WO2022189811A1 - Minimal nephrin promoter - Google Patents

Minimal nephrin promoter Download PDF

Info

Publication number
WO2022189811A1
WO2022189811A1 PCT/GB2022/050649 GB2022050649W WO2022189811A1 WO 2022189811 A1 WO2022189811 A1 WO 2022189811A1 GB 2022050649 W GB2022050649 W GB 2022050649W WO 2022189811 A1 WO2022189811 A1 WO 2022189811A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
promoter
nucleotide sequence
vector
identity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2022/050649
Other languages
English (en)
French (fr)
Inventor
Moin SALEEM-UDDIN
Gavin WELSH
Valeryia KUZMUK
Alan William GRIFFITH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Bristol
Syncona Investment Management Ltd
Purespring Therapeutics Ltd
Original Assignee
University of Bristol
Syncona Investment Management Ltd
Purespring Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/GB2021/050633 external-priority patent/WO2021181118A1/en
Priority claimed from PCT/GB2021/051668 external-priority patent/WO2022003357A1/en
Priority to KR1020237034581A priority Critical patent/KR20230161459A/ko
Priority to ES22710714T priority patent/ES3056304T3/es
Priority to EP22710714.1A priority patent/EP4305181B1/en
Priority to DK22710714.1T priority patent/DK4305181T3/da
Priority to CN202280034399.8A priority patent/CN117377770A/zh
Priority to MX2023010573A priority patent/MX2023010573A/es
Priority to US18/280,699 priority patent/US20250283072A1/en
Priority to PL22710714.1T priority patent/PL4305181T3/pl
Application filed by University of Bristol, Syncona Investment Management Ltd, Purespring Therapeutics Ltd filed Critical University of Bristol
Priority to BR112023018362A priority patent/BR112023018362A2/pt
Priority to CA3211566A priority patent/CA3211566A1/en
Priority to EP25181596.5A priority patent/EP4722371A2/en
Priority to HRP20251561TT priority patent/HRP20251561T1/hr
Priority to IL305714A priority patent/IL305714A/en
Priority to FIEP22710714.1T priority patent/FI4305181T3/fi
Priority to AU2022235209A priority patent/AU2022235209A1/en
Priority to JP2023555709A priority patent/JP2024509316A/ja
Publication of WO2022189811A1 publication Critical patent/WO2022189811A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/63—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/63—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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C12N15/8645—Adeno-associated virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination

Definitions

  • the present invention relates to promoters which are capable of driving transgene expression in podocytes.
  • the present invention also relates to polynucleotides and vectors comprising said promoters.
  • the glomerular filtration barrier comprises 3 main layers: the glomerular endothelial cell, the glomerular basement membrane (GBM) and the podocyte.
  • the GBM is made of a highly crosslinked macromolecular meshwork of type IV collagen, proteoglycans, and laminin. Genetic forms of glomerular disease can be caused by genetic defects in these molecular structures. For example, Alport syndrome is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, which result in abnormalities of the collagen IV a345 network of basement membranes. Alport syndrome affects approximately 1 in 5,000-10,000 of all individuals in continental Europe and the USA. The condition usually presents during childhood and is associated with a spectrum of phenotypes that include a progressive loss of kidney function, and can also include hearing loss and eye abnormalities. Other GBM-associated diseases include Pierson syndrome and Nail-patella syndrome (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).
  • the podocyte has also been implicated as a key cell in the progression of glomerular disease.
  • Podocytes are mesodermally derived cells that are highly specialized and found only in the renal glomerulus. They exhibit unique characteristics such as foot processes and slit diaphragms, which are critical for glomerular filtration.
  • Podocyte-associated genetic glomerular diseases include Nephrotic Syndrome, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, and Epstein and Fechtner syndrome. (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).
  • glomerular cells such as podocytes
  • promoter sequences which can drive transgene expression in glomerular cells, such as podocytes, are required.
  • Wong et al. (American Journal of Physiology Renal Physiology; 2000; 279(6); F1027-32) described a 1.25-kb DNA fragment from the human nephrin promoter and 5'-flanking region that is capable of directing podocyte-specific expression.
  • This promoter has been used to achieve kidney-specific expression of GFP using an AAV9 vector (Picconi et al. ] 2014; Molecular Therapy - Methods & Clinical Development; 1 , 14014).
  • vector cargo capacity e.g. AAV cargo capacity
  • non-coding elements such as the nephrin or podocin promotors, WPRE elements and polyadenylation sequence.
  • the present invention is based on the inventors surprising provision of a minimal promoter which is capable of driving transgene expression in kidney cells, in particular glomerular cells, such as podocytes.
  • the present invention provides a promoter comprising (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4 or 17 and wherein the promoter has a length of about 1.1 kb or less.
