be as previously described herein. When administering the gene construct and / or expression vector and / or composition and / or pharmaceutical of the present

[0134] General Information Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly and ordinarily understood by those of ordinary skill in the art to which the present invention

[0135] Sequence Identity / Similarity In the context of the present invention, a nucleic acid molecule such as a nucleic acid molecule encoding insulin is represented by a nucleotide sequence encoding a protein fragment or polypeptide or peptide or derivative peptide. In the context of the present invention, an insulin protein fragment or polypeptide or peptide or derivative peptide is represented by an amino acid sequence. It should be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derivative peptide or construct identified herein by a given sequence identity number (SEQ ID NO) is not limited to this specific sequence shown. Each coding sequence identified herein encodes a given protein fragment or polypeptide or peptide or derivative peptide or construct, or is itself a protein fragment or polypeptide or construct or peptide or derivative peptide. Throughout this application, each time reference is made to a specific nucleotide sequence (taking SEQ ID NO: X as an example) encoding a given protein fragment or polypeptide or peptide or derivative peptide, this is replaced by:

[0136] i. a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: X; ii. a nucleotide sequence whose sequence differs from that of the nucleic acid molecule of (i) due to the degeneracy of the genetic code; or iii. a nucleotide sequence encoding an amino acid sequence having at least 60% amino acid identity or similarity with the amino acid sequence encoded by the nucleotide sequence SEQ ID NO: X.

[0137] ​​​​​​​​​​​​​​​​Another preferred level of sequence identity or similarity is 70%. Another level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another level of sequence identity or similarity is 99%.

[0138] Throughout this application, each time a specific amino acid sequence is referred to by SEQ ID NO (taking SEQ ID NO Y as an example), it can be replaced by a polypeptide comprising an amino acid sequence having at least 60% sequence identity or similarity to amino acid sequence SEQ ID NO Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%. is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0139] Each nucleotide or amino acid sequence described herein by its percent identity or similarity to a given nucleotide or amino acid sequence, further in a preferred embodiment, is at least 6 0%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least also 78%, at least 79%, at least 80%, at least 81%, at least 82% , at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of identity or similarity.

[0140] Each non-coding nucleotide sequence (i.e., of a promoter or another control region) can be replaced by a nucleotide sequence that has at least 60% sequence identity or similarity with a specific nucleotide sequence, e.g., taking SEQ ID NO: A. Preferred nucleotide sequences have at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74% , at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83 %, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: A. . In a preferred embodiment, such non-coding nucleotide sequences, such as promoters, exhibit or exert at least the activity of such non-coding nucleotide sequences, such as the activity of promoters known to those skilled in the art. For example, such activity is the detectable expression of a nucleotide sequence operably linked to a promoter, such as an insulin coding sequence. Induced by. The terms "homology", "sequence identity", "identity", etc. are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid sequences (peptides or polypeptides or proteins) or two or more nucleic acid sequences (polynucleotides) determined by comparing the sequences. "Similarity" or "sequence similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics a nd the Cell: Modern Computational Approac hes in Genomics, Proteomics and transcrip

[0141] tomics, Xia X., Springer International Pub lishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Co ld Spring Harbor Laboratory Press, New Yo rk, 2004. nd the Cell: Modern Computational Approac hes in Genomics, Proteomics and transcrip tomics, Xia X., Springer International Pub lishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Co ld Spring Harbor Laboratory Press, New Yo rk, 2004. It is.

[0142] Sequence identity or similarity can be calculated based on the full length or a part of two given sequence numbers. In some embodiments, the part means at least 5 0%, 60%, 70%, 80%, 90%, 95% or 100% of both sequence numbers. In a preferred embodiment, sequence identity or similarity is determined by comparing the full length of the sequences specified herein. Unless otherwise indicated herein, identity or similarity with a given sequence number means identity or similarity based on the full length of the said sequence (i.e., over its full length or as a whole). In the art, "identity" also sometimes refers to the degree of sequence relatedness between amino acid or nucleotide sequences, which is determined by the match between such strings of sequences. Sequence identity or similarity can be determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their full length, while sequences of substantially different lengths are preferably aligned using a local alignment

[0143] algorithm (e.g., Smith-Waterman). The sequences are then (e.g., when optimally aligned by the program EMBOSS needle or EMBOSS water using default parameters) at least sequence identity or similarity (described below) by alignment. to optimally align over the full length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Then the sequences are (e.g., when optimally aligned by the program EMBOSS needle or EMBOSS water using default parameters) at least sequence identity or similarity (described below) using the program EMBOSS needle or EMBOSS water (when optimally aligned using default parameters) of sequence identity or similarity (described below). When sharing a certain minimum percentage, it can be called "substantially identical" or "essentially similar". It can be called so.

[0144] Global alignment is appropriately used to determine sequence identity or similarity when two sequences have similar lengths. When the full lengths of the sequences are significantly different, local alignment such as using the Smith-Waterman algorithm is preferred. or similarity. When the full lengths of the sequences are significantly different, local alignment such as using the Smith-Waterman algorithm is preferred. ith-Waterman algorithm is preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their full lengths (complete lengths), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the default parameters of EMBOSS needle and EMBOSS water with a gap open penalty = 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein) can be used. For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). algorithm to align two sequences over their full lengths (complete lengths), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the default parameters of EMBOSS needle and EMBOSS water with a gap open penalty = 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein) can be used. For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). algorithm to align two sequences over their full lengths (complete lengths), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the default parameters of EMBOSS needle and EMBOSS water with a gap open penalty = 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein) can be used. For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Smith-Waterman local alignment algorithm is used. Generally, the default parameters of EMBOSS needle and EMBOSS water with a gap open penalty = 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein) can be used. For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). ally, the default parameters of EMBOSS needle and EMBOSS water with a gap open penalty = 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein) can be used. For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). ally, the default parameters of EMBOSS needle and EMBOSS water with a gap open penalty = 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein) can be used. For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). BOSS needle and EMBOSS water default parameters can be used. BOSS needle and EMBOSS water default parameters can be used. rix is DNAfull for nucleotide sequences, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). rix is DNAfull for nucleotide sequences, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). 89, 915-919).

[0145] Alternatively, the percentage of similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Alternatively, the percentage of similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Preferably, without modifying the default parameters, use Homologene can also be (https: / / en.wikipedia.org / wiki / Hom oloGene). Thus, the nucleotide and amino acid sequences of some embodiments of the present invention can be further used as "query sequences" to search public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:40 3-10. The BLAST nucleotide search can be performed using the NBLAST program, score = 100, word length = 12, to obtain a nucleotide sequence homologous to the nucleic acid molecule of the present invention, preferably a nucleotide sequence encoding insulin. The BLAST protein search can be performed using the BLASTx program, score = 50, word length = 3, to obtain an amino acid sequence homologous to the protein molecule of the present invention. To obtain a gapped alignment for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of each program ( e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information, accessible on the World Wide Web at www.ncbi .nlm.nih.gov / .

[0146] ​​​​​When determining the degree of amino acid similarity, one of ordinary skill in the art may also take into account so-called conservative amino acid substitutions. These may be considered.

[0147] As used herein, "conservative" amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are shown below. Examples of classes of amino acid residues for conservative substitutions are shown below. [Table 2]

[0148] Alternative classes of conservative amino acid residue substitutions are as follows: [Table 3]

[0149] Alternative physical and functional classifications of amino acid residues: [Table 4]

[0150] For example, the group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine and isoleucine; the group of amino acids having aliphatic hydroxyl side chains includes serine and threonine; the group of amino acids having amide-containing side chains includes asparagine and gln utamine; the group of amino acids having aromatic side chains includes phenylalanine, tyrosine and tryptophan; the group of amino acids having basic side chains includes lysine, arginine and histidine; the group of amino acids having sulfur-containing side chains includes cysteine and methion ine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenyl alanine-tyrosine, lysine-arginine, alanine-valine and asparagine-gln It is lutamine. The substitution variants of the amino acid sequences disclosed in this specification are those in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted in its place . . Preferably, the amino acid changes are conservative. The preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to A sn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile or Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; S er to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe ; and Val to Ile or Leu.

[0151] Gene or coding sequence A "gene" is a sequence of DNA or RNA nucleotides that encodes a functional molecule. The nucleotide sequence can include "non-coding sequences" as well as "coding sequences". The coding region of a "gene", also known as a CDS (from a coding sequence), is the portion of DNA or RNA of a gene that encodes a protein. Examples of non-coding sequences are the promoter and microRNA target sequences described elsewhere in this specification . The term "gene" refers to a DNA fragment that includes a region (transcription region) that is transcribed into an intracellular RNA molecule (e.g., mRNA) that is operably linked to an appropriate control region (e.g., a promoter). A gene typically includes a promoter, a 5' leader sequence, a coding region, and a 3' untranslated region . ​​​Translation array (3' end) (e.g., including polyadenylation and / or transcription termination sites), etc. comprises several operably linked fragments of . A chimeric or recombinant gene (such as a chimeric or recombinant insulin gene, etc.) is a gene that is not normally found in nature, such as a gene that is essentially unrelated to part or all of the DNA region where the promoter is transcribed. "Gene expression" refers to the process by which a DNA region operably linked to an appropriate control region, particularly a promoter, is biologically active and is transcribed into RNA that can be translated into a biologically active protein or peptide.

[0152] "Transgene" as used herein refers to a gene or coding sequence or nucleic acid molecule represented by a nucleotide sequence newly introduced into a cell (i.e., a molecule encoding insulin), that is, a gene that may be present in a cell but is usually not expressed or is expressed at an insufficient level. In this context, "insufficient" means that the insulin is expressed in the cell but the conditions and / or diseases described herein can still occur. In this case, the present invention enables overexpression of insulin. A transgene may include sequences specific to the cell, sequences not naturally present in the cell, or a combination of both. A transgene may include the insulin and / or additional protein coding sequences previously identified herein that are operably linked to appropriate control sequences for expression of the insulin-encoding sequence in the cell. Preferably, the transgene is not integrated into the genome of the host cell.

[0153] Promoter As used herein, the terms "promoter" or "transcription control sequence" function to control the transcription of one or more coding sequences, are located upstream with respect to the direction of transcription of the coding sequence at the transcription start site and are nucleic acid fragments structurally defined by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences (including but not limited to transcription factor binding sites, repressor and activator protein binding sites), and any other nucleotide sequences known to those skilled in the art to act directly or indirectly to control the amount of transcription from the promoter . A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions . An "inducible" promoter is a promoter that is controlled physiologically or developmentally or in some other way, for example by the application of a chemical inducer . A "ubiquitous promoter" is active in substantially all tissues, organs and cells of an organism . In some embodiments, the ubiquitous promoter drives expression in at least 5, 6, 7, 8 , 9, 10 or more different types of tissues, organs and / or cells . An "organ-specific" or "tissue-specific" promoter is a promoter that is active in a particular type of organ or tissue, respectively . Organ-specific and tissue-specific promoters primarily control the expression of one or more genes (or coding sequences) in one organ or tissue, but may allow for detectable levels of ("leaky") expression in other organs or tissues

[0154]

[0155] It is possible. Leaky expression in other organs or tissues can be evaluated by standard assays known to those skilled in the art (e.g., qPCR, Western blot analysis, ELISA) at the mRNA or protein level, and is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold lower than that compared to organ-specific or tissue-specific expression, but still detectable. The maximum number of organs or tissues in which leaky expression can be detected is 5, 6, 7, or 8.

[0156] A "CNS-specific promoter and / or brain-specific promoter" is a promoter that can initiate transcription in the CNS and / or the brain, while allowing for leaky expression in other (up to 5, 6, 7, or 8) organs and parts of the body. Transcription in the CNS and / or the brain can be detected in relevant regions such as the CNS and / or the brain and / or the hypothalamus and / or the cortex and / or the hippocampus and / or the cerebellum and / or the olfactory bulb, as well as in cells such as neurons and / or glial cells.

[0157] In the context of the present invention, a CNS-specific promoter and / or brain-specific promoter is preferential or dominant (at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher) compared to other organs or tissues. or more)CNS and / or expression of insulin in the brain can be driven It can be a promoter that can. Other organs or tissues can be the liver, pancreas, adipose tissue, skeletal muscle, heart It can be the kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, other Organs are the liver and / or heart. Other organs can also be skeletal muscle. As used herein The CNS brain-specific promoter and / or brain-specific promoter used also includes C A promoter that directs expression in a specific region or cell subset of the NS and / or brain Including. Therefore, CNS-specific promoters as well as / or brain-specific promoters It can also be a hippocampus-specific promoter, cerebellum-specific promoter, cortex-specific promoter Tertiary, hypothalamus-specific promoter and / or olfactory bulb-specific promoter, or It can be selected from any combination of these. Expression is described in the section entitled "General Information" It can be evaluated using techniques such as qPCR, Western blot analysis or ELISA Can be.

[0158] Throughout this application, when CNS-specific and / or brain-specific are referred to in the context of expression Cell type-specific expression of the cell type(s) that make up the CNS and / or brain is also recalled Each.

[0159] Operably linked As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship Refers to. A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid molecule For example, a transcriptional control sequence is the transcription of a coding sequence When affecting, it is operably linked to the coding sequence. When operably linked means that the linked DNA sequences are typically adjacent and, if necessary, adjacent and join two protein-coding regions within the reading frame. The ligation is by ligation with convenient restriction sites or alternatively inserted adapters or linkers or by gene synthesis or any other method known to those skilled in the art.

[0160] MicroRNA As used herein, "microRNA" or "miRNA" or "miR" has its conventional and ordinary meaning as understood by those skilled in the art in view of the present disclosure. MicroRNAs are small non-coding RNA molecules found in plants, animals, and some viruses and can function in RNA silencing and post-transcriptional regulation of gene expression. The target sequence of a microRNA can be designated as "miRT". For example, the target sequence of microRNA-1 or miRNA-1 or miR-1 can be designated as miRT-1.

[0161] Proteins and Amino Acids The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably and refer to a molecule consisting of a chain of amino acids, regardless of its specific mode of action, size, three-dimensional structure or origin. In the amino acid sequences described herein, amino acids or "residues" are shown in three-letter notation. These three-letter notations and the corresponding one-letter notations are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine ​where D(Asp) is aspartic acid, E(Glu) is glutamic acid, F(Phe) is phenylalanine, G(Gly) is glycine, H(His) is histidine, I(Ile) is isoleucine, K(Lys) is lysine , L(Leu) is leucine, M(Met) is methionine, N(Asn) is asparagine, P(Pro) is proline, Q(Gln) is glutamine, R(Arg) is arginine, S(Ser) is serine, T(Thr) is threo nine, V(Val) is valine, W(Trp) is tryptophan, Y( Tyr) is tyrosine. The residue can be any proteinogenic amino acid, but any non-proteinogenic amino acid such as D-amino acids and modified amino acids formed by post-translational modification, as well as any

[0162] Gene construct The gene constructs described herein can be prepared using any cloning and / or recombinant DNA techniques known to those skilled in the art to express the nucleotide sequence encoding the insulin in suitable cells, e.g., cultured cells or cells of a multicellular organism, such as those described in Ausubel et al., ’’Current Proto cols in Molecular Biology’’, Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, supra), which are hereby incorporated by reference in their entirety. Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site - specific mutagenesis) and Rob erts et al. (1987) Nature 328:731 - 734 or We lls, J.A., et al. (1985) Gene 34:315 (describing the cassette mutagenesis method) should also be referred to.

[0163] Expression vector The terms "expression vector" or "vector" generally refer to a nucleotide sequence that can effect the expression of a gene or coding sequence in a host compatible with such a sequence. An expression vector has a genome that can stabilize and maintain episomes within a cell. In the context of the present invention, a cell can mean a cell used to create a construct or a cell to which the construct is administered. Alternatively, a vector can be integrated into the genome of a cell, for example, through homologous recombination or other methods. These expression vectors typically include at least an appropriate promoter sequence and, optionally, a transcription termination signal.

[0164] Additional factors necessary or useful for effecting expression can also be used as described herein. A nucleic acid or DNA or nucleotide sequence encoding insulin is incorporated into a DNA construct that can be introduced and expressed in in vitro cell cultures. Specifically, the DNA construct is suitable for replication in a prokaryotic host such as bacteria, e.g., Escherichia coli (E. coli), or can be introduced into cultured mammalian cells, plants, insects (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.

[0165] ​​​​​DNA constructs prepared for introduction into a particular host must be capable of replicating sequences that are recognized by the host. The system includes a contemplated DNA fragment encoding a desired polypeptide, and a polypeptide encoding The transcription and translation initiation and termination control sequences are operably linked to the coding segment. The term "operably linked" has been previously explained herein. For example, a promoter or enhancer acts on a coding sequence if it stimulates the transcription of the sequence. The DNA of the signal sequence is operably linked to the preparative sequence involved in the secretion of the polypeptide. When expressed as a protein, it is operably linked to DNA encoding the polypeptide. Generally, operably linked DNA sequences are contiguous and include a signal sequence. In some cases, enhancers are contiguous and in reading frame. The sequence need not be contiguous with the coding sequences whose transcription it controls. Linkage may be via convenient restriction sites or Instead, by ligation with inserted adaptors or linkers, is accomplished by gene synthesis, or any other method known to one of skill in the art.

