Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/28—Insulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
  • C12N2310/141—MicroRNAs, miRNAs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/32—Special delivery means, e.g. tissue-specific
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14171—Demonstrated in vivo effect

Definitions

• the expression vector is a viral vector, preferably wherein the expression vector is a viral vector selected from the group consisting of adenoviral vectors, adeno- associated viral vectors, retroviral vectors, and lentiviral vectors, more preferably an adeno- associated viral vector.
• 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 wherein the disease or condition associated with neuroinflammation, neurodegeneration and/or cognitive disorder is selected from the group consisting of: a cognitive disorder, dementia, Alzheimer’s disease, vascular dementia, Lewy body dementia, frontotemporal dementia (FTD), Parkinson’s disease, Parkinson-like disease, Parkinsonism, Huntington’s disease, traumatic brain injury, prion disease, dementia/neurocognitive issues due to HIV infection, dementia/neurocognitive issues due to aging, tauopathy, multiple sclerosis and other neuroinflammatory/neurodegenerative diseases, preferably Alzheimer’s disease, Parkinson’s disease and/or Parkinson-like disease, more preferably Alzheimer’s disease or Parkinson’s disease.
• a cognitive disorder preferably Alzheimer’s disease, Parkinson’s disease and/or Parkinson-like disease, more preferably Alzheimer’s disease or Parkinson’s disease.
• the gene construct and/or expression vector and/or pharmaceutical composition is administered by intra-CSF administration.
• an expression vector comprising a gene construct as defined in the previous aspect, preferably wherein the expression vector is a viral vector, more preferably wherein the expression vector is a viral vector selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors, most preferably wherein the expression vector is an adeno-associated viral vector.
• a target sequence of a microRNA expressed in the heart may be replaced by a nucleotide sequence comprising a nucleotide sequence that 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
• the target sequence of a microRNA expressed in the heart may be replaced by a nucleotide sequence comprising a nucleotide sequence that 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
• a target sequence of a microRNA expressed in the heart as used herein exerts at least a detectable level of activity of a target sequence of a microRNA expressed in the heart as known to a person of skill in the art.
• An activity of a target sequence of a microRNA expressed in the heart is to bind to its cognate microRNA expressed in the heart and, when operatively linked to a transgene, to mediate detargeting of transgene expression in the heart. This activity may be assessed by measuring the levels of transgene expression in the heart on the level of the mRNA or the protein by standard assays known to a person of skill in the art, such as qPCR, Western blot analysis or ELISA.
• one, two, three, four, five, six, seven or eight copies of a nucleotide sequence encoding miRT-122a (SEQ ID NO: 7) and one, two, three, four, five, six, seven or eight copies nucleotide sequence encoding miRT-1 (SEQ ID NO: 8) are combined in the gene construct of the invention.
• four copies of a nucleotide sequence encoding miRT-122a (SEQ ID NO: 7) and four copies of nucleotide sequence encoding miRT-1 (SEQ ID NO: 8) are combined in the gene construct of the invention.
• the nucleotide sequence encoding an insulin present in a gene construct according to the invention encodes an engineered insulin with furin cleavage sites.
• Such engineered insulin with furin cleavage sites is known to be processed in a highly efficient way to produce mature insulin in non-pancreatic tissues.
• the nucleotide sequence encoding an engineered insulin with furin cleavage sites is selected from the group consisting of:
• a nucleotide sequence encoding a human insulin present in a gene construct according to the 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 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 4. In some embodiments, identity may be assessed relative
• a nucleotide sequence encoding canine insulin present in a gene construct according to the 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 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 6.
• identity may be assessed relative to
• a nucleotide sequence encoding human insulin present in a gene construct according to the 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 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 45 or 46. In some embodiments, identity may be assessed
• a Nestin promoter comprises, consists essentially of, or consists of a nucleotide sequence that 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
• a tyrosine hydroxylase (TH) promoter comprises, consists essentially of, or consists of a nucleotide sequence that 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
• identity may be assessed relative to a part of SEQ ID NO: 34, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of SEQ ID NO: 34.