  • the present invention provides a promoter which comprises or consists of a nucleotide sequence according to SEQ ID NO: 1, but wherein:
  • the present invention provides a promoter which comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a nucleotide sequence according to SEQ ID NO: 1 but wherein:
  • position 493 of SEQ ID NO: 1 is substituted with G
  • position 1080 of SEQ ID NO: 1 is substituted with T
  • position 1169 of SEQ ID NO: 1 is substituted with C
  • position 1249 of SEQ ID NO: 1 is substituted with G.
  • the promoter may comprise (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4 or 17 and/or the promoter may have a length of about 1.1 kb or less.
  • the promoter may be operable in a kidney cell.
  • the promoter may be capable of driving transgene expression in kidney.
  • the promoter may be a kidney-specific promoter.
  • the promoter may be operable in a podocyte cell.
  • the promoter may be capable of driving transgene expression in podocytes.
  • the promoter may be a podocyte-specific promoter.
  • the promoter further comprises (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 5 or 18, a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 6 or 19, and/or a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 7 or 20.
  • the promoter further comprises (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 6 or 19.
  • the promoter further comprises (iii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 8, or one or more fragments thereof.
  • the promoter comprises from 5’ to 3’: (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7.
  • the promoter consists of from 5’ to 3’: (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7, and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter consists of from 5’ to 3’: (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 17; and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20.
  • the promoter consists of from 5’ to 3’: (i) the nucleotide sequence of SEQ ID NO: 17; and (ii) the nucleotide sequence of SEQ ID NO: 20.
  • the promoter comprises from 5’ to 3’: (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 17; and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20, and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter comprises from 5’ to 3’: (i) the nucleotide sequence of SEQ ID NO: 17; and (ii) the nucleotide sequence of SEQ ID NO: 20, and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter may have a length of about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less.
  • the promoter may have a length of from 0.265 kb to 1.0 kb, from 0.265 kb to 0.9 kb, from 0.265 kb to 0.8 kb, from 0.265 kb to 0.7 kb, from 0.265 kb to 0.6 kb, from 0.265 kb to 0.5 kb, from 0.265 kb to 0.4 kb, or from 0.265 kb to 0.3 kb.
  • the promoter may comprise: (a) a retinoic acid receptor binding site; (b) a WT 1 binding site; (c) an enhancer box; (d) a transcription factor binding region; and/or (e) a transcription initiation site.
  • the retinoic acid receptor binding site comprises or consists of a nucleotide sequence shown as SEQ ID NO: 10 or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 10.
  • the WT1 binding site comprises or consists of a nucleotide sequence shown as SEQ ID NO: 11 or a nucleotide sequence having one, two or three substitutions, deletions, or insertions compared to SEQ ID NO: 11.
  • the enhancer box comprises or consists of a nucleotide sequence shown as SEQ ID NO: 12 or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 12.
  • the transcription factor binding region comprises or consists of a nucleotide sequence shown as SEQ ID NO: 13 or 43 or a nucleotide sequence having one, two, three, four or five substitutions, deletions, or insertions compared to SEQ ID NO: 13 or 43.
  • the transcription initiation site comprises or consists of an “AG” dinucleotide.
  • the transcription factor binding site is operably linked to the transcription initiation site, optionally wherein the transcription factor binding site is directly upstream of the transcription initiation site.
  • the nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4 or 17 may comprise one or more of: (a) a retinoic acid receptor binding site; (b) a WT 1 binding site; and (c) an enhancer box.
  • One or more of the following nucleotide sequences may be present in the nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4 or 17:
  • the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 15, SEQ ID NO: 59, or SEQ ID NO: 47.
  • the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 47. In more preferred embodiments, the promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 47.
  • the present invention provides a promoter which consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 2.
  • the present invention provides a promoter which consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 3.
  • the present invention provides a promoter which consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 15.
  • the present invention provides a promoter which consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 59.
  • the present invention provides a promoter which consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 47.
  • the present invention provides a promoter which consists of the nucleotide sequence of SEQ ID NO: 47.
  • the present invention provides a polynucleotide comprising the promoter according to the present invention.
  • the promoter may be operably linked to a protein coding sequence.
  • the protein coding sequence encodes NPHS2 or a fragment and/or variant thereof; a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof; or CFI, CFH or FHL-1, or a fragment and/or variant thereof.
  • the protein coding sequence encodes NPHS2, or a fragment and/or variant thereof.
  • the protein coding sequence encodes an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 55 ora fragment thereof.
  • the protein coding sequence comprises or consists of a nucleotide sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 56 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 57 or a fragment thereof.
  • the protein coding sequence encodes a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to one of SEQ ID NOs: 21-23 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to one of SEQ ID NOs: 24-26 or a fragment thereof.
  • the protein coding sequence encodes CFI, or a fragment and/or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 49 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 50 or a fragment thereof.