[0166] The selection of an appropriate promoter sequence is generally selected for the expression of the DNA segment. Examples of suitable promoter sequences include those known in the art, such as those described herein for prokaryotic cells. and eukaryotic promoters (see, e.g., Sambrook and d Russell, 2001). Transcriptional regulatory sequences are typically recognized by the host. The selection of an appropriate promoter depends on the host. Depending on the promoter, trp, lac and phage promoters, tRNA promoters and Promoters such as the glycolytic enzyme promoter are known and available (see, for example, Sambrook and Russell, 2001 supra). Expression vectors include a replication system as well as transcriptional and translational control sequences along with an insertion site for a segment encoding a polypeptide. In most cases, the replication system is functional only within the cells used to generate the vector (bacterial cells such as Escherichia coli (E. coli)). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334:31-36. For example, suitable expression vectors can be expressed in yeast, such as yeast (S. cerevisiae), insect cells, such as Sf9 cells, mammalian cells, such as CHO cells, and bacterial cells, such as Escherichia coli (E. coli). Thus, the cells can be prokaryotic or eukaryotic host cells. The cells can be cells suitable for culture in liquid or solid media. See, for example, Sambrook and Russell, supra. Expression vectors include a replication system as well as transcriptional and translational control sequences along with an insertion site for a segment encoding a polypeptide. In most cases, the replication system is functional only within the cells used to generate the vector (bacterial cells such as Escherichia coli (E. coli)). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334:31-36. See, for example, Sambrook and Russell, supra. See, for example, Sambrook and Russell, supra. See, for example, Metzger et al. (1988) Nature 334:31-36. For example, suitable expression vectors can be expressed in yeast, such as yeast (S. cerevisiae), insect cells, such as Sf9 cells, mammalian cells, such as CHO cells, and bacterial cells, such as Escherichia coli (E. coli). Thus, the cells can be prokaryotic or eukaryotic host cells. The cells can be cells suitable for culture in liquid or solid media. Alternatively, the host cells can be cells that are part of a multicellular organism, such as a transgenic plant or animal.

[0167] Alternatively, the host cells can be cells that are part of a multicellular organism, such as a transgenic plant or animal. Alternatively, the host cells can be cells that are part of a multicellular organism, such as a transgenic plant or animal.

[0168] Viral vectors A viral vector or viral expression vector or viral gene therapy vector is a vector that contains the gene constructs described herein. A viral vector or viral gene therapy vector is a vector suitable for gene therapy.

[0169] A viral vector or viral gene therapy vector is a vector suitable for gene therapy. It is. Vectors suitable for gene therapy are described in Anderson 1998, Nature 3 92:25-30; Walther and Stein, 2000, Drugs 60: 249-71; Kay et al., 2001, Nat. Med. 7:33-40; R ussell, 2000, J. Gen. Virol. 81:2573-604; Amad o and Chen, 1999, Science 285:674-6; Federic o, 1999, Curr. Opin. Biotechnol. 10:448-53; Vi gna and Naldini, 2000, J. Gene Med. 2:308-16 ; Marin et al., 1997, Mol. Med. Today 3:396-40 3; Peng and Russell, 1999, Curr. Opin. Biotec hnol. 10:454-7; Sommerfelt, 1999, J. Gen. Viro l. 80:3049-64; Reiser, 2000, Gene Ther. 7:910 -3; and are described in the references cited therein. Additional references describing gene therapy vectors are Naldini 2015, Nature 5526(75 73):351-360; Wang et al. 2019 Nat Rev Drug Discov18(5):358-378; Dunbar et al. 2018 Sc ience 359(6372); Lukashey et al. 2016 Biosch emistry(Mosc) 81(7):700-708. Particularly suitable gene therapy vectors include adenovirus vectors and adeno-associated virus

[0170] vectors. It contains (AAV) vectors. These vectors infect a wide number of dividing and non-dividing cell types, including synoviocytes and hepatocytes. After cell entry, due to the episomal nature of adenovirus and AAV vectors, these vectors become suitable for the therapeutic uses shown above (Russell, 2000, J. Gen. Virol. 81:257 3-2604; Goncalves, 2005, Virol J. 2(1):43). An AAV vector results in a very stable long-term expression of transgene expression (up to 9 years in dogs (Niem eyer et al, Blood. 2009 Jan 22;113(4):797-80 6), about 10 years in humans (Buchlis, G. et al., Blood. 2012 Mar 29;119(13):3038-41)) and is thus even more preferable. As outlined by Russell (2000, supra), preferred adenovirus vectors are modified to reduce the host response. Gene therapy using AAV vectors is described by Wang et al., 2005, J Gen e Med. March 9 (Epub ahead of print), Mande ll et al., 2004, Curr Opin Mol Ther. 6(5):48 2-90 and Martin et al., 2004, Eye18(11):1049 -55, Nathwani et al, N Engl J Med. 2011 Dec 2 2;365(25):2357-65, Apparailly et al, Hum G ene Ther. 2005 Apr;16(4):426-34.

[0171] Other suitable gene therapy vectors include retroviral vectors. In the present invention, a preferred retroviral vector for application is a lentivirus-based expression construct There is. Lentiviral vectors can infect the genomes of both dividing and non-dividing cells and stably Integrate (Amado and Chen, 1999 Science 2 85: 674-6). Methods for constructing and using lentivirus-based expression constructs are described in US Patent Nos. 6,165,782, 6,207,455, 6,21 8,181, 6,277,633 and 6,323,031 Specification, as well as Federico (1999, Curr Opin Biotechn ol10: 448-53) and Vigna et al. (2000, J Gene Med2000; 2: 308-16).

[0172] Other suitable gene therapy vectors include adenoviral vectors, herpesvirus vectors Tars, polyomavirus vectors or vaccinia virus vectors.

[0173] Adeno-associated virus vector (AAV vector) As used herein as synonyms, the terms "adeno-associated virus", "AAV virus", " AAV virion", "AAV virus particle" and "AAV particle" refer to AAV At least one capsid protein (preferably composed of all capsids of a specific AAV serotype Composed of proteins) and an encapsulated polynucleotide of the AAV genome Refers to a virus particle composed of. The particle is a heterologous polynucleotide flanked by AAV terminal inverted repeats (i.e., polynucleotides different from the wild-type AAV genome, such as transgenes delivered to mammalian cells) When included, these are typically known as "AAV vector particles" or "AAV viral vectors" or "AAV vectors". AAV refers to a virus belonging to the genus Dependovirus in the family Parvoviridae. The length of the AAV genome is approximately 4.7 Kb and consists of single-stranded deoxyribonucleic acid (ssDNA) that can be detected as plus or minus strands. The present invention also encompasses the use of double-stranded AAV, also called dsAAV or scAAV. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strands, and two open reading frames (ORFs): rep and cap. The frame rep consists of four overlapping genes that encode the proteins Rep necessary for the AAV life cycle. The frame cap contains nucleotide sequences that overlap with the capsid proteins: VP1, VP2, and VP3, which interact to form an icosahedral symmetric capsid (see Carter and Samu lski, Int J Mol Med 2000, 6(1):17-27 and Gao et al, 2004).

[0174] A preferred viral vector or a preferred gene therapy vector is an AAV vector . As used herein, an AAV vector preferably includes a recombinant AAV vector (rA AV vector). As used herein, an "rAAV vector" refers to a recombinant vector that includes a portion of the AAV genome encapsidated in a protein shell of capsid proteins derived from the AAV serotypes described herein . A portion of the AAV genome is AAV1​ 、derived from adeno-associated virus serotypes such as AAV2, AAV3, AAV4, AAV5 may contain inverted terminal repeats (ITRs). Preferred ITRs include SEQ ID NO: 35 (5’ ITR) and or consist essentially of or consist of a sequence represented by SEQ ID NO: 36 (3’ ITR) of AAV2. The present invention also preferably uses, as the 5’ ITR, a sequence having at least 80% (or at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to SEQ ID NO: 35 and, as the 3’ ITR, a sequence having at least 80% (or at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to SEQ ID NO: 36. The protein shell composed of the capsid protein may be derived from any AAV serotype. The protein shell may also be referred to as the capsid protein shell. The rAAV vector may have one or preferably all wild-type AAV genes deleted, but still

[0175] The protein shell composed of the capsid protein may be derived from any AAV serotype. The protein shell may also be referred to as the capsid protein shell. The rAAV vector may have one or preferably all wild-type AAV genes deleted, but still ​​​​​​​​​​​and may contain a functional ITR nucleotide sequence. The functional ITR sequence is required for replication, rescue, and packaging of the AAV virion. The ITR sequence may be a wild-type sequence, or at least 80%, at least 81%, at least 82% , at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % sequence identity with the wild-type sequence, or may be modified by, for example, nucleotide insertion, mutation, deletion, or substitution, as long as it remains functional. In this context, functionality refers to the ability to direct packaging into the capsid shell of the genome and then enable expression in the host cell or target cell to be infected. In the context of the present invention, the capsid protein shell

[0176] may be of a different serotype than the rAAV vector genome ITR. The nucleotide sequence represented by the selectable nucleotide sequence, preferably encoding insulin, is preferably inserted between the rAAV genome or ITR sequence identified above. For example, the expression construct includes an expression control

[0177] element operably linked to the coding sequence and the 3' termination sequence. The nucleic acid molecule may also be referred to as a transgene. , but lacks the AAV ITRs (provided by the rAAV vector genome), which complement AAV functions not present in the rAAV vector. The AAV helper function includes the two major ORFs of AAV, namely the rep coding region and the cap coding region, or functionally substantially identical sequences thereof. The Rep and Cap regions are well known in the art. For example, see Chiorini et al. (1999, J. of Virology, Vol 73(2):1309 - 1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , and lacks the AAV ITRs (provided by the rAAV vector genome). The AAV helper function includes the two major ORFs of AAV, namely the rep coding region and the cap coding region, or functionally substantially identical sequences thereof. The Rep and Cap regions are well known in the art. For example, see Chiorini et al. (1999, J. of Virology, Vol 73(2):1309 - 1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , namely the rep coding region and the cap coding region, or functionally substantially identical sequences thereof. The Rep and Cap regions are well known in the art. For example, see Chiorini et al. (1999, J. of Virology, Vol 73(2):1309 - 1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , or functionally substantially identical sequences thereof. The Rep and Cap regions are well known in the art. For example, see Chiorini et al. (1999, J. of Virology, Vol 73(2):1309 - 1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , J. of Virology, Vol 73(2):1309 - 1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , J. of Virology, Vol 73(2):1309 - 1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. . The AAV helper function can be provided by an AAV helper construct. Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. . Introduction of the helper construct into the host cell can be carried out, for example, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector as specified in this specification, by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , by transformation, transfection, or transduction. Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. . Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. . Thus, the AAV helper construct of the present invention can be selected to provide, on the one hand, the capsid protein shell of the rAAV vector and, on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. , and on the other hand, the desired combination of serotypes for the rAAV genome present in the replication and packaging of the rAAV vector. .

[0178] An "AAV helper virus" provides additional functions necessary for the replication and packaging of AAV. Suitable AAV helper viruses include adenovirus, herpes simplex virus (such as HSV type 1 and type 2), and vaccinia virus. The additional functions provided by the helper virus can also be introduced into the host cell via a plasmid, as described in U.S. Patent No. 6,531,456, which is incorporated herein by reference. , herpes simplex virus (such as HSV type 1 and type 2), and vaccinia virus. The additional functions provided by the helper virus can also be introduced into the host cell via a plasmid, as described in U.S. Patent No. 6,531,456, which is incorporated herein by reference. , and vaccinia virus. The additional functions provided by the helper virus can also be introduced into the host cell via a plasmid, as described in U.S. Patent No. 6,531,456, which is incorporated herein by reference. . The additional functions provided by the helper virus can also be introduced into the host cell via a plasmid, as described in U.S. Patent No. 6,531,456, which is incorporated herein by reference. , 531,456, which is incorporated herein by reference. .

[0179] "Transduction" refers to the delivery of insulin to a recipient host cell by a viral vector. For example, transduction of target cells by the rAAV vectors of the present invention results in the transfer of the rAAV genome contained in the vector into the transduced cells. "Host cell" or "target cell" refers to a cell into which DNA delivery is performed, such as a muscle cell of interest. AAV vectors can transduce both dividing and non-dividing cells.

[0180] Production of AAV vectors The production of recombinant AAV (rAAV) for vectorizing a transgene has been previously described (see Ayuso E,et al.,Curr.Gene Ther.2010;1 0:423-436, Okada T,et al.,Hum.Gene Ther.2 009;20:1013-1021, Zhang H,et al.,Hum.Gene Ther.2009;20:922-929 and Virag T,et al.,H um.Gene Ther.2009;20:807-817). These protocols can be used or adapted to produce the AAV of the present invention. In one embodiment, a producer cell line is transiently transfected with the polynucleotide of the present invention (including an expression cassette flanked by ITRs) and one or more constructs encoding the rep and cap proteins that provide helper functions. In another embodiment, the cell line stably supplies helper functions and the cell line is transiently transfected with the polynucleotide of the present invention (including an expression cassette flanked by ITRs) and one or more constructs encoding the rep and cap proteins. In another embodiment, the cell line is transiently transfected with the polynucleotide of the present invention (including an expression cassette flanked by ITRs) and one or more constructs encoding the rep and cap proteins. In another embodiment, the cell line is transiently transfected with the polynucleotide of the present invention (including an expression cassette flanked by ITRs) and one or more constructs encoding the rep and cap proteins. In another embodiment, the cell line ​​ Supply the rep and cap proteins and the helper function stably, and transiently transfect the cell line with the polynucleotide of the present invention. In another embodiment, the cell line stably supplies the rep and cap proteins, and transiently transfects the cell line with the polynucleotide of the present invention and the polynucleotide encoding the helper function. In yet another embodiment, the cell line stably supplies the polynucleotide of the present invention, the rep and cap proteins, and the helper function. Methods for making and using these and other AAV production systems are described in the art. See U.S. Patent No. 5,139,941 to Muzyczka N et al., U.S. Patent No. 5,741,683 to Zhou X et al., U.S. Patent No. 6,057,152 to Samulski R et al., U.S. Patent No. 6,204,059 to Samulski R et al., U.S. Patent No. 6,268,213 to Samulski R et al., U.S. Patent No. 6,491,907 to Rabinowitz J et al., U.S. Patent No. 6,660,514 to Zolotukhin S et al., U.S. Patent No. 6,951,753 to Shenk T et al., U.S. Patent No. 7,094,604 to Snyder R et al., U.S. Patent No. 7,172,893 to Rabinowitz J et al., U.S. Patent No. 7,201,898 to Monahan P et al., U.S. Patent No. 7,229,823 to Samulski R et al., and U.S. Patent No. 7,439,065 to Ferrari F et al. The rAAV genome present in the rAAV vector is at least one of the AAV serotypes (preferably

[0181] Alternatively, the terminal inverted repeat region (ITR) (e.g., of serotype AAV2 as previously disclosed herein) nucleotide sequence, or a nucleotide sequence substantially identical thereto or having at least 60% identity thereto, and a nucleotide sequence encoding insulin (under the control of appropriate control elements) inserted between the two ITRs. (under the control of appropriate control elements) The vector genome requires the use of adjacent 5' and 3' ITR sequences to enable efficient packaging of the vector genome into the rAAV capsid.

[0182] The complete genomes of several AAV serotypes and corresponding ITRs have been sequenced ( Chiorini et al. 1999, J. of Virology Vol. 73 , No. 2, p1309-1319). These can be cloned or generated by chemical synthesis known in the art using, for example, an oligonucleotide synthesizer supplied by Applied Biosystems Inc. (Fosters, CA, USA), or by standard molecular biology techniques. The ITR can be cloned from the AAV viral genome or excised from a vector containing the AAV ITR. The ITR nucleotide sequence can be ligated to either end of a nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the AAV sequence between the ITRs can be replaced with a desired nucleotide sequence.

[0183] Preferably, the rAAV genome present in the rAAV vector is the rep (replication) or does not contain a nucleotide sequence encoding a viral protein such as a cap (capsid) gene. This rAAV genome may further contain, for example, an antibiotic resistance gene, a gene encoding a fluorescent protein ( for example, gfp), or a marker or reporter gene such as a gene encoding a product (e.g., lacZ, aph etc.) that is detectable and / or selectable by chemical, enzymatic or other methods known in the art. etc.) that is detectable and / or selectable by chemical, enzymatic

[0184] The rAAV genome present in the rAAV vector further contains a promoter sequence operably linked to a nucleotide sequence encoding insulin. The appropriate 3' untranslated sequence can also be operably linked to a nucleotide sequence encoding insulin. The appropriate 3' untranslated region can be naturally associated with the nucleotide sequence

[0185] or can be derived from a different gene such as, for example, the SV40 polyadenylation signal (SEQ ID NO: 37) and the rabbit β -globin polyadenylation signal (SEQ ID NO: 38). or can be derived from a different gene such as, for example, the SV40 polyadenylation signal (SEQ ID NO: 37) and the rabbit β -globin polyadenylation signal (SEQ ID NO: 38).

[0186] Expression Expression can be evaluated by any method known to those skilled in the art. For example, expression can be determined by standard assays known to those skilled in the art such as qPCR, Western blot analysis, or ELISA to measure the level of transgene expression in the liver at the mRNA or protein level. assays known to those skilled in the art such as qPCR, Western blot analysis, or ELISA to measure the level of transgene expression in the liver at the mRNA or protein level. Expression can be evaluated at any time after administration of the gene construct, expression vector, or composition described herein.

[0187] Expression can be evaluated at any time after administration of the gene construct, expression vector, or composition described herein. Expression can be evaluated at any time after administration of the gene construct, expression vector, or composition described herein.

[0188] In some embodiments of the present specification, the expression can be detected immediately after 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks , 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks. It can be detected.