• CMV enhancer sequence SEQ ID NO: 24
• CMV promoter sequence SEQ ID NO: 23
• SEQ ID NO: 39 each of these additional sequences may be present in a gene construct according to the invention.
• a gene construct comprising a nucleotide sequence encoding insulin as described herein, further comprising one 5’ITR and one 3’ITR, preferably AAV2 ITRs, more preferably the AAV2 ITRs represented by SEQ ID NO: 30 (5’ ITR) and SEQ ID NO: 31 (3’ ITR).
• the level of sequence identity or similarity as 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%.
• compositions as described herein are for use as a medicament.
• compositions as described herein are for use in the treatment and/or prevention of neuroinflammation, neurodegeneration and/or cognitive decline, or a disease or condition associated therewith.
• the composition is a pharmaceutical composition.
• Such compositions as described herein may also be called gene therapy compositions.
• a gene construct for use and/or an expression vector for use and/or a pharmaceutical composition for use as described herein is administered by intra-CSF administration.
• the subject to be treated is a subject at risk of developing neuroinflammation, neurodegeneration and/or cognitive decline, or a disease or condition associated therewith.
• Decreasing neuroinflammation may mean that inflammation of nervous tissue is decreased. This could be assessed using techniques known to a person of skill in the art such as the measurement of (neuro)inflammatory markers, for example as done in the experimental part. Exemplary markers that could be used in this regard are 11-1 b, II-6 and NfkB.
• “decrease” means at least a detectable decrease (respectively a detectable improvement) using an assay known to a person of skill in the art, such as assays as carried out in the experimental part.
• Increasing the number of astrocytes may mean that the number of astrocytes is increased. This could be assessed using techniques known to a person of skill in the art such as the measurement of astrocyte markers, for example as done in the experimental part. Exemplary markers that could be used in this regard are Gfap and S100b.
• “increase” means at least a detectable increase (respectively a detectable improvement) using an assay known to a person of skill in the art, such as assays as carried out in the experimental part.
• Laboratory tests may include both macroscopic and microscopic methods, molecular methods, radiographic methods such as X-rays, biochemical methods, immunohistochemical methods and others.
• Memory and learning may be assed in mice e.g. as described in the experimental part, e.g. by a novel object recognition test and/or a Morris water maze test.
• the alleviation of a symptom may be seen after at least one week, one month, six months, one year or more of treatment using a gene construct and/or an expression vector and/or a composition of the invention. Preferably, the alleviation is observed after a single administration.
• a gene construct and/or an expression vector and/or a pharmaceutical composition and/or a medicament as described herein may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing a me neuroinflammation, neurodegeneration and/or cognitive disorder, or a disease associated therewith, and may be administered in vivo, ex vivo or in vitro.
• Said gene construct and/or expression vector and/or pharmaceutical composition and/or medicament may be directly or indirectly administered to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing neuroinflammation, neurodegeneration and/or cognitive disorder, or a disease associated therewith, and may be administered directly or indirectly in vivo, ex vivo or in vitro.
• each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct as identified herein by a given sequence identity number is not limited to this specific sequence as disclosed.
• Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.
• SEQ ID NO take SEQ ID NO: X as example
• a preferred nucleotide sequence 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% identity with SEQ ID NO: A.
• sequence identity is herein described as a relationship between two or more amino acid sequences (peptide or polypeptide or protein) or two or more nucleic acid sequences (polynucleotide), as determined by comparing the sequences.
• similarity or“sequence similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
• a global alignment is suitably used to determine sequence identity or similarity when the two sequences have similar lengths.
• local alignments such as those using the Smith-Waterman algorithm, are preferred.
• EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences overtheir entire length (full length), maximizing the number of matches and minimizing the number of gaps.