  • the protein coding sequence encodes CFH, or a fragment and/or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 51 ora fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 52 or a fragment thereof.
  • the protein coding sequence encodes FHL-1 , or a fragment and/or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 53 ora fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 54 or a fragment thereof.
  • the protein coding sequence is operably linked to one or more further regulatory elements, such as a post-transcriptional regulatory element and/or a polyadenylation sequence.
  • the present invention provides a vector comprising the polynucleotide according to the present invention.
  • the vector is capable of transducing podocytes, optionally wherein the vector is capable of specifically transducing podocytes.
  • the vector is a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an alphaviral vector, a flaviviral vector, a rhabdoviral vector, a measles viral vector, a Newcastle disease viral vector, a poxviral vector, and a picornaviral vector, preferably wherein the vector is an AAV vector.
  • the viral vector is in the form of a viral vector particle, preferably wherein the viral vector is in the form of an AAV vector particle.
  • the vector is the form of an AAV vector particle encapsidated by AAV3B, LK03, or AAV9 capsid proteins.
  • the present invention provides a cell comprising the polynucleotide according to the present invention, or the vector according to the present invention.
  • the present invention provides a pharmaceutical composition comprising the polynucleotide according to the present invention, the vector according to the present invention, or the cell according to the present invention.
  • the present invention provides a polynucleotide according to the present invention, vector according to the present invention, or cell according to the present invention for use in medicine.
  • the present invention provides for use of the promoter according to the present invention to drive expression of a coding sequence.
  • the expression may be kidney-specific.
  • the expression is podocyte-specific.
  • FIG. 1 Schematic illustration of minimal nephrin promoters
  • An exemplary full-length nephrin promoter may be 1249 bp in length (excluding the start codon), hereafter referred to as the “FL” nephrin promoter.
  • An exemplary minimal nephrin promoter with the 5’ region deleted may be 819 bp in length (excluding the start codon), hereafter referred to as the “midi” nephrin promoter.
  • An exemplary minimal nephrin promoter with the 5’ region deleted and central region deleted may be 265 bp in length (excluding the start codon), hereafter referred to as the “mini” nephrin promoter.
  • D The following regions of the nephrin promoter are indicated: (i) human mouse homology region, (ii) retinoic acid receptor (RAR) binding site, (iii) WT1 binding site, (iv) Enhancer box, (v) transcription factors binding region, and (vi) transcription initiation site.
  • FIG. 2 Schematic of a lentiviral vector comprising GFP operably coupled to midi nephrin promoter
  • pACE_hNPHS1 promoter was used as a template, to introduce BamH1 and Cla1 restriction sites.
  • B Final construct vector comprising GFP operably coupled to midi nephrin promoter.
  • FIG. 3 Schematic of a lentiviral vector comprising GFP operably coupled to mini nephrin promoter
  • pACE_hNPHS1 promoter was used as a template to PCR and gel extract the two sections of the promoter.
  • B Final construct vector comprising GFP operably coupled to mini nephrin promoter.
  • CiPodocytes stably expressing GFP-tagged nephrin promoters were generated using the lentiviral approach. GFP expression was observed by fluorescence microscopy.
  • A Untransduced CiPodocytes.
  • B CiPodocytes stably expressing the GFP-tagged mini nephrin promoter.
  • C CiPodocytes stably expressing the GFP-tagged FL nephrin promoter.
  • Lentiviral vectors comprising GFP operably linked to nephrin promoters were transduced into differentiated conditionally immortalised podocytes (CiPodocytes). Immunoprecipitation (IP) was used to detect GFP expression.
  • Figure 6 Human glomerular cells transduced with Lentivirus - GFP.nephrin promoter (FL or 265)
  • FACS analysis displaying median GFP fluorescence (AFU) of all live singlets of conditionally immortalised human podocytes (LY) and glomerular endothelial cells (GEnC) using a Novocyte Analyser.
  • Untransduced cells (Cell Control) were compared with those transduced with lentivirus constructs harbouring a GFP expression cassette controlled by a full length human nephrin promoter (hNPHSI .GFP) or a mini human nephrin promoter (265. GFP). All cells were differentiated for 10 days, trypsinised (100 uL) and diluted in PBS, 2%FBS, 1:1000 DRAQ7 (150uL). Data and error bars represent 3 technical repeats (100 uL, >2500 cells) ⁇ SEM.
  • FIG. 7 AAV transfer plasmids comprising COL4A3, COL4A4, and COL4A5 coupled to mini nephrin promoter
  • FIG. 8 Podocytes transduced with AAV.COL4.nephrin265.Sv40 virus
  • Col4a5 Confocal images showing immunofluorescence staining of transduced Col4a5 in Human wild-type CiPodocytes/Col4a5 3xFlag AAV CiPodocytes with F-Actin.