[0189] In some embodiments of the present specification, the expression can continue for at least 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks , 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks , 48 weeks, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years , 10 years, 12 years, 15 years, 20 years or more. In other words, this means that the expression can be detected 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 week, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 2 8 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 1 year, 2 years, 3 years, 4 years , 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 20 years or more after administration. It means that it can be detected.

[0190] In some embodiments, this expression is detected after a single administration.

[0191] In the context of the present invention, CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb-specific expression is preferential or dominant (at least 10% higher, at least 20% higher , at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher or higher in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Other organs or tissues can be, for example, the liver, pancreas, adipose tissue, skeletal muscle, heart, etc. In one embodiment, the expression is undetectable in the liver, pancreas, adipose tissue, skeletal muscle and / or heart. In some embodiments, the expression is undetectable in at least 1, at least 2, at least 3, at least 4, or all of the organs selected from the group consisting

[0192] of the liver, pancreas, adipose tissue, skeletal muscle and heart. The expression can be evaluated as described above. Throughout this application, when CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb are mentioned in the context of specific expression, cell-type

[0193] specific expression of the (one or more) cell types that make up the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb is also contemplated. Administration As used herein, "intra-CSF administration" means direct administration It means administration. As used herein, "intracerebroventricular administration" means administration to either of the two cerebral ventricles of the brain. As used herein, "intrathecal administration" means administration to the cerebrospinal fluid (CSF) within the intrathecal space of the spinal column. As used herein, "parenchymal administration" means direct local administration to any region of the brain parenchyma. As used herein, "intranasal administration" means administration through the nasal structure.

[0194] In a preferred embodiment, the gene constructs, expression vectors and compositions according to the invention are administered as a single administration.

[0195] Codon optimization As used herein, "codon optimization" refers to a process employed to modify an existing coding sequence or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of the coding sequence. Codon optimization includes processes that include, but are not limited to, selecting codons for a coding sequence to conform to the codon preference of the expression host organism. For example, to conform to the codon preference of a mammalian, preferably mouse, dog, or human expression host. Codon optimization also eliminates elements that may have an adverse effect on RNA stability and / or translation (e.g., termination sequences, TATA boxes, splice sites, ribosome entry sites, repeats and / or GC-rich sequences, as well as RNA secondary structures or instability motifs). In some embodiments, the codon-optimized sequence is at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more show increased transcription, RNA stability and / or translation.

[0196] CNS and brain As used herein, "central nervous system" or "CNS" refers to the part of the nervous system where sensory impulses are transmitted, from which motor impulses emerge, and which regulates the activity of the entire nervous system, including the brain and spinal cord.

[0197] As used herein, "brain" refers to the central organ of the nervous system and consists of the cerebrum, brainstem and cerebellum. The brain controls most of the body's activities, processes, integrates and coordinates the information received from the sensory organs, and makes decisions about the instructions to be sent to the rest of the body.

[0198] In particular, as used herein, "hypothalamus" refers to the area of the forebrain below the thalamus that regulates both the activity of the autonomic nervous system and the pituitary gland, controls body temperature, thirst in the throat, hunger, and other homeostatic systems, and is involved in sleep and emotional activity. "Hippocampus" as used herein belongs to the limbic system of the cerebrum and plays an important role in the fixation of information from short-term memory to long-term memory and in spatial memory that enables navigation. The hippocampus is located below the cerebral cortex (allocortex) and in the medial temporal lobe in primates. "Cortex" or "cerebral cortex" as used herein is the outer layer of neural tissue of the cerebrum of the brain in humans and other mammals. This plays an important role in memory, attention, perception, recognition, thinking, language and consciousness. " Cerebellum" as used herein refers to the main feature in the hindbrain of all vertebrates. In humans, the cerebellum plays an important role in motor control. The cerebellum also plays a role in some cognitive functions such as attention and language, as well as and may also be involved in the control of fear and pleasure responses. As used herein, "olfactory bulb" refers to the essential structure of the olfactory system (the system dedicated to olfaction). The olfactory bulb transmits information that is further processed by the amygdala, the orbitofrontal cortex (OFC ), as well as the hippocampus, which plays a role in emotion, memory, and learning.

[0199] Memory Memory is generally understood to be the brain's ability to encode, store, and retrieve data or information as needed. Various types or memories have been described. One possible distinction involves sensory memory, short-term memory, and long-term memory. Sensory memory holds sensory information within 1 second after an item is recognized. Short-term (also called working memory) memory typically allows recall for a period of seconds to 1 minute without rehearsal. In contrast, long-term memory can store a much larger amount of information potentially over an unlimited period (up to a full lifetime).

[0200] Another distinction involves procedural memory (or implicit memory) and explicit memory (or declarative memory). Implicit memory is not based on the conscious recollection of information, but rather on latent learning, i.e., remembering how to do something. Explicit (or declarative) memory is the conscious and intentional recall of factual information, previous experiences, and concepts.

[0201] It is also possible to distinguish between recall memory and recognition memory. Recognition refers to the ability to "recognize" an event or information as something familiar, and recall refers to retrieving relevant details from memory.

[0202] Spatial memory is a form of memory that is responsible for recording information regarding one's environment and spatial orientation.​​

[0203] In this document and its claims, the verb "comprising" and its conjugations are used in a non- limiting sense, meaning that the items following the word are included, but items not specifically recited are not excluded. Further, the verb "consisting of" can be replaced by "consisting essentially of", meaning that the gene constructs, expression vectors, or compositions described herein may include (one or more) additional components other than those specifically identified, provided that the (one or more) said additional components do not change the unique features of the present invention.

[0204] References to an element by the indefinite article "a" or "an" do not exclude the possibility that more than one element is present, unless the context clearly requires that there be only one and only one element. Thus, the indefinite article "a" or "an" typically means "at least one".

[0205] As used herein, "at least" a particular value means that value or more. For example, "at least 2" is understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and so on.

[0206] Individual numerical values are stated as approximations, as if the word "about" or "approximately" preceded the value. Similarly, numerical values for various ranges specified in this application are stated as approximations, as if the word "about" or "approximately" preceded both the minimum and maximum values of the stated range, unless otherwise specified. As used herein, a When referring to a value, the terms "about" and "approximately" mean that the disclosed subject matter is apparent and ordinary to a person of ordinary skill in the most closely related field or the field relevant to the scope or elements in question. The amount of spread from a precise numerical boundary depends on many factors. For example, some of the factors that may be considered include the importance of the element and / or the effect that a given amount of variation has on the performance of the claimed subject matter, as well as other considerations known to those of ordinary skill in the art. In the absence of contrary considerations, the words "about" or "approximately", when used in relation to a numerical value (e.g., about 10), preferably mean that the value can be 1% more or less than a given value (of 10).

[0207] As used herein, the term "and / or" means that one or more of the stated cases can occur alone or in combination with at least one of the stated cases to all of the stated cases.

[0208] Each embodiment described herein can be combined with other embodiments described herein, unless otherwise indicated.

[0209] All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for definitions, disclaimers or denials of the subject matter, and except where the incorporated material is inconsistent with the explicit disclosure of this specification (in which case, the language of this disclosure prevails).

[0210] A person of ordinary skill in the art will recognize that materials similar to those described herein can be used in the practice of the invention. ​​​​will recognize many equivalent methods and materials. In fact, the present invention is in no way limited to the methods and materials described.

[0211] The present invention will be further illustrated by the following examples, which should not be construed as limiting the scope of the present invention. by.

Brief Description of the Drawings

[0212]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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Figure 8A

Figure 8B

Figure 9

Figure 10A

Figure 10B

Figure 11

[0213] [Examples] Use an AAV vector to test the effect of insulin on the brain when overexpressed in the brain. Three different experiments were conducted: · Treatment of SAMP8 mice with AAV9-Ins. Dosage: 5x10 10 vg / mouse (Example 1).

[0214] · Treatment of db / db mice with AAV9-Ins. Dosage: 5x10 10 vg / mouse (Example 2).

[0215] · Treatment of SAMP8 mice with AAV1-CAG-hInsAsp and AAV1-CAG-h InsWt. Dosage: 5x10 10 vg / mouse (Example 5).

[0216] Furthermore, the brain transduction efficiency of AAV1-hIns, AAV2-hIns, and AAV9-hIns vectors after intracerebrospinal fluid (CSF) administration in wild-type mice was also examined (Example 3).

[0217] General procedure of the examples Characteristics of the subjects Male SAMP8 / TaHsd (SAMP8), BKS.Cg-+Lepr db / +Lep r db OlaHsd (db / db), and C57Bl / 6J (wild-type) mice were used. For Example 5, SAMR1 / TaHsd (SAMR1) was used. Mice were given ad libitum a standard diet (2018S Teklad Global Diets (registered trademark), Harlan Labs., Inc., Madison, WI, USA) and maintained under a 12-hour light / dark cycle (lights on at 8:00 am) and a stable temperature (22 °C ± 2). For tissue sampling, mice were anesthetized with the inhalation anesthetic isoflurane (IsoFlo (registered trademark), Abbott Laboratories, Abbott Park, IL, USA) and decapitated. The tissues of interest were excised and stored at -80 °C until analysis. All experimental procedures were approved by the Ethics Committee for Animal and Human Experiments of the Universitat Autònoma de Barcelona. Recombinant AAV vectors Single-stranded AAV vectors of serotypes 1, 2, and 9 were produced by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36). For Examples 1-3, cells were cultured in DMEM 10% FBS in 10 roller bottles (850 cm

[0218] (flat bottom; Corning (trademark), Sigma-Aldrich Co., Saint Louis, MO, USA) to 80% confluence and transfected with a plasmid having an expression cassette flanked by AAV2 ITR (SEQ ID NO: 40), the AAV2 rep gene and a helper plasmid having the cap gene of AAV of serotype 1, 2, or 9, respectively, and a plasmid having adenoviral helper functions using calcium phosphate For Examples 1-3, cells were cultured in DMEM 10% FBS in 10 roller bottles (850 cm 2 (flat bottom; Corning (trademark), Sigma-Aldrich Co., Saint Louis, MO, USA) until 80% confluent in DMEM 10% FBS, and transfected with a plasmid having an expression cassette flanked by AAV2 ITR (SEQ ID NO: 40), the AAV2 rep gene and a helper plasmid having the cap gene of AAV of serotype 1, 2, or 9, respectively, and a plasmid having adenoviral helper functions using calcium phosphate For Examples 1-3, cells were cultured in DMEM 10% FBS in 10 roller bottles (850 cm (flat bottom; Corning Transfected by the mu method. The transgenes used were the miR-122a sequence (5'CAAACACCATTGT CACACTCCA3', SEQ ID NO: 7) of four tandem repeats and the miR-1 sequence cloned into the 3' untranslated region of the expression cassette (5'TTACATACTTCTTTACATTCCA3', SEQ ID NO: 8) of four tandem repeats added to the initial enhancer / avian β-actin (CAG) promoter (SEQ ID NO: 22) driven by the human insulin-encoding sequence (SEQ ID NO: 46). For Example 5, cells were cotransfected with a plasmid having an expression cassette flanked by AAV2 ITR (SEQ ID NO: 49 or SEQ ID NO: 50), an AAV2 rep gene and a helper plasmid having serotype 1 of AAV, and a plasmid having adenovirus helper function. The transgenes used were, respectively, driven by the initial enhancer / avian β-actin (CAG) promoter (SEQ ID NO: 22), the human insulin aspartic acid-encoding sequence (SEQ ID NO: 46) containing a furin cleavage site or the human insulin wild-type encoding sequence (SEQ ID NO: 45) containing a furin cleavage site. AAV was purified by an optimized method based on polyethylene glycol precipitation steps and two consecutive cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol dramatically reduced empty AAV capsids as well as DNA and protein impurities (Ayuso, E. et al., 2010. Curr Gene Ther .10(6):423-36). The purified AAV vector was dialyzed against PBS, filtered and stored at -80°C. The titer of the viral genome was determined using a linearized plasmid D as a standard curve .10(6):423-36). The purified AAV vector was dialyzed against PBS, filtered and stored at -80°C. The titer of the viral genome was determined using a linearized plasmid D as a Using NA, quantitative P was performed according to the protocol described for the AAV2 reference standard as determined by CR (Lock M., et al., Hum. Gene Ther. 2010;21:1273-1285). The vector was constructed according to molecular biology techniques well known in the art .

[0219] In vivo CSF administration of AAV vector Mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg), and the skin of the posterior head was shaved from behind the ear to approximately between the scapulae and rinsed with ethanol . The mice were kept in the prone position with their heads tilted slightly downward. A 2 mm rostrocaudal incision was made, and a Hamilton syringe was introduced into the cisterna magna at an angle of 45-55° between the posterior head and the C1 vertebra, and 5 μl of vector diluent was administered. Considering that the CNS is the main target compartment for vector delivery, the same number of vector genomes (vg) / mouse were administered to the mice regardless of body weight (5x10 vg / mouse). 10 .

[0220] RNA analysis Total RNA was obtained from the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb using Tripure isolation reagent ( Roche Diagnostics Corp., Indianapolis, IN, USA), and the RNeasy Mini Kit or RNeasy Mi cro Kit (Qiagen NV, Venlo, NL) for hippocampal samples. To eliminate residual viral genomes, total RNA was treated with DNaseI (Qiagen NV, Ven lo, NL). For RT-PCR analysis, Transcriptor Firs ​​​t Strand cDNA Synthesis Kit (04379012001, Roche, Cal ifornia, USA) was used to reverse-transcribe 1 μg of RNA sample. Real-time quantitative PCR was performed on a LightCycler 480 II (Roche, Mannheim, Ge rmany) using TB Green Premix Ex TaqII (Takara Bio Europe, France). Data were normalized with the Rplp0 value and analyzed as described previously (Pfaffl, M., Nucleic Acids Re s. 2001;29(9):e45).

[0221] Vector biodistribution The hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb were digested overnight with proteinase K (0.2 mg / mL). Total DNA was isolated using the MasterPure DNA Purification Kit (Epicenter Biotechnologies, Madison, WI , USA). The vector genomic copy number was determined by TaqMan qPCR using primers and probes specific for human insulin on 20 ng of genomic D NA. The vector genome per sample was interpolated from a standard curve generated by serial dilution of a linearized plasmid with a target sequence spiked with 20 ng of non-transduced genomic DNA.

[0222] Novel object recognition test. The novel object recognition test was performed in an open-field box. The open-field test was used to acclimatize the mice to the box. The next day, for the first trial, two identical objects (A and B) were placed in the upper right and upper left quadrants of the box, and then the mice were allowed to explore both objects ​​​​​​It was placed behind. After 10 minutes of exploration, the mouse was taken out of the box and allowed to rest for 10 minutes. In the second trial, one of the same objects (A or B) was replaced with object C (a new object). Then, the mouse was put back into the box to be explored for another 10 minutes to evaluate short-term memory . 24 hours after the second trial, object C was replaced with a new object (D) to conduct the third trial. Then, the mouse was put back into the box to be explored for another 10 minutes to evaluate long-term memory . The time spent by the animal exploring the new object was recorded and evaluated using a video tracking system (SMART Junior; Panlab). The evaluation of memory in the novel object recognition test was expressed as the percentage of the discrimination ratio calculated according to the following formula. Discrimination ratio (%) = (N - F) / (N + F) × 100% (where N represents the time spent exploring the new object and F represents the time spent exploring the same object).

[0223] Morris water maze. The mouse was trained to find a submerged platform (10 cm in diameter) in a water tank (1 m in diameter, temperature 26 - 28 °C) by swimming and relying on external visual cues. 5-day procedure: Familiarization (day 1), the mouse was placed on the visible platform and then allowed to move freely for 30 seconds. Then, in two consecutive trials, the mouse was placed into the maze from two different starting points. If the mouse did not reach the platform within 60 seconds, it was guided to the platform. The latency to reach the visible platform was measured; in training (days 2 - 4), the mouse was randomly placed in various maze quadrants. The latency to reach the hidden platform (placed in the "correct" quadrant) was measured using a 60-second cutoff. That is, it was measured twice per session in two sessions per day (with one hour between sessions). In the test (on the 5th day), a probe trial was conducted following the last session of training. The hidden platform was removed, the mouse was placed in the center of the pool, and the latency until it crossed the area where the platform was located was measured using a video tracking system (Viewpoint, France); In Examples 1 to 4, the nucleotide sequence of the human (H. sapiens) insulin mutant His - B10 - Asp with a furin cleavage site (hInsAsp; SEQ ID NO: 46) was used. In Example 5, both the nucleotide sequence of the human (H. sapiens) insulin mutant His - B10 - Asp with a furin cleavage site (hInsAsp; SEQ ID NO: 46) and the nucleotide sequence of the human (H. sapiens) insulin wild - type with a furin cleavage site (hInswt; SEQ ID NO: 45) were used. The genetically engineered furin endoprotease cleavage site enables very efficient production of mature insulin in non - pancreatic tissues; 85 - 93% of the total insulin production is mature insulin

[0224] (Gros et al., Hum Gene Ther. 1997 Dec 10; 8(18):2249 - 59; Gros et al. Hum Gene The r. 1999 May 1; 10(7):1207 - 17 and Riu et al. Dia betes. 2002 Mar; 51(3):704 - 11). Furin is known to be present in various brain regions (Foti et al. Gene Ther. 200 9 November; 16(11):1314 - 1319), and furin cleavage in this organ ​​​It becomes possible to efficiently produce mature insulin from an array containing the site.