• EMBOSS water uses the Smith-Waterman local alignment algorithm.
• the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
• a "transgene” is herein described as a gene or a coding sequence or a nucleic acid molecule represented by a nucleotide sequence (i.e. a molecule encoding an insulin) that has been newly introduced into a cell, i.e. a gene that may be present but may normally not be expressed or expressed at an insufficient level in a cell.
• “insufficient” means that although said insulin is expressed in a cell, a condition and/or disease as described herein could still be developed.
• the invention allows the over-expression of an insulin.
• the transgene may comprise sequences that are native to the cell, sequences that naturally do not occur in the cell and it may comprise combinations of both.
• CNS- and/or brain- -specific promoters may be promoters that are capable of driving the preferential or predominant (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 or more) expression of insulin in the CNS and/or the brain as compared to other organs or tissues.
• Other organs or tissues may be the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis and others.
• operably linked refers to a linkage of polynucleotide elements in a functional relationship.
• a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid molecule.
• a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
• Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis, or any other method known to a person skilled in the art.
• microRNA or“miRNA” or“miR” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure.
• a microRNA is a small non-coding RNA molecule found in plants, animals and some viruses, that may function in RNA silencing and post-transcriptional regulation of gene expression.
• a target sequence of a microRNA may be denoted as“miRT”.
• miRT-1 a target sequence of microRNA-1 or miRNA-1 or miR-1.
• protein or “polypeptide” or“amino acid sequence” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin.
• amino acids or“residues” are denoted by three-letter symbols.
• expression vector generally refers to a nucleotide sequence that is capable of effecting expression of a gene or a coding sequence in a host compatible with such sequences.
• An expression vector carries a genome that is able to stabilize and remain episomal in a cell.
• a cell may mean to encompass a cell used to make the construct or a cell wherein the construct will be administered.
• a vector is capable of integrating into a cell's genome, for example through homologous recombination or otherwise.
• suitable expression vectors can be expressed in, yeast, e.g. S. cerevisiae, insect cells, e.g. Sf9 cells, mammalian cells, e.g., CHO cells, and bacterial cells, e.g., E. coli.
• yeast e.g. S. cerevisiae
• insect cells e.g. Sf9 cells
• mammalian cells e.g., CHO cells
• bacterial cells e.g., E. coli.
• a cell may thus be a prokaryotic or eukaryotic host cell.
• a cell may be a cell that is suitable for culture in liquid or on solid media.
• a viral vector or a viral expression vector or a viral gene therapy vector is a vector that comprises a gene construct as described herein.
• a particularly suitable gene therapy vector includes an adenoviral and adeno-associated virus (AAV) vector. These vectors infect a wide number of dividing and non-dividing cell types including synovial cells and liver cells. The episomal nature of the adenoviral and AAV vectors after cell entry makes these vectors suited for therapeutic applications (Russell, 2000, J. Gen. Virol.
• AAV vectors are even more preferred since they are known to result in very stable long-term expression of transgene expression (up to 9 years in dog (Niemeyer et al, Blood. 2009 Jan 22;1 13(4)797-806) and ⁇ 10 years in human (Buchlis, G. et al., Blood. 2012 Mar 29;1 19(13):3038-41 ).
• Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, supra). Gene therapy methods using AAV vectors are described by Wang et al., 2005, J Gene Med.
• AAV refers to a virus that belongs to the genus Dependovirus family Parvoviridae.
• the AAV genome is approximately 4.7 Kb in length and it consists of single strand deoxyribonucleic acid (ssDNA) that can be positive or negative detected.
• ssDNA single strand deoxyribonucleic acid
• the invention also encompasses the use of double stranded AAV also called dsAAV or scAAV.
• AAV helper functions generally refers to the corresponding AAV functions required for rAAV replication and packaging supplied to the rAAV vector in trans.