  • Col4a5 is present at the cytosolic level in the human differentiated podocytes infected with Col4a5 3xFlag AAV virus in comparison to the wild-type counterpart.
  • HEK293T cells Protein expression analysis by western blot in 293T Human Embryonic Kidney cells.
  • Cells were transfected with pAAV-265-CFH, pAAV-FL-CFI or pAAV-FL-CFHL1.
  • NT non- transfected 293T HEK cells. Expression of each of the transgenes is demonstrated in the cell lysate and/or media using protein-specific antibodies or anti-FLAG-tag or anti-MYC-tag antibodies.
  • HEK293T cells transfected with plasmid expressing the CFH transgene under the control of the 265bp minimal nephrin promoter expressed and secreted human Factor H.
  • FIG. 10 Transduction of Factor H mutated podocytes (“Human early disease (ED) podocytes”) with AAV2/9 265-CFH or transfection with plasmid encoding CFH
  • A Analysis of human Factor H concentration using an ELISA assay. Podocytes transduced with AAV2/9 virus containing the CFH transgene demonstrated higher concentrations of human Factor H in the culture media than the non-transduced control.
  • B The average human Factor H concentration from (A).
  • C Analysis of human Factor H concentration using an ELISA assay. Podocytes transfected with plasmid expressing the CFH transgene under the control of the 265bp minimal nephrin promoter demonstrated higher concentrations of human Factor H than the non-transfected control.
  • eGFP expressed in cells transduced with AAV particles incorporating transgene cassettes containing Full Length (PS0281) and 265bp (PS0282) minimal nephrin promoters driving eGFP.
  • A Transduction results in human Podocytes.
  • B Transduction results in Proximal Tube Epithelial cells (PTECs).
  • C Transduction results in Glomerular Endothelial cells (GENCs).
  • D Transduction results in Mesangial cells.
  • (A) Protein expression analysis by FACs in Lenti-X293T Human Embryonic Kidney cells. Cells were transfected with 3ug of plasmid with PEI at ratio of 1:3. Each plasmid expresses human Podocin-HA driven by either full length (FL.NPSH1-Podocin-HA) or 265bp (265.hNPSH1- Podocin-HA) minimal nephrin promoter. Expression of human Podocin is demonstrated by anti-HA antibody on Day 2 post transfection. N 3 data expressed as mean values ⁇ SD.
  • Cells were transduced with AAV particles, incorporating human Podocin-HA transgene cassette driven by either full length (FL.NPSH1-Podocin-HA) or 265bp (265.hNPSH1-Podocin-HA) minimal nephrin promoter. Expression of human Podocin is demonstrated by anti-HA antibody on Day 9 post transduction and differentiation.
  • the present invention provides a promoter for glomerular gene therapy.
  • a “promoter” is used herein according to its typical meaning to refer to a sequence of DNA which regulates and/or initiates transcription of a RNA from a DNA sequence.
  • the promoter is operable in mammalian cells, e.g. human cells.
  • the promoter may be capable of driving transgene expression in mammalian cells, e.g. human cells.
  • the promoter may be a mammalian promoter, e.g. a human promoter.
  • the promoter may be operable in kidney cells.
  • the promoter of the invention may be capable of driving transgene expression in the kidney. Examples of kidney cells in which the promoter may be operable include, but are not limited to glomerular cells.
  • the promoter may be operable in glomerular cells.
  • the promoter of the invention may be capable of driving transgene expression in the glomerulus.
  • the mature glomerulus contains four cell types: Parietal epithelial cells that form Bowman's capsule, podocytes that cover the outermost layer of the glomerular filtration barrier, glycocalyx-coated fenestrated endothelial cells that are in direct contact with blood, and mesangial cells that sit between the capillary loops (Vaughan, M.R. and Quaggin, S.E., 2008. Journal of the American Society of Nephrology, 19(1), pp.24-33).
  • the promoter may be operable in a podocyte cell.
  • the promoter of the invention may be capable of driving transgene expression in podocytes.
  • the promoter of the invention may be a tissue-specific promoter.
  • tissue-specific promoter is a promoter which preferentially facilitates expression of a transgene in a specific type of cells or tissue.
  • a tissue-specific promoter may facilitate higher expression of a transgene in one cell-type as compared to other cell-types.
  • tissue-specific promoter may be a promoter which facilitates transgene expression levels at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in one cell-type as compared to expression levels in other cell-types.
  • the promoter is a kidney-specific promoter. In some embodiments, the promoter is a glomerular-specific promoter. In some embodiments, the promoter is a podocyte-specific promoter.
  • Transgene expression may be measured by any suitable method known in the art. For example by measuring the expression of a reporter transgene, e.g. Green fluorescent protein (GFP), operably linked to the promoter, wherein expression of the reporter transgene correlates with the ability of the promoter to facilitate expression of a gene.