[0225] [Example 1] Reduction of neuroinflammation and increase of neurogenesis in SAMP8 mice by intracerebrospinal fluid (CSF) administration of AAV9-CAG-hIns-dmiRT vector Therapeutic potential of AAV-mediated gene engineering of the brain by insulin on neuroinflammation and neurogenesis was evaluated. For this purpose, senescence-accelerated mouse-prone 8 (SAMP8) mice, a widely used mouse model of aging with age-related brain pathologies such as neuroinflammation, were used (Takeda T., Neurochem.Res. 2009, 34(4):639-659; Grinan-Ferre C. et al. Mol.Neurobiol. 2016, 53(4):2435-2450).

[0226] Seven-week-old male SAMP8 mice were locally administered an AAV9 vector encoding human insulin at 5x10^12 vector genomes (vg) / mouse into the CSF through a cisterna magna under the control of a CAG ubiquitous promoter (AAV9-CAG-hIns-dmiRT) containing target sites for liver-specific miR122 and heart-specific miR1. As a control, untreated SAMP8 animals were used. At 21 weeks of age, the animals were euthanized and tissue samples were collected for analysis. 10

[0227] Intracerebrospinal fluid (CSF) administration of the AAV9-CAG-hIns-dmiRT vector mediated widespread overexpression of insulin in the brain, as evidenced by increased expression levels of human insulin in various regions of the brain such as the hypothalamus, cortex, hippocampus, and cerebellum of SAMP8 mice. ​​ Figure 1).

[0228] Neuroinflammation was analyzed through the expression of the inflammatory molecules Nfkb, Il1b, and Il6 in various regions of the brain. Notably, the expression of these inflammatory molecules decreased in all brain regions analyzed (Figure 2). throughout all of the brain regions analyzed decreased (Figure 2).

[0229] The expression of the astrocyte markers Gfap and S100b was analyzed. SAMP8 mice treated intracerebroventricularly with the AAV9-CAG-h Ins-dmiRT vector showed increased expression of Gfap in the hypothalamus, cortex, hippocampus, and cerebellum (Figure 3), as well as increased expression of S100b in the cortex (Figure 3). Astrocytes can secrete neurotransmitters and ATP, which can regulate the activity of nearby neurons (Cai W. et al. Journal of Clinical Investigation 2018, 128(7) :2914-2926). Therefore, an increase in the number of astrocytes can support neuronal activity and result in an anxiolytic effect and antidepressant action. Furthermore, the decrease in inflammatory markers accompanying the increase in astrocyte markers further indicates that the population of astrocytes that increases after insulin gene therapy treatment is a population of "beneficial astrocytes" also called "A2 astrocytes". an anxiolytic effect and antidepressant action a population of "beneficial astrocytes" also called "A2 astrocytes". Furthermore, the decrease in inflammatory markers accompanying the increase in astrocyte markers further indicates that the population of astrocytes that increases after insulin gene therapy treatment is .

[0230] To test neurogenesis in SAMP8-treated mice, real-time PCR of neuronal markers was performed. The expression of doublecortin (Dcx), neural cell adhesion molecule (Ncam), and sex-determining region Y-box 2 (Sox2) was increased in the cortex of AAV9-CAG-hIns-dmiRT-treated mice (Figure 4). mice (Figure 4).

[0231] [Example 2] Reduction of neuroinflammation in db / db mice by intracerebrospinal fluid (CSF) administration of AAV9-CAG-hIns-dmiRT vector The effect of insulin on obesity and / or diabetes-related neuroinflammation in db / db mice was evaluated. db / db mice are a widely used genetic mouse model of obesity and diabetes characterized by a deficiency in leptin signaling. Furthermore, these mice exhibit inflammation not only in peripheral tissues such as adipose tissue and liver but also in the brain (Dey et al, J.Neuroimmmunol. 2014). For this purpose, 7-week-old male db / db mice were administered 5x10 vg / mouse of AAV9-CAG-hIns-dmiRT vector through the cisterna magna into the CSF. As a control, untreated db / db animals were used. At 19 weeks of age, the animals were euthanized and tissue samples were collected for analysis.

[0232] 10 vg / mouse of AAV9-CAG-hIns-dmiRT vector through the cisterna magna into the CSF. As a control, untreated db / db animals were used. At 19 weeks of age, the animals were euthanized and tissue samples were collected for analysis.

[0233] Similar to the observations made in SAMP8 mice, intracerebrospinal fluid (CSF) administration of the AAV9-CAG-hIns-dmiRT vector mediated strong overexpression of insulin in the hypothalamus, cortex, hippocampus and cerebellum of db / db mice (Figure 5).

[0234] In db / db mice treated with the vector encoding insulin, the expression of the pro-inflammatory molecules Nfkb, Il1b and Il6 was reduced in all brain regions analyzed (Figure 6). Furthermore, db / db treated mice showed increased expression of the astrocyte marker Gfap in the hypothalamus, cortex and hippocampus, as well as increased expression of the astrocyte marker S100b in the cortex. ​​​​​​​​​​as shown in (Figure 7). Astrocytes can secrete neurotransmitters and ATP, which can regulate the activity of nearby neurons (Cai W.et al. Journal of Clinical Investigation 2018,128(7):29 14-2926). Therefore, an increase in the number of astrocytes can support neuronal activity and bring about anxiolytic and antidepressant effects. Furthermore, the decrease in inflammatory

[0235] [Example 3] AAV1-CAG-hIns-dmiRT, AAV2-CAG-hIns-dmiRT and brain transduction after intracerebrospinal fluid (CSF) administration of the AAV9-CAG-hIns-dmiRT vector . To investigate whether various AAV serotypes can efficiently transduce the brain, wild-type mice were intracerebrospinal fluid (CSF)-treated with AAV1, AAV2, and AAV9 vectors encoding the human insulin coding sequence under the control of the CAG ubiquitin promoter containing the target sites for liver-specific miR-122a and heart-specific miR-1 at 5x10 10 vg / mouse (AAV1-CAG-hIns-dmiRT, AAV2-CAG-hIns-dmiRT, and AAV9- CAG-hIns-dmiRT, respectively). As a control, untreated wild-type mice were used. Three weeks after intracerebrospinal fluid (CSF) administration of the AAV vector, brain samples were collected and vector genome copy number and human insulin expression were measured. As shown in Figure 8, AAV1, AAV2, and

[0236] AAV9 efficiently transduced the brain, as measured by both vector genome copy number and human insulin expression. After intracerebroventricular administration of AAV9, transduction in the hypothalamus, cortex, hippocampus, and cerebellum (Figure 8A), as well as expression of hIns in the same brain regions (Figure 8B), were observed.

[0237] [Example 4] Intracerebroventricular administration of the AAV1-CAG-hIns vector in an Alzheimer's disease mouse model into the cerebrospinal fluid. To evaluate the therapeutic potential of insulin-induced AAV-mediated gene engineering of the brain for Alzheimer's disease, 3xTg-AD (B6;129Tg(APPSwe, TauP301L) 1Lfa Psen1 tm1Mpm ) mouse models are used. 3xTg-AD is a widely used mouse model of Alzheimer's disease that is homozygous for all three mutant alleles, homozygous for the Psen1 mutation, and homozygous for the co-injected APPSwe and tauP301 L transgenes (Belfiore, R., Aging Cell. 2 019, 18(1):e12873).

[0238] 3xTg-AD mice are locally administered, through the cisterna magna, with an AAV1 vector encoding human insulin at 5x10 10 v g / mouse into the cerebrospinal fluid. As a control, untreated 3xTg-AD animals are used. Several behavioral tests, such as the Y-maze, open field and Morris water maze, are performed on these mice. At 1 2 months of age, the animals are euthanized and serum and tissue samples are collected for analysis.

[0239] Analysis of these samples includes neurogenesis (expression of neuronal markers such as Sox2, NeuN, and Dcx), neuroinflammation (GFAP, Iba1, and several cytokine levels ), ​​Tests on the expression of amyloid β (soluble amyloid and plaques), and the levels of amyloid β synaptic degeneration (protein levels of synaptophysin and spine density), and tests on the level of taurin phosphorylation are included.

[0240] [Example 5] Short-term and long-term memory and learning ability improvement in SAMP8 mice by intracerebroventricular administration of AAV1-CAG-hInsAsp and AAV1-CAG-hInsWt vectors The potential of insulin-mediated brain AAV gene engineering treatment for cognitive function decline was evaluated. For this purpose, an SAMP8 mouse model showing cognitive function decline by 8 - 12 months of age was used (Miyamoto, M., Physiol Behav. 1986;3 8(3):399 - 406; Markowska, A.L., Physiol Behav .1998;64(1):15 - 26). Male SAMP8 mice at 7 weeks of age were locally administered with AAV1 vectors encoding the human insulin aspartic acid or human insulin wild-type coding sequence (AAV1-CAG-hInsAsp and AAV1- CAG-hInsWt vectors) at 5×10 10 vg / mouse through the cerebral ventricle under the control of the CAG ubiquitously promoter. As controls, untreated SAMP8 animals and untreated SAM Resistant 1 (SAMR1) animals were used.

[0241] 10 vg / mouse of the human insulin aspartic acid or human insulin wild-type coding sequence under the control of the CAG ubiquitously promoter. As controls, untreated SAMP8 animals and untreated SAM Resistant 1 (SAMR1) animals were used. CAG-hInsWt vectors) through the cerebral ventricle under the control of the CAG ubiquitously promoter. As controls, untreated SAMP8 animals and untreated SAM Resistant 1 (SAMR1) animals were used. CAG-hInsWt vectors) were locally administered into the CSF through the cerebral ventricle. As controls, untreated SAMP8 animals and untreated SAM Resistant 1 (SAMR1) animals were used. Untreated SAMP8 animals and untreated SAM / Resistant 1 (SAMR1) animals were used as controls.

[0242] As demonstrated by the increased expression levels of human insulin in various regions of the brain such as the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb of 41-week-old SAMP8 mice, AAV1-C As demonstrated by the increased expression levels of human insulin in various regions of the brain such as the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb of 41-week-old SAMP8 mice, AAV1-C CSF of the AG-hInsAsp vector and the AAV1-CAG-hInswt vector Intracerebroventricular administration mediated widespread overexpression of insulin in the brain (Figure 9).

[0243] To test the effect of CSF treatment with a viral vector encoding insulin on memory, a novel object recognition test was performed at 33 weeks of age. SAMP8 mice treated with either the vector encoding AAV1-CAG-hInsAsp or the vector encoding AAV1-CAG-hInsWt functioned significantly better than the untreated SAMP8 cohort (Figure 10), and their discrimination indices were similar to those of SAMR1 untreated control mice both 10 minutes (Figure 10A) and 24 hours (Figure 10B) after the first trial, indicating increased short-term and long-term memory after gene therapy. When learning ability was evaluated using the Morris water maze test in 39-week-old AAV1-CAG-hInsWt mice, the latency until the treated mice first entered the target home was shortened (Figure 11) in SAMP8 mice after gene therapy treatment, indicating that administration of AAV1-CAG -hInsWt into the CNS enhances the learning ability of SAMP8 mice.

[0244] When learning ability was evaluated using the Morris water maze test in 39-week-old AAV1-CAG-hInsWt mice, the latency until the treated mice first entered the target home was shortened (Figure 11) in SAMP8 mice after gene therapy treatment, indicating that administration of AAV1-CAG -hInsWt into the CNS enhances the learning ability of SAMP8 mice. -hInsWt into the CNS enhances the learning ability of SAMP8 mice.

Table 5

[0245] Nucleotide sequence of human (H. sapiens) insulin (SEQ ID NO: 4) ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTG CTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGA ​​​ACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTA CCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAG ACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGG AGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTT GGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAA CAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGA ACTACTGCAACTAG Amino acid sequence of human (H. sapiens) insulin (SEQ ID NO: 1) MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEAL YLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQP LALEGSLQKRGIVEQCCTSICSLYQLENYCN Nucleotide sequence of human (H. sapiens) insulin having a furin cleavage site (SEQ ID NO: 45) sequence(SEQ ID NO: 45) ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTG CTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGA ACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTA CCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAGG ACCAAGCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGG AGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTT GGCCCTGGAGGGGTCGCGACAGAAGCGTGGCATTGTGGAA CAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGA ACTACTGCAACTAG Amino acid sequence of human (H. sapiens) insulin with a furin cleavage site ( SEQ ID NO: 41) MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEAL YLVCGERGFFYTPRTKREAEDLQVGQVELGGGPGAGSLQP LALEGSRQKRGIVEQCCTSICSLYQLENYCN Amino acid sequence of human (H. sapiens) insulin mutant (His- B10-Asp) with a furin cleavage site (SEQ ID NO: 46) ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTG CTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGA ACCAACACCTGTGCGGCTCAGATCTGGTGGAAGCTCTCTA CCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAGG ACCAAGCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGG AGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTT GGCCCTGGAGGGGTCGCGACAGAAGCGTGGCATTGTGGAA CAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGA ACTACTGCAACTAG Amino acid sequence (SEQ ID NO: 42) of a human (H. sapiens) insulin variant (His- B10-Asp) having a furin cleavage site MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSDLVEAL YLVCGERGFFYTPRTKREAEDLQVGQVELGGGPGAGSLQP LALEGSRQKRGIVEQCCTSICSLYQLENYCN Nucleotide sequence of mouse (M. musculus) insulin (SEQ ID NO: 5) ATGGCCCTGTGGATGCGCTTCCTGCCCCTGCTGGCCCTG CTCTTCCTCTGGGAGTCCCACCCCACCCAGGCTTTTGTCA AGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTA CCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATG TCCCGCCGTGAAGTGGAGGACCCACAAGTGGCACAACTGG AGCTGGGTGGAGGCCCGGGAGCAGGTGACCTTCAGACCTT GGCACTGGAGGTGGCCCAGCAGAAGCGTGGCATTGTAGAT CAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGA ACTACTGCAACTAG Amino acid sequence of mouse (M. musculus) insulin (SEQ ID NO: 2) MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEAL YLVCGERGFFYTPMSRREVEDPQVAQLELGGGPGAGDLQT LALEVAQQKRGIVDQCCTSICSLYQLENYCN Nucleotide sequence of rat (R. norvegicus) insulin (SEQ ID NO: 47) atggccctgtggatccgcttcctgcccctgctggccctg ctcatcctctgggagccccgccctgcccaggcttttgtca aacagcacctttgtggttctcacttggtggaagctctcta cctggtgtgtggggagcgtggattcttctacacacccatg tcccgccgcgaagtggaggacccacaagtggcacaactgg agctgggtggaggcccgggggcaggtgaccttcagacctt ggcactggaggtggcccggcagaagcgcggcatcgtggat cagtgctgcaccagcatctgctctctctaccaactggaga actactgcaactag Amino acid sequence of rat (R. norvegicus) insulin (SEQ ID NO: 43) MALWIRFLPLLALLILWEPRPAQAFVKQHLCGSHLVEAL YLVCGERGFFYTPMSRREVEDPQVAQLELGGGPGAGDLQT LALEVARQKRGIVDQCCTSICSLYQLENYCN Nucleotide sequence of canine (C. lupus familiaris) insulin (SEQ ID NO: 6) atggccctctggatgcgcctcctgcccctgctggccctg ctggccctctgggcgcccgcgcccacccgagccttcgtta accagcacctgtgtggctcccacctggtagaggctctgta cctggtgtgcggggagcgcggcttcttctacacgcctaag gcccgccgggaggtggaggacctgcaggtgagggacgtgg agctggccggggcgcctggcgagggcggcctgcagcccct ggccctggagggggccctgcagaagcgaggcatcgtggag cagtgctgcaccagcatctgctccctctaccagctggaga attactgcaactag Amino acid sequence of canine (C. lupus familiaris) insulin (SEQ ID NO: 3) MALWMRLLPLLALLALWAPAPTRAFVNQHLCGSHLVEAL YLVCGERGFFYTPKARREVEDLQVRDVELAGAPGEGGLQP LALEGALQKRGIVEQCCTSICSLYQLENYCN Nucleotide sequence of chimpanzee (P. troglodytes) insulin (SEQ ID No. 48) atggccctgtggatgcgcctcctgcccctgctggtgctg ctggccctctggggacctgacccagcctcggcctttgtga accaacacctgtgcggctcccacctggtggaagctctcta cctagtgtgcggggaacgaggcttcttctacacacccaag acccgccgggaggcagaggacctgcaggtggggcaggtgg agctgggcgggggccctggtgcaggcagcctgcagccctt ggccctggaggggtccctgcagaagcgtggtatcgtggaa caatgctgtaccagcatctgctccctctaccagctggaga actactgcaactag Amino acid sequence of chimpanzee (P. troglodytes) insulin (SEQ ID No. 44) MALWMRLLPLLVLLALWGPDPASAFVNQHLCGSHLVEAL YLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQP LALEGSLQKRGIVEQCCTSICSLYQLENYCN Nucleotide sequence of CAG promoter (SEQ ID No. 22) gacattgattattgactagttattaatagtaatcaatta cggggtcattagttcatagcccatatatggagttccgcgt tacataacttacggtaaatggcccgcctggctgaccgccc aacgacccccgcccattgacgtcaataatgacgtatgttc ccatagtaacgccaatagggactttccattgacgtcaatg ggtggagtatttacggtaaactgcccacttggcagtacat caagtgtatcatatgccaagtacgccccctattgacgtca atgacggtaaatggcccgcctggcattatgcccagtacat gaccttatgggactttcctacttggcagtacatctacgta ttagtcatcgctattaccatggtcgaggtgagccccacgt tctgcttcactctccccatctcccccccctccccaccccc aattttgtatttatttattttttaattattttgtgcagcg atgggggcggggggggggggggggcgcgcgccaggcgggg cggggcggggcgaggggcggggcggggcgaggcggagagg tgcggcggcagccaatcagagcggcgcgctccgaaagttt ccttttatggcgaggcggcggcggcggcggccctataaaa agcgaagcgcgcggcgggcgggagtcgctgcgttgccttc gccccgtgccccgctccgcgccgcctcgcgccgcccgccc cggctctgactgaccgcgttactcccacaggtgagcgggc gggacggcccttctcctccgggctgtaattagcgcttggt ttaatgacggcttgtttcttttctgtggctgcgtgaaagc cttgaggggctccgggagggccctttgtgcggggggagcg gctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgc cgcgtgcggctccgcgctgcccggcggctgtgagcgctgc gggcgcggcgcggggctttgtgcgctccgcagtgtgcgcg aggggagcgcggccgggggcggtgccccgcggtgcggggg gctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgt gggggggtgagcagggggtgtgggcgcgtcggtcgggctg caaccccccctgcacccccctccccgagttgctgagcacg gcccggcttcgggtgcggggctccgtacggggcgtggcgc ggggctcgccgtgccgggcggggggtggcggcaggtgggg gtgccgggcggggcggggccgcctcgggccggggagggct cgggggaggggcgcggcggcccccggagcgccggcggctg tcgaggcgcggcgagccgcagccattgccttttatggtaa tcgtgcgagagggcgcagggacttcctttgtcccaaatct gtgcggagccgaaatctgggaggcgccgccgcaccccctc tagcgggcgcggggcgaagcggtgcggcgccggcaggaag gaaatgggcggggagggccttcgtgcgtcgccgcgccgcc gtccccttctccctctccagcctcggggctgtccgcgggg ggacggctgccttcgggggggacggggcagggcggggttc ggcttctggcgtgtgaccggcggctctagagcctctgcta accatgttcatgccttcttctttttcctacag Nucleotide sequence of CMV promoter (SEQ ID NO: 23) gtgatgcggttttggcagtacaccaatgggcgtggatag cggtttgactcacggggatttccaagtctccaccccattg acgtcaatgggagtttgttttggcaccaaaatcaacggga ctttccaaaatgtcgtaacaactgcgatcgcccgccccgt tgacgcaaatgggcggtaggcgtgtacggtgggaggtcta tataagcagagct Nucleotide sequence of CMV enhancer (SEQ ID NO: 24) ggcattgattattgactagttattaatagtaatcaatta cggggtcattagttcatagcccatatatggagttccgcgt tacataacttacggtaaatggcccgcctggctgaccgccc aacgacccccgcccattgacgtcaataatgacgtatgttc ccatagtaacgccaatagggactttccattgacgtcaatg ggtggagtatttacggtaaactgcccacttggcagtacat caagtgtatcatatgccaagtccgccccctattgacgtca atgacggtaaatggcccgcctggcattatgcccagtacat gaccttacgggactttcctacttggcagtacatctacgta ttagtcatcgctattaccatg CMV promoter and CMV enhancer sequence (SEQ ID NO: 39) ggcattgattattgactagttattaatagtaatcaatta cggggtcattagttcatagcccatatatggagttccgcgt tacataacttacggtaaatggcccgcctggctgaccgccc aacgacccccgcccattgacgtcaataatgacgtatgttc ccatagtaacgccaatagggactttccattgacgtcaatg ggtggagtatttacggtaaactgcccacttggcagtacat caagtgtatcatatgccaagtccgccccctattgacgtca atgacggtaaatggcccgcctggcattatgcccagtacat gaccttacgggactttcctacttggcagtacatctacgta ttagtcatcgctattaccatggtgatgcggttttggcagt acaccaatgggcgtggatagcggtttgactcacggggatt tccaagtctccaccccattgacgtcaatgggagtttgttt tggcaccaaaatcaacgggactttccaaaatgtcgtaaca actgcgatcgcccgccccgttgacgcaaatgggcggtagg cgtgtacggtgggaggtctatataagcagagct Truncated AAV2 5’ ITR (SEQ ID NO: 35) gcgcgctc gctcgctcac tgaggccgcc cgggcaaa gc ccgggcgtcg ggcgaccttt ggtcgcccgg cctc agtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct Cleaved AAV2 3’ ITR (SEQ ID NO: 36) aggaacccct agtgatggag ttggccactc cctctc tgcg cgctcgctcg ctcactgagg ccgggcgacc aa aggtcgcc cgacgcccgg gctttgcccg ggcggcctc a gtgagcgagc gagcgcgc Rabbit β-globin polyadenylation signal (including 3’ UTR and polyA signal) Adjacent region of rabbit β-globin (SEQ ID NO: 38) gatctttttccctctgccaaaaattatggggacatcatg aagccccttgagcatctgacttctggctaataaaggaaat ttattttcattgcaatagtgtgttggaattttttgtgtct ctcactcggaaggacatatgggagggcaaatcatttaaaa catcagaatgagtatttggtttagagtttggcaacatatg cccatatgctggctgccatgaacaaaggttggctataaag aggtcatcagtatatgaaacagccccctgctgtccattcc ttattccatagaaaagccttgacttgaggttagatttttt ttatattttgttttgtgttatttttttctttaacatccct aaaattttccttacatgttttactagccagatttttcctc ctctcctgactactcccagtcatagctgtccctcttctct tatggagatc miRT sequence miRT-122a (Accession No. 7): 5’ CAAACACCATTGTCACACT CCA 3’, target of microRNA-122a (miRBase database accession number MI0000442) expressed in the liver.