• AAV helper functions complement the AAV functions which are missing in the rAAV vector, but they lack AAV ITRs (which are provided by the rAAV vector genome).
• AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are well known in the art, see e.g. Chiorini et al. (1999, J. of Virology, Vol 73(2): 1309-1319) or US 5,139,941 , incorporated herein by reference.
• Transduction refers to the delivery of an insulin into a recipient host cell by a viral vector.
• transduction of a target cell by a rAAV vector of the invention leads to transfer of the rAAV genome contained in that vector into the transduced cell.
• “Host cell” or“target cell” refers to the cell into which the DNA delivery takes place, such as the muscle cells of a subject.
• AAV vectors are able to transduce both dividing and non-dividing cells.
• the rAAV genome as present in a rAAV vector does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV.
• This rAAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art.
• CNS- and/or brain- and/or hypothalamus and/or cortex- and/or hippocampus- and/or cerebellum- and/or olfactory bulb-specific expression refers to the preferential or predominant (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 or more) expression of insulin in 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 compared to other organs or tissues.
• organs or tissues may be the liver, pancreas, adipose tissue, skeletal muscle, heart, and others.
• expression is not detectable in the liver, pancreas, adipose tissue, skeletal muscle and/or heart.
• expression is not detectable in at least one, at least two, at least three, at least four or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle and heart. Expression may be assessed as described above.
• central nervous system or“CNS” refers to the part of the nervous system that comprises the brain and the spinal cord, to which sensory impulses are transmitted and from which motor impulses pass out, and which coordinates the activity of the entire nervous system.
• Spatial memory is a form of memory responsible for the recording of information about one's environment and spatial orientation.
• the word“about” or“approximately” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 1 % of the value.
• FIG. 3 Increased expression of astrocyte markers in the brain of SAMP8 mice treated with AAV9-hlns vectors.
• Figure 5 Expression of hlns in the brain of db/db mice.
• Figure 6 Reduction of brain inflammation in db/db mice treated with AAV9-hlns vectors.
• Nfkb nuclear factor kappa B
• 111b interleukin-1 beta
• 116 interleukin-6.
• FIG. 10 Amelioration of short-term and long-term memory in SAMP8 mice after intra- CSF gene therapy with AAV1 -CAG-hlnsWt and AAV1 -CAG-hlnsAsp vectors.
• (A) Short-term and (B) long-term discrimination index was measured during a novel object recognition test in SAMR1 non-treated, SAMP8 non-treated, SAMP8 AAV1 -CAG-hlnsWt-treated and SAMP8 AAV1 -CAG-hlnsAsp-treated mice, at 33 weeks of age, and calculated as explained in the General Procedures of the Examples. Results are expressed as the mean ⁇ SEM, n 5 animals/group. *p ⁇ 0.05, ** p ⁇ 0.01 vs SAMP8 non-treated mice.
• FIG. 11 Amelioration of learning capacity in SAMP8 mice after intra-CSF gene therapy with AAV1 -CAG-hlnsWt.
• mice we also examined brain transduction efficiency by AAV1 -hlns, AAV2-hlns and AAV9-hlns vectors after intra-CSF administration of wild-type mice (Example 3).
• SAMP8/TaHsd SAMP8
• BKS.Cg -+Lepr db /+Lepr db OlaHsd db/db
• C57BI/6J wild- type mice mice
• SAMR1/TaHsd SAMR1 were used. Mice were fed ad libitum with a standard diet (2018S Teklad Global Diets®, Harlan Labs., Inc., Madison, Wl, US) and kept under a light-dark cycle of 12 h (lights on at 8:00 a.m.) and stable temperature (22°C ⁇ 2).
• mice were anesthetized by means of inhalational anesthetic isoflurane (IsoFlo®, Abbott Laboratories, Abbott Park, IL, US) and decapitated. Tissues of interest were excised and kept at -80°C until analysis. All experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation of the Universitat Autonoma de Barcelona. Recombinant AA V vectors
• Single-stranded AAV vectors of serotype 1 , 2 and 9 were produced by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et a!., 2010. Curr Gene Ther. 10(6):423- 36).