  • a reporter transgene e.g. Green fluorescent protein (GFP)
  • GFP Green fluorescent protein
  • Expression of the reporter transgene, e.g. GFP may be determined by any suitable method e.g. FACS.
  • a kidney-specific promoter may facilitate higher expression of a reporter transgene in kidney cells compared to other cell types (e.g. CNS, retina, lung, pancreas, heart, or muscle cells).
  • a podocyte-specific promoter may facilitate higher expression of a reporter transgene in conditionally immortalised podocytes compared to other cell-types.
  • Suitable podocyte cell lines will be well known to those of skill in the art, for example CIHP-1.
  • Methods to generate immortalized podocytes will be well known to those of skill in the art. Suitable methods are described in Ni, L, et al., 2012. Nephrology, 17(6), pp.525-531.
  • the promoter is a minimal kidney-specific promoter. In some embodiments, the promoter is a minimal glomerular-specific promoter. In some embodiments, the promoter is a minimal podocyte-specific promoter.
  • the promoter may have a length of about 1.2 kb or less.
  • the promoter has a length of about 1.18 kb or less, about 1.17 kb or less, about 1.16 kb or less, about 1.15 kb or less, about 1.14 kb or less, about 1.13 kb or less, about 1.12 kb or less, about 1.11 kb or less, or about 1.10 kb or less.
  • the promoter has a length of about 1.15 kb or less.
  • the promoter may have a length of about 1.1 kb or less. In some embodiments, the promoter has a length of about 1.1 kb or less, 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less.
  • the promoter has a length of about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. In some embodiments, the promoter has a length of 818 bp or less. In some embodiments, the promoter has a length of 800 bp or less.
  • the promoter has a length of about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. In some embodiments, the promoter has a length of about 0.3 kb or less.
  • the promoter may have a length of about 250 bp or more. In some embodiments, the promoter has a length of about 250-1100 bp, 250-1000 bp, 250-900 bp, 250-800 bp, 250-700 bp, 250- 600 bp, 250-500 bp, 250-400 bp, 250-300 bp.
  • the promoter may have a length of about 265 bp or more. In some embodiments, the promoter has a length of about 265-1100 bp, 265-1000 bp, 265-900 bp, 265-800 bp, 265-700 bp, 265- 600 bp, 265-500 bp, 265-400 bp, 265-300 bp.
  • the promoter has a length of 250-300 bp, 250-280 bp, 255-275 bp, 260- 270 bp, or about 265 bp. In one embodiment, the promoter has a length of 800-850 bp, 800- 840 bp, 810-830 bp, 815-825 bp, about 819 bp, or about 818 bp.
  • Nephrin promoters The promoter may be a synthetic promoter.
  • a “synthetic promoter” is a promoter which does not occur naturally.
  • a synthetic promoter will be produced by genetic modification of a pre-existing promoter.
  • a synthetic promoter may be derived from a pre-existing promoter, i.e. identical to a pre-existing promoter, except comprising one or more genetic modifications (e.g. substitutions, deletions, or insertions).
  • the promoter may be derived from the nephrin (NPHS1) promoter.
  • the promoter may be a minimal nephrin promoter.
  • the NPHS1 gene encodes nephrin, which is selectively expressed in podocytes.
  • NPHS1 promoter A human NPHS1 promoter has been described in Moeller et al. 2002 J Am Soc Nephrol, 13(6): 1561-7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6): F1027-32.
  • This NPHS1 promoter is a 1.2kb fragment and appears to be podocyte-specific.
  • the 1.2kb promoter region lacks a TATA box, but has recognition motifs for other transcription factors e.g. PAX-2 binding element, E-box and GATA consensus sequences.
  • nephrin promoters are shown as SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 44, and SEQ ID NO: 45.
  • nephrin promoter- 1,249 bp SEQ ID NO: 1
  • nephrin promoter- 1, 192 bp SEQ ID NO: 14
  • the promoter of the present invention is derived from SEQ ID NO: 1 or a variant that has at least 70% identity to SEQ ID NO: 1.
  • the promoter of the present invention has one or more deletions compared to SEQ ID NO: 1 or a variant that has at least 70% identity to SEQ ID NO: 1.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 1.
  • An exemplary variant is SEQ ID NO: 14, SEQ ID NO: 44, or SEQ ID NO: 45.
  • the variant of SEQ ID NO: 1 comprises one or more of the following substitutions: A493G, C1080T, G1169C, and C1249G.
  • the variant of SEQ ID NO: 1 comprises each of the substitutions A493G, C1080T, G1169C, and C1249G (see SEQ ID NO: 14).
  • the promoter of the present invention is derived from SEQ ID NO: 14, SEQ ID NO: 44, or SEQ ID NO: 45, or a variant that has at least 70% identity to SEQ ID NO: 14, SEQ ID NO: 44, or SEQ ID NO: 45.