[0246] miRT-152 (Accession No. 9): 5’ CCAAGTTCTGTCATGCACTG A 3’, target of microRNA-152 (MI0000462) expressed in the liver.

[0247] miRT-199a-5p (Accession No. 10): 5’ GAACAGGTAGTCTGA ACACTGGG 3’, target of microRNA 199a (MI00002 42) expressed in the liver.

[0248] miRT-199a-3p (Accession No. 11): 5’ TAACCAATGTGCAGA CTACTGT 3’, target of microRNA-199a (MI000024 2) expressed in the liver.

[0249] miRT-215 (Accession No. 12): 5’ GTCTGTCAATTCATAGGTC AT 3’, target of microRNA-215 (MI0000291) expressed in the liver.

[0250] miRT-192 (Accession No. 13): 5’ GGCTGTCAATTCATAGGTC AG 3’, target of microRNA-192 (MI0000234) expressed in the liver.

[0251] miRT-148a (Accession No. 14): 5’ ACAAAGTTCTGTAGTGCA Target of CTGA 3’ and microRNA-148a (MI0000253) expressed in the liver Target.

[0252] miRT-194 (SEQ ID NO: 15): 5’ TCCACATGGAGTTGCTGTT ACA 3’, target of microRNA-194 (MI0000488) expressed in the liver .

[0253] miRT-133a (SEQ ID NO: 16): 5’ CAGCTGGTTGAAGGGGAC CAAA 3’, target of microRNA-133a (MI0000450) expressed in the heart Target.

[0254] miRT-206 (SEQ ID NO: 17): 5’ CCACACACTTCCTTACATT CCA 3’, target of microRNA-206 (MI0000490) expressed in the heart .

[0255] miRT-1 (SEQ ID NO: 8): 5’ TTACATACTTCTTTACATTCCA 3’, target of microRNA-1 (MI0000651) expressed in the heart.

[0256] miRT-208a-5p (SEQ ID NO: 18): 5’ GTATAACCCGGGCCA AAAGCTC 3’, target of microRNA-208a (MI000025 1) expressed in the heart

[0257] miRT-208a-3p (SEQ ID NO: 19): 5’ ACAAGCTTTTTGCTC GTCTTAT 3’, target of microRNA-208a (MI000025 1) expressed in the heart

[0258] miRT-499-5p (SEQ ID NO: 20): 5’ AAACATCACTGCAAGT CTTAA 3’, a target of microRNA-499 (MI0003183) expressed in the heart Target