• cells were cultured in 10 roller bottles (850 cm 2 , flat; CorningTM, Sigma- Aldrich Co., Saint Louis, MO, US) in DMEM 10% FBS to 80% confluence and co-transfected by calcium phosphate method with a plasmid carrying the expression cassette flanked by the AAV2 ITRs (SEQ ID NO: 40), a helper plasmid carrying the AAV2 rep gene and the AAV of serotype 1 , 2 or 9 cap gene, respectively, and a plasmid carrying the adenovirus helper functions.
• the transgene used was the human insulin coding-sequence (SEQ ID NO: 46) driven by the early enhancer/chicken beta actin (CAG) promoter (SEQ ID NO: 22), with the addition of four tandem repeats of the miRT-122a sequence (5OAAACACCATTGTCACACTCCA3’, SEQ ID NO: 7) and four tandems repeats of the miRT-1 sequence (5TTACATACTTCTTTACATTCCA3’, SEQ ID NO: 8) cloned in the 3’ untranslated region of the expression cassette.
• CAG early enhancer/chicken beta actin
• cells were cotransfected with a plasmid carrying the expression cassette flanked by the AAV2 ITRs (SEQ ID NO: 49 or SEQ ID NO: 50), a helper plasmid carrying the AAV2 rep gene and the AAV of serotype 1 , and a plasmid carrying the adenovirus helper functions.
• the transgene used was either the human insulin aspartic coding-sequence containing the furin cleaving sites (SEQ ID NO: 46) or the human insulin wild-type coding-sequence containing the furin cleaving sites (SEQ ID NO: 45), respectively, driven by the early enhancer/chicken beta actin (CAG) promoter (SEQ ID NO: 22).
• AAVs were purified with an optimized method based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsCI) gradients. This second-generation CsCI-based protocol reduced empty AAV capsids and DNA and protein impurities dramatically (Ayuso, E. et a/., 2010. Curr Gene Ther. 10(6):423-36).
• Purified AAV vectors were dialyzed against PBS, filtered and stored at -80°C. Titers of viral genomes were determined by quantitative PCR following the protocol described for the AAV2 reference standard material using linearized plasmid DNA as standard curve (Lock M., et a/., Hum. Gene Ther. 2010; 21 :1273-1285). The vectors were constructed according to molecular biology techniques well known in the art.
• mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and the skin of the posterior part of the head, from behind the ears to approximately between the scapulas, was shaved and rinsed with ethanol. Mice were held in prone position, with the head at a slightly downward inclination. A 2-mm rostro-caudal incision was made to introduce a Hamilton syringe at an angle of 45-55° into the cisterna magna, between the occiput and the C1 -vertebra and 5 pi of vector dilution was administered. Given that the CNS is the main target compartment for vector delivery, mice were dosed with the same number of vector genomes (vg)/mouse irrespective of body weight (5x10 10 vg/mice).
• Discrimination ratio (%) (N-F)/(N+F)x100%, where N represents the time spent in exploring the new object and F represents the time spent in exploring the same object.
• furin endoprotease cleavage sites allow highly efficient production of mature insulin in non-pancreatic tissues; between 85-93% of the total insulin production is mature insulin (Gros et al., Hum Gene Ther. 1997 Dec 10;8(18):2249-59; Gros et al. Hum Gene Ther. 1999 May 1 ;10(7): 1207-17 and Riu et al. Diabetes. 2002 Mar;51 (3):704-11).
• Furin is known to be present in different brain areas (Foti et al. Gene Ther. 2009 November;16(11):1314-1319), allowing the efficient production of mature insulin from a sequence containing furin cleavage sites in this organ.