  • the promoter of the present invention has one or more deletions compared to SEQ ID NO: 14, SEQ ID NO: 44, or SEQ ID NO: 45, or a variant that has at least 70% identity to SEQ ID NO: 14, SEQ ID NO: 44, or SEQ ID NO: 45.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 14, SEQ ID NO: 44, or SEQ ID NO: 45.
  • the promoter of the present invention is derived from SEQ ID NO: 14, or a variant that has at least 70% identity to SEQ ID NO: 14.
  • the promoter of the present invention has one or more deletions compared to SEQ ID NO: 14, or a variant that has at least 70% identity to SEQ ID NO: 14.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 14.
  • the promoter of the present invention comprises or consists of a nucleotide sequence according to SEQ ID NO: 1 and having one or more deletions, e.g. one or two deletions, or nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • the promoter of the present invention comprises or consists of a nucleotide sequence according to SEQ ID NO: 1 and having two or more deletions, or nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • the deletions may be any size.
  • the deletions are each at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, or at least 400 bp in size.
  • the deletions are each from 50 to 500 bp, from 100 to 500 bp, from 150 to 500 bp, from 200 to 500 bp, from 250 to 500 bp, from 300 to 500 bp, from 350 to 500 bp, or from 400 to 500 bp in size.
  • the promoter of the present invention comprises or consists of a nucleotide sequence according to SEQ ID NO: 1 , but wherein:
  • n2 position n2 to position n3 of SEQ ID NO: 1 is deleted, wherein n3 3 n2, n2 is an integer from 508 to 1061 , and n3 is an integer from 508 to 1061 ; or a nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • nucleotide positions above are referred to by reference to a position in SEQ ID NO: 1 , the skilled person would be able to readily identify the corresponding nucleotide location in variants thereof, for example by aligning SEQ ID NO: 1 and the variant sequence.
  • position 493 of SEQ ID NO: 1 corresponds to position 436 of SEQ ID NO: 14
  • position 1080 of SEQ ID NO: 1 corresponds to position 1023 of SEQ ID NO: 14
  • position 1169 of SEQ ID NO: 1 corresponds to position 1112 of SEQ ID NO: 14
  • position 1249 of SEQ ID NO: 1 corresponds to position 1192 of SEQ ID NO: 14.
  • the promoter of the present invention may comprise or consist of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a nucleotide sequence according to SEQ ID NO: 1 , but wherein:
  • position 493 of SEQ ID NO: 1 is substituted with G
  • position 1080 of SEQ ID NO: 1 is substituted with T
  • position 1169 of SEQ ID NO: 1 is substituted with C
  • position 1249 of SEQ ID NO: 1 is substituted with G.
  • position 493 of SEQ ID NO: 1 is substituted with G
  • position 1080 of SEQ ID NO: 1 is substituted with T
  • position 1169 of SEQ ID NO: 1 is substituted with C
  • position 1249 of SEQ ID NO: 1 is substituted with G.
  • n3 and n2 specifies the size of the deletion.
  • n3 3 n2 + 49 is an integer portion of the deletion.
  • n2 and n3 are each integers from 550 to 1050, n2 and n3 are each integers from 600 to 1000, n2 and n3 are each integers from 650 to 950, n2 and n3 are each integers from 700 to 900, n2 and n3 are integers from 750 to 850.
  • Promoter regions The present inventors have determined the regions of the nephrin promoter which drive transgene expression.
  • a promoter typically comprises a “core” and a “proximal” region.
  • the “core promoter region” may comprise a transcription start site, a RNA polymerase binding sites and a general transcription factor binding site.
  • the “proximal promoter region” may comprise primary regulatory elements and specific transcription factor binding sites which are required, for example, to facilitate effective and controllable transcription. The size and components of both the core and proximal promoter regions typically vary in a gene specific manner.
  • a promoter may also comprise a 5' untranslated region (5' UTR) (also known as a leader sequence) downstream of the core promoter region and upstream from the initiation codon. (See e.g. Figure 1).
  • 5' UTR also known as a leader sequence
  • the promoter may be a hybrid promoter.
  • a “hybrid promoter” comprises a combination of elements derived from different promoters.
  • a hybrid promoter may comprise a proximal promoter region derived from one pre-existing promoter and a core promoter from another pre-existing promoter to achieve the desired transgene expression.
  • a muscle hybrid promoter is described in Piekarowicz, K., et al. (2019). Methods & clinical development, 15, 157-169.
  • the promoter of the present invention comprises (i) the nucleotide sequence shown as SEQ ID NO: 4, or a variant which is at least 70% identical to SEQ ID NO: 4.
  • a nucleotide sequence having at least about 70% identity to SEQ ID NO: 4 may provide a proximal promoter region.
  • proximal promoter region SEQ ID NO: 4.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 4.