[0259] Mini CMV: cmv intermediate early promoter (SEQ ID NO: 25) tatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgg gactttcctacttggcagtacatctacgtattagtcatcg ctattaccatggtgatgcggttttggcagtacatcaatgg gcgtggatagcggtttgactcacggggatttccaagtctc caccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgcccc attgacgcaaatgggcggtaggcgtgtacggtgggaggtc tatataagcagagctctctggctaactagagaacccactg cttaactggcttatcgaaattaatacgactcactataggg agacccaagctt Nucleotide sequence of the EF1α promoter (SEQ ID NO: 26) ggctccggtgcccgtcagtgggcagagcgcacatcgccc acagtccccgagaagttggggggaggggtcggcaattgaa ccggtgcctagagaaggtggcgcggggtaaactgggaaag tgatgtcgtgtactggctccgcctttttcccgagggtggg ggagaaccgtatataagtgcagtagtcgccgtgaacgttc tttttcgcaacgggtttgccgccagaacacaggtaagtgc cgtgtgtggttcccgcgggcctggcctctttacgggttat ggcccttgcgtgccttgaattacttccactggctgcagta cgtgattcttgatcccgagcttcgggttggaagtgggtgg gagagttcgaggccttgcgcttaaggagccccttcgcctc gtgcttgagttgaggcctggcctgggcgctggggccgccg cgtgcgaatctggtggcaccttcgcgcctgtctcgctgct ttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgc gggccaagatctgcacactggtatttcggtttttggggcc gcgggcggcgacggggcccgtgcgtcccagcgcacatgtt cggcgaggcggggcctgcgagcgcggccaccgagaatcgg acgggggtagtctcaagctggccggcctgctctggtgcct ggcctcgcgccgccgtgtatcgccccgccctgggcggcaa ggctggcccggtcggcaccagttgcgtgagcggaaagatg gccgcttcccggccctgctgcagggagctcaaaatggagg acgcggcgctcgggagagcgggcgggtgagtcacccacac aaaggaaaagggcctttccgtcctcagccgtcgcttcatg tgactccacggagtaccgggcgccgtccaggcacctcgat tagttctcgagcttttggagtacgtcgtctttaggttggg gggaggggttttatgcgatggagtttccccacactgagtg ggtggagactgaagttaggccagcttggcacttgatgtaa ttctccttggaatttgccctttttgagtttggatcttggt tcattctcaagcctcagacagtggttcaaagtttttttct tccatttcaggtgtcgtga Nucleotide sequence of the RSV promoter (SEQ ID NO: 27) catgtttgacagcttatcatcgcagatccgtatggtgca ctctcagtacaatctgctctgatgccgcatagttaagcca gtatctgctccctgcttgtgtgttggaggtcgctgagtag tgcgcgagcaaaatttaagctacaacaaggcaaggcttga ccgacaattgcatgaagaatctgcttagggttaggcgttt tgcgctgcttcgcgatgtacgggccagatattcgcgtatc tgaggggactagggtgtgtttaggcgaaaagcggggcttc ggttgtacgcggttaggagtcccctcaggatatagtagtt tcgcttttgcatagggagggggaaatgtagtcttatgcaa tactcttgtagtcttgcaacatggtaacgatgagttagca acatgccttacaaggagagaaaaagcaccgtgcatgccga ttggtggaagtaaggtggtacgatcgtgccttattaggaa ggcaacagacgggtctgacatggattggacgaaccactaa attccgcattgcagagatattgtatttaagtgcctagctc gatacaataaacgccatttgaccattcaccacattggtgt gcacctccaagctgggtaccagct Synapsin 1 promoter (SEQ ID NO: 28) ctgcgctctcaggcacgacacgactcctccgctgcccac cgcagactgaggcagcgctgagtcgccggcgccgcagcgc agatggtcgcgcccgtgcccccctatctcgcgcctcgcgt ggtgcggtccggctgggccggcggcggcgcggacgcgacc aaggtggccgggaaggggagtttgcgggggaccggcgagt gacgtcagcgcgccttcagtgctgaggcggcggtggcgcg cgccgccaggcgggggcgaaggcactgtccgcggtgctga agctggcagtgcgcacgcgcctcgccgcatcctgtttccc ctccccctctctgataggggatgcgcaatttggggaatgg gggttgggtgcttgtccagtgggtcggggtcggtcgtcag gtaggcacccccaccccgcctcatcctggtcctaaaaccc acttgcact Calcium / calmodulin-dependent protein kinase II (CaMKII) promoter (SEQ ID NO: 29) taacattatggccttaggtcacttcatctccatggggtt cttcttctgattttctagaaaatgagatgggggtgcagag agcttcctcagtgacctgcccagggtcacatcagaaatgt cagagctagaacttgaactcagattactaatcttaaattc catgccttgggggcatgcaagtacgatatacagaaggagt gaactcattagggcagatgaccaatgagtttaggaaagaa gagtccagggcagggtacatctacaccacccgcccagccc tgggtgagtccagccacgttcacctcattatagttgcctc tctccagtcctaccttgacgggaagcacaagcagaaactg ggacaggagccccaggagaccaaatcttcatggtccctct gggaggatgggtggggagagctgtggcagaggcctcagga ggggccctgctgctcagtggtgacagataggggtgagaaa gcagacagagtcattccgtcagcattctgggtctgtttgg tacttcttctcacgctaaggtggcggtgtgatatgcacaa tggctaaaaagcagggagagctggaaagaaacaaggacag agacagaggccaagtcaaccagaccaattcccagaggaag caaagaaaccattacagagactacaagggggaagggaagg agagatgaattagcttcccctgtaaaccttagaacccagc tgttgccagggcaacggggcaatacctgtctcttcagagg agatgaagttgccagggtaactacatcctgtctttctcaa ggaccatcccagaatgtggcacccactagccgttaccata gcaactgcctctttgccccacttaatcccatcccgtctgt taaaagggccctatagttggaggtgggggaggtaggaaga gcgatgatcacttgtggactaagtttgttcgcatcccctt ctccaaccccctcagtacatcaccctgggggaacagggtc cacttgctcctgggcccacacagtcctgcagtattgtgta tataaggccagggcaaagaggagcaggttttaaagtgaaa ggcaggcaggtgttggggaggcagttaccggggcaacggg aacagggcgtttcggaggtggttgccatggggacctggat gctgacgaaggctcgcgaggctgtgagcagccacagtgcc ctgctcagaagccccaagctcgtcagtcaagccggttctc cgtttgcactcaggagcacgggcaggcgagtggcccctag ttctgggggcagcgggg Glial fibrillary acidic protein (GFAP) promoter (SEQ ID NO: 30) cgcgtgatctaacatatcctggtgtggagtaggggacgc tgctctgacagaggctcgggggcctgagctggctctgtga gctggggaggaggcagacagccaggccttgtctgcaagca gacctggcagcattgggctggccgccccccagggcctcct cttcatgcccagtgaatgactcaccttggcacagacacaa tgttcggggtgggcacagtgcctgcttcccgccgcacccc agcccccctcaaatgccttccgagaagcccattgagcagg gggcttgcattgcaccccagcctgacagcctggcatcttg ggataaaagcagcacagccccctaggggctgcccttgctg tgtggcgccaccggcggtggagaacaaggctctattcagc ctgtgcccaggaaaggggatcaggggatgcccaggcatgg acagtgggtggcagggggggagaggagggctgtctgcttc ccagaagtccaaggacacaaatgggtgaggggagagctct ccccatagctgggctgcggcccaaccccaccccctcaggc tatgccagggggtgttgccaggggcacccgggcatcgcca gtctagcccactccttcataaagccctcgcatcccaggag cgagcagagccagagcaggttggagaggagacgcatcacc tccgctgctcgcggggtctagagtcga Nestin promoter (SEQ ID NO: 31) gaaggcagcccccggaggtcaaaggctgggcacgcggga ggagaggccagagtcagaggctgcgggtatctcagatatg aaggaaagatgagagaggctcaggaagaggtaagaaaaga cacaagagaccagagaagggagaagaattagagagggagg cagaggaccgctgtctctacagacatagctggtagagact gggaggaagggatgaaccctgagcgcatgaagggaaggag gtggctggtggtatatggaggatgtagctgggccagggaa aagatcctgcactaaaaatctgaagctaaaaataacagga cacggggtggagaggcgaaaggagggcagattgaggcaga gagactgagaggcctggggatgtgggcattccggtagggc acacagttcacttgtcttctctttttccaggaggccaaag atgctgacctcaagaactcataataccccagtggggacca ccgcattcatagccctgttacaagaagtgggagatgttcc tttttgtcccagactggaaatccattacatcccgaggctc aggttctgtggtggtcatctctgtgtggcttgttctgtgg gcctacctaaagtcctaagcacagctctcaagcagatccg aggcgactaagatgctagtaggggttgtctggagagaaga gccgaggaggtgggctgtgatggatcagttcagctttcaa ataaaaaggcgtttttatattctgtgtcgagttcgtgaac ccctgtggtgggcttctccatctgtctgggttagtacctg ccactatactggaataaggagacgcctgcttccctcgagt tggctggacaaggttatgagcatccgtgtacttatggggt tgccagcttggtcctggatcgcccgggcccttcccccacc cgttcggttccccaccaccacccgcgctcgtacgtgcgtc tccgcctgcagctcttgactcatcggggcccccgggtcac atgcgctcgctcggctctataggcgccgccccctgcccac cccccgcccgcgctgggagccgcagccgccgccactcctg ctctctctgcgccgccgccgtcaccaccgccaccgccacc ggctgagtctgcagtcctccgaaacgggccctct Homeobox protein 9 promoter (HB9) promoter (SEQ ID NO: 32) tgaataaatttaagcaggctaattaatatataaactagc tcaatttgtcaagttgatttgtattttagttaattgtgaa agtaattaccacatggtcaaattaacagctttctggaaat gaccaagcctgaggttttatttccttcctgggtgaagaaa attcatttttccaagctcttgatgtgatgaataaaagtca taaatctgggtgattggtgcaggcagagtctaaatggctt catatttcattttaggtttaatagaaatattcatgctctg ttttaatgaaattaaattgaagggggatggggctagagtg gttagctgatgaattgacaaaaactaatcagctttattgg gaaacaggtttaagggcacggacgtgtcaataacgctcag cctgaccccctcttccattagctaggcaggctgattaga Tyrosine hydroxylase (TH) promoter (SEQ ID NO: 33) ctgctaggggctgcttcccagctactcctcttggctccg tggcttgccttccagcctgtgtgctgtctggagagccttt aaagcctcacttccaccaactagaagtctctccccaaccc tgccctgacctcaagtgcacctcttcaaagtcaggtttag cagctgcagctgggggccctgaatcccacccctgctgtct tccttgaagacagaagtgttgggagctgaggatctgggct agagactggctgtatgatccagagaagtagtgtgcttctg ggcctcagatttcccttctgtagaacaggtttgtctgaaa tggagaggttggtgctcctctgcagggcctagtgggagtc accatgagtggttaaaagatccagcttgtcttttggtgag ctttgagaggaggtaacagggctgagttctggaagcctga ccaagggcagacttaaggggcctcttggagttgttctcat caaatggggatgggacacagctaaagtgcccagggcttct ctgtgcccacagatgctttagatcttggcacagtgtggtc taccagctgtctctctctgtgtatatatatgtatttcata gacagtgtacagtggcctggtttgtgctatcaggctggat atggacagaggcaagagtttgtggcagcagttatctccca agagagtccaaagacatcatgttttcaagtttaggccagg tgctacttgagagagctcagacacagacaaaggtctggag agcacatgtcctccacccccacctagcttctgttgcaagc acctccagccgagacaagagaacgaattaaaaagcaatat ttgtgtcagtgtaagacatttgccgaaaggttaaatccac attcgtgttgctgcagagcagccccctatgcaggatttgt tagatacagctccgtcctaccctgtgccagctgagcaaac gccaggctgggtggggtggaacccagcctgggtttgcctc accctgcaatccccccagcaccctctaaaggaggaccctg tggtgggcatgcagacctagggactgggcatagataacct ttgggtttgggcaacagcccccactcctcaggattgaagg ctaaggtgcagccagctctgccttcatggtgggaatgtct ccacgtgacccctttctgggctgtggagaacactcagaga agagtcctgggatgccaggcaggccagggatgtgctgggc atgttgagacaggagtgggctaagccagcagagttgctga cccaggaagagttcagaaaggggcatggaacatggggagg ggtccatagtgagagagagcaggcagtgcagagtaaatag tccctgagctgggggttatgggatttgcaggagcttgctc agagaaggcagaggagagatgctgcgccaagctgggtatc acagagcctcagactcctggaacaggaactgtgggggtca ggtcagcaggggaggttagggagtgttccctttgtactga cttagcatttatcctgcttctaggggggaaggggggccag tgggggatgcacagcaaggcagtgatgtggcaggcagcct gcgggagctcctggttcctggtgtgaaaaagctgggaagg aagagggctgggtctggtaagtacagcaggcagttggctc ctgagagtccaagccctgtctagagggtggagtgagattt cagagggagagctaaacggggtgggggctggggagtccag gcttctggctcctgctaatactcagtgtgctgggtcctca gaacctcagggtggccattttcagggtgagagctctgtcc tttggcacttctgcagactccagtatccagaggaataaag atggtactcttcctcagttcccttagtgagaggacacctt tctctgaagggcttgggcagttgtcctgaaccattgcctg aaggaaggacttgactccagggacatagaatgggctcagc ataagtcccctgtagtagagaaaggtcccctctctggtct ccttagagatcctgtttccttggctgaggaagctagggtg gatctttgtgtaagtgggtgtggatgctcactggaaatca aaaggccccttggtgttagaccttggggtgccatgggaga gttgatcactgagtgcgcccttacatgggggccagctgag aatggggctgcctctagctcgagaccatgatgcagggagt gagtgggggagttcaggatactcttaactaaagcagaggt ctgtccccccagggaggggaggtcagaagaccctagggag atgccaaaggctagggttggcaccatgttgcaggctgtgt cttcaaggagatgataatcagaggaatcgaacctgcaaaa gtgggccagtcttagatacactatagaggaataatcttct gaaacattctgtgtctcataggacctgcctgaggacccag ccccagtgccagcacatacactggggcagtgagtagatag tatactttgttacatgggctggggggacatggcctgtgcc ctggaggggacttgaagacatccaaaaagctagtgagagg gctcctagatttatttgtctccaagggctatatatagcct tcctaacatgaacccttgggtaatccagcatgggcgctcc catatgccctggtttgattagagagctctagatgtctcct gtcccagaacaccagccagcccctgtcttcatgtcgtgtc tagggcggagggtgattcagaggcaggtgcctgcgacagt ggatgcaattagatctaatgggacggaggcctctctcgtc cgtcgccctcgctctgtgcccacccccgcctccctcaggc acagcaggcgtggagaggatgcgcaggaggtaggaggtgg gggacccagaggggctttgacgtcagcctggcctttaaga ggccgcctgcctggcaagggccgtggagacagaactcggg accaccagcttgcact Myelin basic protein (MBP) promoter (SEQ ID NO: 34) caccgtggctttaacacttagagaaaatgcatcccctct aatcaataagtcatcgacagtgggtagatggaggaacggc agtgcgtagtaggatgcgtgcaagcatagtctcgtgcatg ggtgcatagatcgctgggcaggtggacaaggtgggggtgg ataaagaagtgggtagatgattgatgttaggtaaatatca ctgggtggacagatgggtggtaggtggatggatggttaga atagtcagaagagggatggattgataaggtgaacagatga taaatgggtgatagactggaagggttgtcaaaagaggata agggaagtgtgagctagccgtatttctaaggtcagtaata gagttgggagaagaggttaagttacatccatttaaacctc acacgaagctgagagggaatggacttgctgccgttggtga ggaaagcgttgcatttcccgtgtgcttggttgtgaagtgc tcaggtcccacatgaagcagtcaggttactgcggcttaca gaggagccagatccaaatgccccgagtaagcacgtccccg agccagaggcctccagcggaatccgggagagggattgctc agtgccctgcttccctggactgtaagctgcagaaagatgt gggaagtcctgttctccactgagaacactaaaagcacctt ttgtcaaacgaccgcttcacatctggggcttgtgcactgg tggccttttaaaccagagacaacccacaagatacctaacc tgcggggctctctggtacagtgagcaactcaggaaatgct ttggcttgattgctgtgggctctcaggccatcgccctctg gagtggttcttttaatgagaacctgaagattggcccctga gccatgtataccaagcaagctcaatccaggttagctccct ctggttggggcaagctaacgtgctccttgggccccgcgcg taactgtgcgttttataggagacagctagttcaagacccc aggaagaaagcggctttgtccccctctaggcctcgtacag gcccacattcatatctcattgttgttgcaggggaggcaga tgcgatccagaacaatgggacctcggctgaggacacggcg gtgacagactccaagcacacagcagacccaaagaataact ggcaaggcgcccacccagctgacccagggaaccgccccca cttgatccgcctcttttcccgagatgccccgggaagggag gacaacaccttcaaagacaggccctcagagtccgacgagc ttcagaccatccaagaagatcccacagcagcttccgaaga attctgcagtcgacggtaccgcgggcccgggatc SV40 polyA signal (SEQ ID NO: 37) taagatacat tgatgagttt ggacaaacca caacta gaat gcagtgaaaatttgtgaaat ttgtgatgct att gctttat ttgtaaccat tataagctgc aataaacaag tt Chimeric intron composed of the intron of human β - globin and the immunoglobulin heavy chain gene intron (SEQ ID NO: 21) gtaagtatca aggttacaag acaggtttaa ggagac caat agaaactggg cttgtcgagacagagaagac tct tgcgttt ctgataggca cctattggtc ttactgacat ccactttgcctttctctcca cag pAAV - CAG - hIns - dmiRT (SEQ ID NO: 40) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAA TCGGCC AACGCGCGGG 51 GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGC TTCCTCG CTCACTGACT 101 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CG GTATCAGC TCACTCAAAG 151 GCGGTAATAC GGTTATCCAC AGAATCAGGG GA TAACGCAG GAAAGAACAT 201 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CC GTAAAAAG GCCGCGTTGC 251 TGGCGTTTTT CCATAGGCTC CGCCCCCCTG AC GAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GG ACTATAAA GATACCAGGC 351 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TC CTGTTCCG ACCCTGCCGC 401 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CG GGAAGCGT GGCGCTTTCT 451 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GT GTAGGTCG TTCGCTCCAA 501 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GC CCGACCGC TGCGCCTTAT 551 CCGGTAACTA TCGTCTTGAG TCCAACCCGG TA AGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AG AGCGAGGT ATGTAGGCGG 651 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CT ACGGCTAC ACTAGAAGAA 701 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CA GTTACCTT CGGAAAAAGA 751 GTTGGTAGCT CTTGATCCGG CAAACAAACC AC CGCTGGTA GCGGTGGTTT 801 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AA AAAAAGGA TCTCAAGAAG 851 ATCCTTTGAT CTTTTCTACG GGGTCTGACG CT CAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AA AAGGATCT TCACCTAGAT 951 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AA TCTAAAGT ATATATGAGT 1001 AAACTTGGTC TGACAGTTAC CAATGCTTAA T CAGTGAGGC ACCTATCTCA 1051 GCGATCTGTC TATTTCGTTC ATCCATAGTT G CCTGACTCC CCGTCGTGTA 1101 GATAACTACG ATACGGGAGG GCTTACCATC T GGCCCCAGT GCTGCAATGA 1151 TACCGCGAGA CCCACGCTCA CCGGCTCCAG A TTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT C CTGCAACTT TATCCGCCTC 1251 CATCCAGTCT ATTAATTGTT GCCGGGAAGC T AGAGTAAGT AGTTCGCCAG 1301 TTAATAGTTT GCGCAACGTT GTTGCCATTG C TACAGGCAT CGTGGTGTCA 1351 CGCTCGTCGT TTGGTATGGC TTCATTCAGC T CCGGTTCCC AACGATCAAG 1401 GCGAGTTACA TGATCCCCCA TGTTGTGCAA A AAAGCGGTT AGCTCCTTCG 1451 GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG C CGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT G TCATGCCAT CCGTAAGATG 1551 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA G TCATTCTGA GAATAGTGTA 1601 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT C AATACGGGA TAATACCGCG 1651 CCACATAGCA GAACTTTAAA AGTGCTCATC A TTGGAAAAC GTTCTTCGGG 1701 GCGAAAACTC TCAAGGATCT TACCGCTGTT G AGATCCAGT TCGATGTAAC 1751 CCACTCGTGC ACCCAACTGA TCTTCAGCAT C TTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT G CCGCAAAAA AGGGAATAAG 1851 GGCGACACGG AAATGTTGAA TACTCATACT C TTCCTTTTT CAATATTATT 1901 GAAGCATTTA TCAGGGTTAT TGTCTCATGA G CGGATACAT ATTTGAATGT 1951 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG C GCACATTTC CCCGAAAAGT 2001 GCCACCTGAC GTCTAAGAAA CCATTATTAT C ATGACATTA ACCTATAAAA 2051 ATAGGCGTAT CACGAGGCCC TTTCGTCTCG C GCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG A CGGTCACAG CTTGTCTGTA 2151 AGCGGATGCC GGGAGCAGAC AAGCCCGTCA G GGCGCGTCA GCGGGTGTTG 2201 GCGGGTGTCG GGGCTGGCTT AACTATGCGG C ATCAGAGCA GATTGTACTG 2251 AGAGTGCACC ATATGCGGTG TGAAATACCG C ACAGATGCG TAAGGAGAAA 2301 ATACCGCATC AGGCGATTCC AACATCCAAT A AATCATACA GGCAAGGCAA 2351 AGAATTAGCA AAATTAAGCA ATAAAGCCTC A GAGCATAAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA C TTTTGCGGG AGAAGCCTTT 2451 ATTTCAACGC AAGGATAAAA ATTTTTAGAA C CCTCATATA TTTTAAATGC 2501 AATGCCTGAG TAATGTGTAG GTAAAGATTC A AACGGGTGA GAAAGGCCGG 2551 AGACAGTCAA ATCACCATCA ATATGATATT C AACCGTTCT AGCTGATAAA 2601 TTCATGCCGG AGAGGGTAGC TATTTTTGAG A GGTCTCTAC AAAGGCTATC 2651 AGGTCATTGC CTGAGAGTCT GGAGCAAACA A GAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC C CCGGTTGAT AATCAGAAAA 2751 GCCCCAAAAA CAGGAAGATT GTATAAGCAA A TATTTAAAT TGTAAGCGTT 2801 AATATTTTGT TAAAATTCGC GTTAAATTTT T GTTAAATCA GCTCATTTTT 2851 TAACCAATAG GCCGAAATCG GCAAAATCCC T TATAAATCA AAAGAATAGA 2901 CCGAGATAGG GTTGAGTGTT GTTCCAGTTT G GAACAAGAG TCCACTATTA 2951 AAGAACGTGG ACTCCAACGT CAAAGGGCGA A AAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC A AGTTTTTTG GGGTCGAGGT 3051 GCCGTAAAGC ACTAAATCGG AACCCTAAAG G GAGCCCCCG ATTTAGAGCT 3101 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA A AGGAAGGGA AGAAAGCGAA 3151 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT A GCGGTCACG CTGCGCGTAA 3201 CCACCACACC CGCCGCGCTT AATGCGCCGC T ACAGGGCGC GTACTATGGT 3251 TGCTTTGACG AGCACGTATA ACGTGCTTTC C TCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT T AGACAGGAA CGGTACGCCA 3351 GAATCCTGAG AAGTGTTTTT ATAATCAGTG A GGCCACCGA GTAAAAGAGT 3401 CTGTCCATCA CGCAAATTAA CCGTTGTCGC A ATACTTCTT TGATTAGTAA 3451 TAACATCACT TGCCTGAGTA GAAGAACTCA A ACTATCGGC CTTGCTGGTA 3501 ATATCCAGAA CAATATTACC GCCAGCCATT G CAACGGAAT CGCCATTCGC 3551 CATTCAGGCT GCGCAACTGT TGGGAAGGGC G ATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC A CTGAGGCCG CCCGGGCAAA 3651 GCCCGGGCGT CGGGCGACCT TTGGTCGCCC G GCCTCAGTG AGCGAGCGAG 3701 CGCGCAGAGA GGGAGTGGCC AACTCCATCA C TAGGGGTTC CTTGTAGTTA 3751 ATGATTAACC CGCCATGCTA CTTATCTACT C GACATTGAT TATTGACTAG 3801 TTATTAATAG TAATCAATTA CGGGGTCATT A GTTCATAGC CCATATATGG 3851 AGTTCCGCGT TACATAACTT ACGGTAAATG G CCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG A CGTATGTTC CCATAGTAAC 3951 GCCAATAGGG ACTTTCCATT GACGTCAATG G GTGGAGTAT TTACGGTAAA 4001 CTGCCCACTT GGCAGTACAT CAAGTGTATC A TATGCCAAG TACGCCCCCT 4051 ATTGACGTCA ATGACGGTAA ATGGCCCGCC T GGCATTATG CCCAGTACAT 4101 GACCTTATGG GACTTTCCTA CTTGGCAGTA C ATCTACGTA TTAGTCATCG 4151 CTATTACCAT GGTCGAGGTG AGCCCCACGT T CTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT T TATTTATTT TTTAATTATT 4251 TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG G GGGCGCGCG CCAGGCGGGG 4301 CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA G GCGGAGAGG TGCGGCGGCA 4351 GCCAATCAGA GCGGCGCGCT CCGAAAGTTT C CTTTTATGG CGAGGCGGCG 4401 GCGGCGGCGG CCCTATAAAA AGCGAAGCGC G CGGCGGGCG GGAGTCGCTG 4451 CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG C CGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG G TGAGCGGGC GGGACGGCCC 4551 TTCTCCTCCG GGCTGTAATT AGCGCTTGGT T TAATGACGG CTTGTTTCTT 4601 TTCTGTGGCT GCGTGAAAGC CTTGAGGGGC T CCGGGAGGG CCCTTTGTGC 4651 GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG T GTGTGTGCG TGGGGAGCGC 4701 CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG T GAGCGCTGC GGGCGCGGCG 4751 CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG A GGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG G AACAAAGGC TGCGTGCGGG 4851 GTGTGTGCGT GGGGGGGTGA GCAGGGGGTG T GGGCGCGTC GGTCGGGCTG 4901 CAACCCCCCC TGCACCCCCC TCCCCGAGTT G CTGAGCACG GCCCGGCTTC 4951 GGGTGCGGGG CTCCGTACGG GGCGTGGCGC G GGGCTCGCC GTGCCGGGCG 5001 GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG G GGCGGGGCC GCCTCGGGCC 5051 GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC C CCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT T TTATGGTAA TCGTGCGAGA 5151 GGGCGCAGGG ACTTCCTTTG TCCCAAATCT G TGCGGAGCC GAAATCTGGG 5201 AGGCGCCGCC GCACCCCCTC TAGCGGGCGC G GGGCGAAGC GGTGCGGCGC 5251 CGGCAGGAAG GAAATGGGCG GGGAGGGCCT T CGTGCGTCG CCGCGCCGCC 5301 GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT G TCCGCGGGG GGACGGCTGC 5351 CTTCGGGGGG GACGGGGCAG GGCGGGGTTC G GCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA T GCCTTCTTC TTTTTCCTAC 5451 AGCTCCTGGG CAACGTGCTG GTTATTGTGC T GTCTCATCA TTTTGGCAAA 5501 GAATTGATTA ATTCGAGCGA ACGCGTCGAG T CGCTCGGTA CGATTTAAAT 5551 TGAATTGGCC TCGAGCGCAA GCTTGAGCTA G GACCTTCTG CCATGGCCCT 5601 GTGGATGCGC CTCCTGCCCC TGCTGGCGCT G CTGGCCCTC TGGGGACCTG 5651 