• Intra-CSF administration of AAV9-CAG-hlns-dmiRT vectors mediated widespread overexpression of insulin in the brain, as evidenced by the increased expression levels of human insulin in different areas of the brain such as hypothalamus, cortex, hippocampus and cerebellum of SAMP8 mice (FIG 1).
• 3xTg-AD mice are administered locally intra-CSF, through the cisterna magna, with 5x10 10 vg/mouse of AAV1 vectors encoding human insulin under the control of the CAG ubiquitous promoter.
• control non-treated 3xTg-AD animals are used.
• Several behavioural tests as Y- Maze, Open-Field and Morris Water Maze are performed in these mice. At 12 months of age, animals are euthanized and serum and tissue samples are taken for analysis.
• Nucleotide sequence H. sapiens insulin with furin cleaving sites (SEQ ID NO: 45)
• Amino acid sequence H. sapiens insulin with furin cleaving sites (SEQ ID NO: 41)
• Amino acid sequence C. lupus familiaris insulin (SEQ ID NO : 3)
• miRT-122a (SEQ ID NO: 7): 5’ CAAACACCATTGTCACACTCCA 3’, target for the microRNA- 122a (Accession Number to the miRBase database MI0000442), which is expressed in the liver.
• miRT-152 (SEQ ID NO: 9): 5’ CCAAGTTCTGTCATGCACTGA 3’, target for the microRNA-152 (MI0000462), which is expressed in the liver.
• miRT-199a-5p (SEQ ID NO: 10): 5’ GAACAGGTAGTCTGAACACTGGG 3’, target for the microRNA 199a (MI0000242), which is expressed in the liver.
• miRT-199a-3p SEQ ID NO: 1 1): 5’ TAACCAATGTGCAGACT ACTGT 3’, target for the microRNA-199a (MI0000242), which is expressed in the liver.
• miRT-215 (SEQ ID NO: 12): 5’ GTCTGT C AATT CAT AG GT CAT 3’, target for the microRNA-215 (MI0000291 ), which is expressed in the liver.
• miRT-192 (SEQ ID NO: 13): 5’ GGCTGTCAATTCATAGGTCAG 3’, target for the microRNA-192 (MI0000234), which is expressed in the liver.
• miRT-148a (SEQ ID NO: 14): 5’ AC AAAGTT CTGT AGTG C ACT G A 3’, target for the microRNA- 148a (MI0000253), which is expressed in the liver.
• miRT-194 (SEQ ID NO: 15): 5’ TCCACATGGAGTTGCTGTTACA 3’, target for the microRNA-194 (MI0000488), which is expressed in the liver.
• miRT-206 (SEQ ID NO: 17): 5’ CCACACACTTCCTT ACATTCCA 3’, target for the microRNA-206 (MI0000490), which is expressed in the heart.
• miRT-1 (SEQ ID NO: 8): 5’ TT AC AT ACTT CTTT ACATTCCA 3’, target for the microRNA-1 (MI0000651), which is expressed in the heart.
• miRT-208a-3p (SEQ ID NO: 19): 5’ ACAAGCTTTTTGCTCGTCTTAT 3’, target for the microRNA- 208a (MI0000251), which is expressed in the heart.
• miRT-499-5p (SEQ ID NO: 20): 5’ AAAC AT C ACTG C AAGTCTT AA 3’, target for the microRNA- 499 (MI0003183), which is expressed in the heart.
• CaMKIO promoter SEQ ID NO: 291
• MRR Myelin basic protein promoter
• Rabbit b-globin polyA signal (3' UTR and 3’ flanking region of rabbit beta-globin, including polyA signal): 6293-681 1 bp
• CAG promoter 3779-5423 bp
• hlnsulin Aspartic (hlnsAsp) 5590-5936 bp
• CAG promoter 3779-5423 bp
• Rabbit b-globin polyA signal (3' UTR and 3’ flanking region of rabbit beta-globin, including polyA signal): 6025-6543 bp

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