  • the promoter may comprise a variant of SEQ ID NO: 4 shown as SEQ ID NO: 17.
  • a variant of SEQ ID NO: 4 comprises the substitution A63G (see SEQ ID NO: 17).
  • the promoter of the present invention comprises (i) the nucleotide sequence shown as SEQ ID NO: 17, or a variant which is at least 70% identical to SEQ ID NO: 17.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 17.
  • Exemplary variant proximal promoter region SEQ ID NO: 17
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 5 or 18, or a variant which is at least 70% identical to SEQ ID NO: 5 or 18; the nucleotide sequence shown as SEQ ID NO: 6 or 19, or a variant which is at least 70% identical to SEQ ID NO: 6 or 19; and/or the nucleotide sequence shown as SEQ I D NO: 7 or 20, or a variant which is at least 70% identical to SEQ I D NO: 7 or 20.
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 5, or a variant which is at least 70% identical to SEQ ID NO:
  • nucleotide sequence having at least about 70% identity to SEQ ID NO: 5 may provide a core promoter region.
  • Exemplary core promoter region (SEQ ID NO: 5)
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 5.
  • the promoter may comprise a variant of SEQ ID NO: 5 shown as SEQ ID NO: 18.
  • a variant of SEQ ID NO: 5 comprises the substitution C19T (see SEQ ID NO: 18).
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 18, or a variant which is at least 70% identical to SEQ ID NO: 18.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 18.
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 6, or a variant which is at least 70% identical to SEQ ID NO:
  • nucleotide sequence having at least about 70% identity to SEQ ID NO: 6 may provide a 5’UTR.
  • Exemplary 5’UTR (SEQ ID NO: 6) AGGGGAAGAGGAAAACGAGAAAGGGAGGAGAGTAACGGAAAGAGATAAAAAAGAAAAGCAGGTGGCAG AGACACAGAGAGGGACCCAGAGAAAGCCAGACAGACGCAGGTGGCTGGCAGCGGGCGCTGTGGGGG T CACAGT AGGGGGACCT GT C
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 6.
  • the promoter may comprise a variant of SEQ ID NO: 6 shown as SEQ ID NO: 19.
  • a variant of SEQ ID NO: 6 comprises the substitutions G76C and/or C156G (see SEQ ID NO: 19).
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 19, or a variant which is at least 70% identical to SEQ ID NO: 19.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 19.
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 7, or a variant which is at least 70% identical to SEQ ID NO: 7.
  • a nucleotide sequence having at least about 70% identity to SEQ ID NO: 7 may provide a core promoter region and a 5’UTR.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 7.
  • the promoter may comprise a variant of SEQ ID NO: 7 shown as SEQ ID NO: 20.
  • a variant of SEQ ID NO: 7 comprises the substitutions C19T, G108C and/or C188G (see SEQ ID NO: 20).
  • a variant of SEQ ID NO: 7 comprises the substitutions C19T, G108C and C188G (see SEQ ID NO: 20).
  • the promoter of the present invention comprises (ii) the nucleotide sequence shown as SEQ ID NO: 20, or a variant which is at least 70% identical to SEQ ID NO: 20.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 20.
  • the promoter of the present invention comprises (iii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 8, or one or more fragments thereof.
  • the promoter comprises a nucleotide sequence having at least about 70% identity to SEQ ID NO: 8, or one or more fragments thereof, immediately downstream of the proximal promoter region and/or immediately upstream of the core promoter region.
  • the promoter may comprise a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 8, or one or more fragments thereof.
  • the promoter may comprise the nucleotide sequence of SEQ ID NO: 8, or one or more fragments thereof.
  • Exemplary optional promoter region (SEQ ID NO: 8)
  • the one or more fragments are (a) a 5’ terminal fragment; and/or (b) a 3’ terminal fragment.
  • the 5’ terminal fragment may be immediately downstream of the proximal promoter region.
  • the 3’ terminal fragment may be immediately upstream of the core promoter region.
  • the promoter of the present invention may comprise:
  • the fragment(s) of SEQ ID NO: 8 may be any length.
  • the fragment(s) may have a length of about 500 bp or less, 450 bp or less, 400 bp or less, 350 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less.
  • the promoter does not comprise SEQ ID NO: 8.
  • the promoter of the present invention comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 9, or a fragment thereof.
  • the promoter comprises a nucleotide sequence having at least about 70% identity to SEQ ID NO: 9, or a fragment thereof, immediately upstream of the proximal promoter region.
  • the promoter may comprise a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 9, or a fragment thereof.
  • the promoter may comprise the nucleotide sequence of SEQ ID NO: 9, or one or more fragments thereof.
  • Exemplary optional upstream promoter region (SEQ ID NO: 9)
  • the fragment is a 3’ terminal fragment.
  • the promoter of the present invention may comprise a nucleotide sequence having at least 70% identity to positions z to 430 of SEQ ID NO: 9, wherein z is an integer.