ACCCAGCCGC AGCCTTTGTG AACCAACACC T GTGCGGCTC AGATCTGGTG 5701 GAAGCTCTCT ACCTAGTGTG CGGGGAACGA G GCTTCTTCT ACACACCCAG 5751 GACCAAGCGG GAGGCAGAGG ACCTGCAGGT G GGGCAGGTG GAGCTGGGCG 5801 GGGGCCCTGG TGCAGGCAGC CTGCAGCCCT T GGCCCTGGA GGGGTCGCGA 5851 CAGAAGCGTG GCATTGTGGA ACAATGCTGT A CCAGCATCT GCTCCCTCTA 5901 CCAGCTGGAG AACTACTGCA ACTAGACGCA G CCGTCGGCC GCTAATTCTA 5951 GATCGCGAAC AAACACCATT GTCACACTCC A GTATACACA AACACCATTG 6001 TCACACTCCA GATATCACAA ACACCATTGT C ACACTCCAA GGCGAACAAA 6051 CACCATTGTC ACACTCCAAG GCTATTCTAG A TCGCGAATT ACATACTTCT 6101 TTACATTCCA GTATACATTA CATACTTCTT T ACATTCCAG ATATCATTAC 6151 ATACTTCTTT ACATTCCAAG GCGAATTACA T ACTTCTTTA CATTCCAAGG 6201 CTACCTGAGG CCCGGGGGTA CCTCTTAATT A ACTGGCCTC ATGGGCCTTC 6251 CGCTCACTGC CCGCTTTCCA GTCGGGAAAC C TGTCGTGCC AGTCAGGTGC 6301 AGGCTGCCTA TCAGAAGGTG GTGGCTGGTG T GGCCAATGC CCTGGCTCAC 6351 AAATACCACT GAGATCTTTT TCCCTCTGCC A AAAATTATG GGGACATCAT 6401 GAAGCCCCTT GAGCATCTGA CTTCTGGCTA A TAAAGGAAA TTTATTTTCA 6451 TTGCAATAGT GTGTTGGAAT TTTTTGTGTC T CTCACTCGG AAGGACATAT 6501 GGGAGGGCAA ATCATTTAAA ACATCAGAAT G AGTATTTGG TTTAGAGTTT 6551 GGCAACATAT GCCCATATGC TGGCTGCCAT G AACAAAGGT TGGCTATAAA 6601 GAGGTCATCA GTATATGAAA CAGCCCCCTG C TGTCCATTC CTTATTCCAT 6651 AGAAAAGCCT TGACTTGAGG TTAGATTTTT T TTATATTTT GTTTTGTGTT 6701 ATTTTTTTCT TTAACATCCC TAAAATTTTC C TTACATGTT TTACTAGCCA 6751 GATTTTTCCT CCTCTCCTGA CTACTCCCAG T CATAGCTGT CCCTCTTCTC 6801 TTATGGAGAT CCCTCGACCT GCAGCCCAAG C TGTAGATAA GTAGCATGGC 6851 GGGTTAATCA TTAACTACAA GGAACCCCTA G TGATGGAGT TGGCCACTCC 6901 CTCTCTGCGC GCTCGCTCGC TCACTGAGGC C GGGCGACCA AAGGTCGCCC 6951 GACGCCCGGG CTTTGCCCGG GCGGCCTCAG T GAGCGAGCG AGCGCGCAGC 7001 TGGCGTAA AAV2 5’ ITR: 3612 - 3742 bp CAG promoter: 3779 - 5423 bp h insulin (hIns): 5586 - 5932 bp dmiRT (4 copies of miR - 122a and 4 copies of miR - 1) : 5943 - 6203 bp Rabbit β - globin polyA signal (3’ UTR and the 3’ flanking region of rabbit β globin including the polyA signal): 6293 - 6811 bp AAV2 3’ ITR: 6870 - 7000 bp pAAV - CAG - hInsAsp (SEQ ID NO: 49) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAA TCGGCC AACGCGCGGG GAGAGGCGGT 61 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTC ACTGACT CGCTGCGCTC GGTCGTTCGG 121 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GC GGTAATAC GGTTATCCAC AGAATCAGGG 181 GATAACGCAG GAAAGAACAT GTGAGCAAAA GG CCAGCAAA AGGCCAGGAA CCGTAAAAAG 241 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CG CCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GG ACTATAAA GATACCAGGC GTTTCCCCCT 361 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG AC CCTGCCGC TTACCGGATA CCTGTCCGCC 421 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CA TAGCTCAC GCTGTAGGTA TCTCAGTTCG 481 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GT GCACGAAC CCCCCGTTCA GCCCGACCGC 541 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TC CAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AG AGCGAGGT ATGTAGGCGG TGCTACAGAG 661 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC AC TAGAAGAA CAGTATTTGG TATCTGCGCT 721 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GT TGGTAGCT CTTGATCCGG CAAACAAACC 781 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AA GCAGCAGA TTACGCGCAG AAAAAAAGGA 841 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GG GTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AA AAGGATCT TCACCTAGAT CCTTTTAAAT 961 TAAAAATGAA GTTTTAAATC AATCTAAAGT AT ATATGAGT AAACTTGGTC TGACAGTTAC 1021 CAATGCTTAA TCAGTGAGGC ACCTATCTCA G CGATCTGTC TATTTCGTTC ATCCATAGTT 1081 GCCTGACTCC CCGTCGTGTA GATAACTACG A TACGGGAGG GCTTACCATC TGGCCCCAGT 1141 GCTGCAATGA TACCGCGAGA CCCACGCTCA C CGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT C CTGCAACTT TATCCGCCTC CATCCAGTCT 1261 ATTAATTGTT GCCGGGAAGC TAGAGTAAGT A GTTCGCCAG TTAATAGTTT GCGCAACGTT 1321 GTTGCCATTG CTACAGGCAT CGTGGTGTCA C GCTCGTCGT TTGGTATGGC TTCATTCAGC 1381 TCCGGTTCCC AACGATCAAG GCGAGTTACA T GATCCCCCA TGTTGTGCAA AAAAGCGGTT 1441 AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA A GTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT G TCATGCCAT CCGTAAGATG CTTTTCTGTG 1561 ACTGGTGAGT ACTCAACCAA GTCATTCTGA G AATAGTGTA TGCGGCGACC GAGTTGCTCT 1621 TGCCCGGCGT CAATACGGGA TAATACCGCG C CACATAGCA GAACTTTAAA AGTGCTCATC 1681 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC T CAAGGATCT TACCGCTGTT GAGATCCAGT 1741 TCGATGTAAC CCACTCGTGC ACCCAACTGA T CTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT G CCGCAAAAA AGGGAATAAG GGCGACACGG 1861 AAATGTTGAA TACTCATACT CTTCCTTTTT C AATATTATT GAAGCATTTA TCAGGGTTAT 1921 TGTCTCATGA GCGGATACAT ATTTGAATGT A TTTAGAAAA ATAAACAAAT AGGGGTTCCG 1981 CGCACATTTC CCCGAAAAGT GCCACCTGAC G TCTAAGAAA CCATTATTAT CATGACATTA 2041 ACCTATAAAA ATAGGCGTAT CACGAGGCCC T TTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG A CGGTCACAG CTTGTCTGTA AGCGGATGCC 2161 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA G CGGGTGTTG GCGGGTGTCG GGGCTGGCTT 2221 AACTATGCGG CATCAGAGCA GATTGTACTG A GAGTGCACC ATATGCGGTG TGAAATACCG 2281 CACAGATGCG TAAGGAGAAA ATACCGCATC A GGCGATTCC AACATCCAAT AAATCATACA 2341 GGCAAGGCAA AGAATTAGCA AAATTAAGCA A TAAAGCCTC AGAGCATAAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA C TTTTGCGGG AGAAGCCTTT ATTTCAACGC 2461 AAGGATAAAA ATTTTTAGAA CCCTCATATA T TTTAAATGC AATGCCTGAG TAATGTGTAG 2521 GTAAAGATTC AAACGGGTGA GAAAGGCCGG A GACAGTCAA ATCACCATCA ATATGATATT 2581 CAACCGTTCT AGCTGATAAA TTCATGCCGG A GAGGGTAGC TATTTTTGAG AGGTCTCTAC 2641 AAAGGCTATC AGGTCATTGC CTGAGAGTCT G GAGCAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC C CCGGTTGAT AATCAGAAAA GCCCCAAAAA 2761 CAGGAAGATT GTATAAGCAA ATATTTAAAT T GTAAGCGTT AATATTTTGT TAAAATTCGC 2821 GTTAAATTTT TGTTAAATCA GCTCATTTTT T AACCAATAG GCCGAAATCG GCAAAATCCC 2881 TTATAAATCA AAAGAATAGA CCGAGATAGG G TTGAGTGTT GTTCCAGTTT GGAACAAGAG 2941 TCCACTATTA AAGAACGTGG ACTCCAACGT C AAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC A AGTTTTTTG GGGTCGAGGT GCCGTAAAGC 3061 ACTAAATCGG AACCCTAAAG GGAGCCCCCG A TTTAGAGCT TGACGGGGAA AGCCGGCGAA 3121 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA A GGAGCGGGC GCTAGGGCGC TGGCAAGTGT 3181 AGCGGTCACG CTGCGCGTAA CCACCACACC C GCCGCGCTT AATGCGCCGC TACAGGGCGC 3241 GTACTATGGT TGCTTTGACG AGCACGTATA A CGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT T AGACAGGAA CGGTACGCCA GAATCCTGAG 3361 AAGTGTTTTT ATAATCAGTG AGGCCACCGA G TAAAAGAGT CTGTCCATCA CGCAAATTAA 3421 CCGTTGTCGC AATACTTCTT TGATTAGTAA T AACATCACT TGCCTGAGTA GAAGAACTCA 3481 AACTATCGGC CTTGCTGGTA ATATCCAGAA C AATATTACC GCCAGCCATT GCAACGGAAT 3541 CGCCATTCGC CATTCAGGCT GCGCAACTGT T GGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC A CTGAGGCCG CCCGGGCAAA GCCCGGGCGT 3661 CGGGCGACCT TTGGTCGCCC GGCCTCAGTG A GCGAGCGAG CGCGCAGAGA GGGAGTGGCC 3721 AACTCCATCA CTAGGGGTTC CTTGTAGTTA A TGATTAACC CGCCATGCTA CTTATCTACT 3781 CGACATTGAT TATTGACTAG TTATTAATAG T AATCAATTA CGGGGTCATT AGTTCATAGC 3841 CCATATATGG AGTTCCGCGT TACATAACTT A CGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG A CGTATGTTC CCATAGTAAC GCCAATAGGG 3961 ACTTTCCATT GACGTCAATG GGTGGAGTAT T TACGGTAAA CTGCCCACTT GGCAGTACAT 4021 CAAGTGTATC ATATGCCAAG TACGCCCCCT A TTGACGTCA ATGACGGTAA ATGGCCCGCC 4081 TGGCATTATG CCCAGTACAT GACCTTATGG G ACTTTCCTA CTTGGCAGTA CATCTACGTA 4141 TTAGTCATCG CTATTACCAT GGTCGAGGTG A GCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT T TATTTATTT TTTAATTATT TTGTGCAGCG 4261 ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG C CAGGCGGGG CGGGGCGGGG CGAGGGGCGG 4321 GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA G CCAATCAGA GCGGCGCGCT CCGAAAGTTT 4381 CCTTTTATGG CGAGGCGGCG GCGGCGGCGG C CCTATAAAA AGCGAAGCGC GCGGCGGGCG 4441 GGAGTCGCTG CGTTGCCTTC GCCCCGTGCC C CGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG G TGAGCGGGC GGGACGGCCC TTCTCCTCCG 4561 GGCTGTAATT AGCGCTTGGT TTAATGACGG C TTGTTTCTT TTCTGTGGCT GCGTGAAAGC 4621 CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC G GGGGGAGCG GCTCGGGGGG TGCGTGCGTG 4681 TGTGTGTGCG TGGGGAGCGC CGCGTGCGGC T CCGCGCTGC CCGGCGGCTG TGAGCGCTGC 4741 GGGCGCGGCG CGGGGCTTTG TGCGCTCCGC A GTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG G AACAAAGGC TGCGTGCGGG GTGTGTGCGT 4861 GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC G GTCGGGCTG CAACCCCCCC TGCACCCCCC 4921 TCCCCGAGTT GCTGAGCACG GCCCGGCTTC G GGTGCGGGG CTCCGTACGG GGCGTGGCGC 4981 GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG G CAGGTGGGG GTGCCGGGCG GGGCGGGGCC 5041 GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG G CGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT T TTATGGTAA TCGTGCGAGA GGGCGCAGGG 5161 ACTTCCTTTG TCCCAAATCT GTGCGGAGCC G AAATCTGGG AGGCGCCGCC GCACCCCCTC 5221 TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC C GGCAGGAAG GAAATGGGCG GGGAGGGCCT 5281 TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT C CCTCTCCAG CCTCGGGGCT GTCCGCGGGG 5341 GGACGGCTGC CTTCGGGGGG GACGGGGCAG G GCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA T GCCTTCTTC TTTTTCCTAC AGCTCCTGGG 5461 CAACGTGCTG GTTATTGTGC TGTCTCATCA T TTTGGCAAA GAATTGATTA ATTCGAGCGA 5521 ACGCGTCGAG TCGCTCGGTA CGATTTAAAT T GAATTGGCC TCGAGCGCAA GCTTGAGCTA 5581 GCGTCGACCT TCTGCCATGG CCCTGTGGAT G CGCCTCCTG CCCCTGCTGG CGCTGCTGGC 5641 CCTCTGGGGA CCTGACCCAG CCGCAGCCTT T GTGAACCAA CACCTGTGCG GCTCAGATCT 5701 GGTGGAAGCT CTCTACCTAG TGTGCGGGGA A CGAGGCTTC TTCTACACAC CCAGGACCAA 5761 GCGGGAGGCA GAGGACCTGC AGGTGGGGCA G GTGGAGCTG GGCGGGGGCC CTGGTGCAGG 5821 CAGCCTGCAG CCCTTGGCCC TGGAGGGGTC G CGACAGAAG CGTGGCATTG TGGAACAATG 5881 CTGTACCAGC ATCTGCTCCC TCTACCAGCT G GAGAACTAC TGCAACTAGA CGCAGCCGTC 5941 GACGGTACCC CCGACGCGGC CTAACTGGCC T CATGGGCCT TCCGCTCACT GCCCGCTTTC 6001 CAGTCGGGAA ACCTGTCGTG CCAGTCAGGT G CAGGCTGCC TATCAGAAGG TGGTGGCTGG 6061 TGTGGCCAAT GCCCTGGCTC ACAAATACCA C TGAGATCTT TTTCCCTCTG CCAAAAATTA 6121 TGGGGACATC ATGAAGCCCC TTGAGCATCT G ACTTCTGGC TAATAAAGGA AATTTATTTT 6181 CATTGCAATA GTGTGTTGGA ATTTTTTGTG T CTCTCACTC GGAAGGACAT ATGGGAGGGC 6241 AAATCATTTA AAACATCAGA ATGAGTATTT G GTTTAGAGT TTGGCAACAT ATGCCCATAT 6301 GCTGGCTGCC ATGAACAAAG GTTGGCTATA A AGAGGTCAT CAGTATATGA AACAGCCCCC 6361 TGCTGTCCAT TCCTTATTCC ATAGAAAAGC C TTGACTTGA GGTTAGATTT TTTTTATATT 6421 TTGTTTTGTG TTATTTTTTT CTTTAACATC C CTAAAATTT TCCTTACATG TTTTACTAGC 6481 CAGATTTTTC CTCCTCTCCT GACTACTCCC A GTCATAGCT GTCCCTCTTC TCTTATGGAG 6541 ATCCCTCGAC CTGCAGCCCA AGCTGTAGAT A AGTAGCATG GCGGGTTAAT CATTAACTAC 6601 AAGGAACCCC TAGTGATGGA GTTGGCCACT C CCTCTCTGC GCGCTCGCTC GCTCACTGAG 6661 GCCGGGCGAC CAAAGGTCGC CCGACGCCCG G GCTTTGCCC GGGCGGCCTC AGTGAGCGAG 6721 CGAGCGCGCA GCTGGCGTAA AAV2 5’ ITR: 3601 - 3742bp CAG promoter: 3779 - 5423bp h-insulin aspartic acid (hInsAsp): 5590 - 5936 bp Rabbit β-globin polyA signal (rabbit β globin 3' adjacent region including 3'UTR and polyA signal): 6025 - 6543 bp AAV2 3' ITR: 6602 - 6743 bp pAAV-CAG-hInsWt (SEQ ID NO: 50) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAA TCGGCC AACGCGCGGG GAGAGGCGGT 61 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTC ACTGACT CGCTGCGCTC GGTCGTTCGG 121 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GC GGTAATAC GGTTATCCAC AGAATCAGGG 181 GATAACGCAG GAAAGAACAT GTGAGCAAAA GG CCAGCAAA AGGCCAGGAA CCGTAAAAAG 241 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CG CCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GG ACTATAAA GATACCAGGC GTTTCCCCCT 361 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG AC CCTGCCGC TTACCGGATA CCTGTCCGCC 421 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CA TAGCTCAC GCTGTAGGTA TCTCAGTTCG ​481 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GT GCACGAAC CCCCCGTTCA GCCCGACCGC 541 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TC CAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AG AGCGAGGT ATGTAGGCGG TGCTACAGAG 661 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC AC TAGAAGAA CAGTATTTGG TATCTGCGCT 721 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GT TGGTAGCT CTTGATCCGG CAAACAAACC 781 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AA GCAGCAGA TTACGCGCAG AAAAAAAGGA 841 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GG GTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AA AAGGATCT TCACCTAGAT CCTTTTAAAT 961 TAAAAATGAA GTTTTAAATC AATCTAAAGT AT ATATGAGT AAACTTGGTC TGACAGTTAC 1021 CAATGCTTAA TCAGTGAGGC ACCTATCTCA G CGATCTGTC TATTTCGTTC ATCCATAGTT 1081 GCCTGACTCC CCGTCGTGTA GATAACTACG A TACGGGAGG GCTTACCATC TGGCCCCAGT 1141 GCTGCAATGA TACCGCGAGA CCCACGCTCA C CGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT C CTGCAACTT TATCCGCCTC CATCCAGTCT 1261 ATTAATTGTT GCCGGGAAGC TAGAGTAAGT A GTTCGCCAG TTAATAGTTT GCGCAACGTT 1321 GTTGCCATTG CTACAGGCAT CGTGGTGTCA C GCTCGTCGT TTGGTATGGC TTCATTCAGC 1381 TCCGGTTCCC AACGATCAAG GCGAGTTACA T GATCCCCCA TGTTGTGCAA AAAAGCGGTT 1441 AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA A GTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT G TCATGCCAT CCGTAAGATG CTTTTCTGTG 1561 ACTGGTGAGT ACTCAACCAA GTCATTCTGA G AATAGTGTA TGCGGCGACC GAGTTGCTCT 1621 TGCCCGGCGT CAATACGGGA TAATACCGCG C CACATAGCA GAACTTTAAA AGTGCTCATC 1681 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC T CAAGGATCT TACCGCTGTT GAGATCCAGT 1741 TCGATGTAAC CCACTCGTGC ACCCAACTGA T CTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT G CCGCAAAAA AGGGAATAAG GGCGACACGG 1861 AAATGTTGAA TACTCATACT CTTCCTTTTT C AATATTATT GAAGCATTTA TCAGGGTTAT 1921 TGTCTCATGA GCGGATACAT ATTTGAATGT A TTTAGAAAA ATAAACAAAT AGGGGTTCCG 1981 CGCACATTTC CCCGAAAAGT GCCACCTGAC G TCTAAGAAA CCATTATTAT CATGACATTA 2041 ACCTATAAAA ATAGGCGTAT CACGAGGCCC T TTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG A CGGTCACAG CTTGTCTGTA AGCGGATGCC 2161 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA G CGGGTGTTG GCGGGTGTCG GGGCTGGCTT 2221 AACTATGCGG CATCAGAGCA GATTGTACTG A GAGTGCACC ATATGCGGTG TGAAATACCG 2281 CACAGATGCG TAAGGAGAAA ATACCGCATC A GGCGATTCC AACATCCAAT AAATCATACA 2341 GGCAAGGCAA AGAATTAGCA AAATTAAGCA A TAAAGCCTC AGAGCATAAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA C TTTTGCGGG AGAAGCCTTT ATTTCAACGC 2461 AAGGATAAAA ATTTTTAGAA CCCTCATATA T TTTAAATGC AATGCCTGAG TAATGTGTAG 2521 GTAAAGATTC AAACGGGTGA GAAAGGCCGG A GACAGTCAA ATCACCATCA ATATGATATT 2581 CAACCGTTCT AGCTGATAAA TTCATGCCGG A GAGGGTAGC TATTTTTGAG AGGTCTCTAC 2641 AAAGGCTATC AGGTCATTGC CTGAGAGTCT G GAGCAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC C CCGGTTGAT AATCAGAAAA GCCCCAAAAA 2761 CAGGAAGATT GTATAAGCAA ATATTTAAAT T GTAAGCGTT AATATTTTGT TAAAATTCGC 2821 GTTAAATTTT TGTTAAATCA GCTCATTTTT T AACCAATAG GCCGAAATCG GCAAAATCCC 2881 TTATAAATCA AAAGAATAGA CCGAGATAGG G TTGAGTGTT GTTCCAGTTT GGAACAAGAG 2941 TCCACTATTA AAGAACGTGG ACTCCAACGT C AAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC A AGTTTTTTG GGGTCGAGGT GCCGTAAAGC 3061 ACTAAATCGG AACCCTAAAG GGAGCCCCCG A TTTAGAGCT TGACGGGGAA AGCCGGCGAA 3121 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA A GGAGCGGGC GCTAGGGCGC TGGCAAGTGT 3181 AGCGGTCACG CTGCGCGTAA CCACCACACC C GCCGCGCTT AATGCGCCGC TACAGGGCGC 3241 GTACTATGGT TGCTTTGACG AGCACGTATA A CGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT T AGACAGGAA CGGTACGCCA GAATCCTGAG 3361 AAGTGTTTTT ATAATCAGTG AGGCCACCGA G TAAAAGAGT CTGTCCATCA CGCAAATTAA 3421 CCGTTGTCGC AATACTTCTT TGATTAGTAA T AACATCACT TGCCTGAGTA GAAGAACTCA 3481 AACTATCGGC CTTGCTGGTA ATATCCAGAA C AATATTACC GCCAGCCATT GCAACGGAAT 3541 CGCCATTCGC CATTCAGGCT GCGCAACTGT T GGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC A CTGAGGCCG CCCGGGCAAA GCCCGGGCGT 3661 CGGGCGACCT TTGGTCGCCC GGCCTCAGTG A GCGAGCGAG CGCGCAGAGA GGGAGTGGCC 3721 AACTCCATCA CTAGGGGTTC CTTGTAGTTA A TGATTAACC CGCCATGCTA CTTATCTACT 3781 CGACATTGAT TATTGACTAG TTATTAATAG T AATCAATTA CGGGGTCATT AGTTCATAGC 3841 CCATATATGG AGTTCCGCGT TACATAACTT A CGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG A CGTATGTTC CCATAGTAAC GCCAATAGGG 3961 ACTTTCCATT GACGTCAATG GGTGGAGTAT T TACGGTAAA CTGCCCACTT GGCAGTACAT 4021 CAAGTGTATC ATATGCCAAG TACGCCCCCT A TTGACGTCA ATGACGGTAA ATGGCCCGCC 4081 TGGCATTATG CCCAGTACAT GACCTTATGG G ACTTTCCTA CTTGGCAGTA CATCTACGTA 4141 TTAGTCATCG CTATTACCAT GGTCGAGGTG A GCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT T TATTTATTT TTTAATTATT TTGTGCAGCG 4261 ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG C CAGGCGGGG CGGGGCGGGG CGAGGGGCGG 4321 GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA G CCAATCAGA GCGGCGCGCT CCGAAAGTTT 4381 CCTTTTATGG CGAGGCGGCG GCGGCGGCGG C CCTATAAAA AGCGAAGCGC GCGGCGGGCG 4441 GGAGTCGCTG CGTTGCCTTC GCCCCGTGCC C CGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG G TGAGCGGGC GGGACGGCCC TTCTCCTCCG 4561 GGCTGTAATT AGCGCTTGGT TTAATGACGG C TTGTTTCTT TTCTGTGGCT GCGTGAAAGC 4621 CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC G GGGGGAGCG GCTCGGGGGG TGCGTGCGTG 4681 TGTGTGTGCG TGGGGAGCGC CGCGTGCGGC T CCGCGCTGC CCGGCGGCTG TGAGCGCTGC 4741 GGGCGCGGCG CGGGGCTTTG TGCGCTCCGC A GTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG G AACAAAGGC TGCGTGCGGG GTGTGTGCGT 4861 GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC G GTCGGGCTG CAACCCCCCC TGCACCCCCC 4921 TCCCCGAGTT GCTGAGCACG GCCCGGCTTC G GGTGCGGGG CTCCGTACGG GGCGTGGCGC 4981 GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG G CAGGTGGGG GTGCCGGGCG GGGCGGGGCC 5041 GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG G CGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT T TTATGGTAA TCGTGCGAGA GGGCGCAGGG 5161 ACTTCCTTTG TCCCAAATCT GTGCGGAGCC G AAATCTGGG AGGCGCCGCC GCACCCCCTC 5221 TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC C GGCAGGAAG GAAATGGGCG GGGAGGGCCT 5281 TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT C CCTCTCCAG CCTCGGGGCT GTCCGCGGGG 5341 GGACGGCTGC CTTCGGGGGG GACGGGGCAG G GCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA T GCCTTCTTC TTTTTCCTAC AGCTCCTGGG 5461 CAACGTGCTG GTTATTGTGC TGTCTCATCA T TTTGGCAAA GAATTGATTA ATTCGAGCGA 5521 ACGCGTCGAG TCGCTCGGTA CGATTTAAAT T GAATTGGCC TCGAGCGCAA GCTTGAGCTA 5581 GCGTCGAGGG GTCGACATGG CCCTGTGGAT G CGCCTCCTG CCCCTGCTGG CGCTGCTGGC 5641 CCTCTGGGGA CCTGACCCAG CCGCAGCCTT T GTGAACCAA CACCTGTGCG GCTCACACCT 5701 GGTGGAAGCT CTCTACCTAG TGTGCGGGGA A CGAGGCTTC TTCTACACAC CCAGGACCAA 5761 GCGGGAGGCA GAGGACCTGC AGGTGGGGCA G GTGGAGCTG GGCGGGGGCC CTGGTGCAGG 5821 CAGCCTGCAG CCCTTGGCCC TGGAGGGGTC G CGACAGAAG CGTGGCATTG TGGAACAATG 5881 CTGTACCAGC ATCTGCTCCC TCTACCAGCT G GAGAACTAC TGCAACTAGG TCGACCCCTC 5941 GACGGTACCC CCGACGCGGC CTAACTGGCC T CATGGGCCT TCCGCTCACT GCCCGCTTTC 6001 CAGTCGGGAA ACCTGTCGTG CCAGTCAGGT G CAGGCTGCC TATCAGAAGG TGGTGGCTGG 6061 TGTGGCCAAT GCCCTGGCTC ACAAATACCA C TGAGATCTT TTTCCCTCTG CCAAAAATTA 6121 TGGGGACATC ATGAAGCCCC TTGAGCATCT G ACTTCTGGC TAATAAAGGA AATTTATTTT 6181 CATTGCAATA GTGTGTTGGA ATTTTTTGTG T CTCTCACTC GGAAGGACAT ATGGGAGGGC 6241 AAATCATTTA AAACATCAGA ATGAGTATTT G GTTTAGAGT TTGGCAACAT ATGCCCATAT 6301 GCTGGCTGCC ATGAACAAAG GTTGGCTATA A AGAGGTCAT CAGTATATGA AACAGCCCCC 6361 TGCTGTCCAT TCCTTATTCC ATAGAAAAGC C TTGACTTGA GGTTAGATTT TTTTTATATT 6421 TTGTTTTGTG TTATTTTTTT CTTTAACATC C CTAAAATTT TCCTTACATG TTTTACTAGC 6481 CAGATTTTTC CTCCTCTCCT GACTACTCCC A GTCATAGCT GTCCCTCTTC TCTTATGGAG 6541 ATCCCTCGAC CTGCAGCCCA AGCTGTAGAT A AGTAGCATG GCGGGTTAAT CATTAACTAC 6601 AAGGAACCCC TAGTGATGGA GTTGGCCACT C CCTCTCTGC GCGCTCGCTC GCTCACTGAG 6661 GCCGGGCGAC CAAAGGTCGC CCGACGCCCG G GCTTTGCCC GGGCGGCCTC AGTGAGCGAG 6721 CGAGCGCGCA GCTGGCGTAA AAV2 5’ ITR: 3601 - 3742 bp CAG promoter: 3779 - 5423 bp h - insulin wild - type (hInsWt): 5597 - 5929 bp Rabbit β - globin polyA signal (rabbit β globin 3’ flanking region including 3’UTR and polyA signal) : 6025 - 6543 bp AAV2 3’ ITR: 6602 - 6743 bp

Claims

1. A gene construct containing a nucleotide sequence encoding insulin for use in the treatment and / or prevention of neuroinflammation, neurodegeneration and / or cognitive decline, or diseases or conditions related thereto.

2. The gene construct for use according to claim 1, wherein the nucleotide sequence encoding insulin is operably linked to a ubiquitous promoter.

3. The gene construct for use according to claim 1 or 2, wherein the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter, preferably the ubiquitous promoter is a CAG promoter.

4. The gene construct for use according to any one of claims 1 to 3, wherein the gene construct contains at least one target sequence of a microRNA expressed in a tissue where insulin expression is desired to be prevented, preferably at least one target sequence of the microRNA is selected from target sequences that bind to microRNAs expressed in the mammalian heart and / or liver.

5. The gene construct for use according to claim 4, wherein the gene construct contains at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart, preferably the target sequence of the microRNA expressed in the heart is selected from SEQ ID NO: 8 and 16 - 20, and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NO: 7 and 9 - 15, more preferably the gene construct contains a target sequence of microRNA - 122a (SEQ ID NO: 7) and a target sequence of microRNA - 1 (SEQ ID NO: 8).

6. The nucleotide sequence encoding insulin is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2 or 3; (b) a nucleotide sequence having at least 60% sequence identity with the nucleotide sequence of SEQ ID NO: 4, 5 or 6; and (c) a nucleotide sequence that differs from the sequence of the nucleotide sequence in (b) due to the degeneracy of the genetic code. The gene construct for use according to any one of claims 1 to 5.

7. Neuroinflammation, neurodegeneration and / or cognitive decline, or diseases or conditions related thereto. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ An expression vector containing the gene construct defined in any one of claims 1 to 6 for use in treating and / or preventing a condition. **Claim 8** A viral vector, preferably a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and a lentiviral vector, more preferably an adeno-associated viral vector, for use according to claim 7. The expression vector for use according to claim 7. **Claim 9** An adeno-associated viral vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, rh8, Cb4, rh74, DJ, 2 / 5, 2 / 1, 1 / 2 or Anc80, preferably an adeno-associated viral vector of serotype 1, 2 or 9, more preferably an adeno-associated viral vector of serotype 1 or 9, for use according to claim 8. The expression vector for use according to claim 8. **Claim 10** For use in treating and / or preventing neuroinflammation, neurodegeneration and / or cognitive decline, or a disease or condition associated therewith, an expression vector containing the gene construct defined in any one of claims 1 to 6 and / or the gene construct defined in any one of claims 7 to 9, in combination with one or more pharmaceutically acceptable components. **Claim 11** Wherein the disease or condition associated with the neuroinflammation, neurodegeneration and / or cognitive impairment is cognitive impairment, dementia, Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia (FTD), Parkinson's disease, Parkinsonian disorders, Parkinson's syndrome, Huntington's disease, traumatic brain injury, prion disease, dementia / neurocognitive problems due to HIV infection, dementia / neurocognitive problems due to aging, tauopathy, multiple sclerosis and other neuroinflammatory / neurodegenerative diseases, preferably Alzheimer's disease, Parkinson's disease and / or Parkinsonian disorders, more preferably Alzheimer's disease or Parkinson's disease, The gene construct for use according to any one of claims 1 to 7 and / or the expression vector for use according to any one of claims 7 to 9 and / or the pharmaceutical composition for use according to claim 10. **Claim 12** Wherein the gene construct and / or the expression vector and / or the pharmaceutical composition is administered into the CSF ​ ​ ​ The genetic agent for use according to any one of claims 1 to 7 and 11, which is administered by For the child construct and / or use according to any one of claims 7 to 9 and 11 An expression vector and / or a pharmaceutical composition for use according to claim 10 or 11.

13. A genetic construct comprising a nucleotide sequence encoding insulin, said insulin being A nucleotide sequence encoding the gene is operably linked to a ubiquitous promoter. The gene construct is designed to block the expression of a microRNA in a tissue in which insulin expression is desired to be prevented. At least one target sequence, preferably at least one target of said microRNA. The target sequence binds to a microRNA expressed in the mammalian heart and / or liver. A genetic construct, wherein the sequence is selected from the group consisting of target sequences.

14. The gene construct comprises at least one target sequence of a microRNA expressed in the liver, and At least one target sequence of a microRNA expressed in the heart, preferably The target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 8 and 16 to 20; The target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 7 and 9 to 15, More preferably, the gene construct comprises a target sequence of microRNA-122a (SEQ ID NO: 7) and and a target sequence of microRNA-1 (SEQ ID NO: 8). building.

15. Preferably, the expression vector is a viral vector, more preferably, the expression vector Adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and and lentiviral vectors, most preferably Preferably, the expression vector is an adeno-associated virus vector. An expression vector comprising a defined genetic construct.

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