  • the fragment of SEQ ID NO: 9 may be any length.
  • the fragment may have a length of about 400 bp or less, 350 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less.
  • the promoter does not comprise SEQ ID NO: 9.
  • the promoter of the present invention comprises or consists of from 5’ to 3’: (i) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4 or 17;
  • nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8, or one or more fragments thereof;
  • the promoter of the present invention comprises or consists of from 5’ to 3’:
  • the promoter of the present invention comprises or consists of from 5’ to 3’:
  • nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4 or 17;
  • the promoter of the present invention comprises or consists of from 5’ to 3’:
  • the present inventors have determined the functional elements of the nephrin promoter which drive transgene expression.
  • the promoter of the invention may comprise one or more of the following elements: (a) a retinoic acid receptor binding site; (b) a WT1 binding site; (c) an enhancer box; (d) a transcription factor binding region; and (e) a transcription initiation site.
  • the promoter of the invention comprises all of the following elements: (a) a retinoic acid receptor binding site; (b) a WT1 binding site; (c) an enhancer box; (d) a transcription factor binding region; and (e) a transcription initiation site.
  • a retinoic acid receptor (RAR) binding site refers to a polynucleotide sequence which is capable of binding RAR alpha, RAR beta, and/or RAR gamma.
  • the RAR binding site may comprise or consist of a nucleotide sequence shown as SEQ ID NO: 10, or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 10.
  • the substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the RAR binding site retains at least one of its endogenous functions.
  • RAR binding site SEQ ID NO: 10.
  • a WT 1 binding site refers to a polynucleotide sequence which is capable of binding the zinc finger polypeptide encoded by the Wilms' tumor suppressor gene, WT1.
  • the WT1 binding site may comprise or consist of a nucleotide sequence shown as SEQ ID NO: 11 , or a nucleotide sequence having one, two or three substitutions, deletions, or insertions compared to SEQ ID NO: 11.
  • the substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the WT1 binding region retains at least one of its endogenous functions.
  • An enhancer box refers to a DNA response element found in some eukaryotes that acts as a protein-binding site.
  • the enhancer box may comprise or consist of a nucleotide sequence shown as SEQ ID NO: 12, ora nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 12.
  • the substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the enhancer box retains at least one of its endogenous functions.
  • a retinoic acid receptor binding site may be present in the proximal promoter region.
  • an enhancer box may be present in the proximal promoter region.
  • each of (a) a retinoic acid receptor binding site; (b) a WT1 binding site; and (c) an enhancer box are present in the proximal promoter region
  • one or more of the following elements is present in (i) the nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17: (a) a RAR binding site at a position corresponding approximately to position 7 to position 13 of SEQ ID NO: 4 or 17; (b) a WT1 binding site at a position corresponding approximately to position 14 to position 30 of SEQ ID NO: 4 or 17; and (c) an enhancer box at a position corresponding approximately to position 49 to position 53 of SEQ ID NO: 4 or 17.
  • each of the elements are present in (i) the nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17.
  • one or more of the following nucleotide sequences is present in (i) the nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17: (a) GGGGTCA at a position corresponding to position 7 to position 13 of SEQ ID NO: 4 or 17; (b) CGGAGGCTGGGGAGGCA at a position corresponding to position 14 to position 30 of SEQ ID NO: 4 or 17; and (c) ATGTG at a position corresponding to position 49 to position 53 of SEQ ID NO: 4 or 17.
  • each of the nucleotide sequences are present in (i) the nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17.
  • the promoter may comprise a transcription factor binding region comprising or consisting of a nucleotide sequence shown as SEQ ID NO: 13 or a nucleotide sequence having one, two, three, four or five substitutions, deletions, or insertions compared to SEQ ID NO: 13.
  • the substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the transcription factor binding region retains at least one of its endogenous functions.
  • An exemplary variant is shown in SEQ ID NO: 43.
  • a variant of SEQ ID NO: 13 comprises the substitution C19T (see SEQ ID NO: 43).
  • the promoter may comprise a transcription factor binding region comprising or consisting of a nucleotide sequence shown as SEQ ID NO: 43 or a nucleotide sequence having one, two, three, four or five substitutions, deletions, or insertions compared to SEQ ID NO: 43.
  • the substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the transcription factor binding region retains at least one of its endogenous functions.
  • TACGAT SEQ ID NO: 37
  • TATAAT SEQ ID NO: 38
  • GATACT SEQ ID NO: 39
  • TATGAT SEQ ID NO: 40
  • TATGTT SEQ ID NO: 41
  • the promoter may comprise a transcription initiation site which comprises or consists of an “AG” dinucleotide.
  • the transcription factor binding site is operably linked to the transcription initiation site.
  • the transcription factor binding site may be directly upstream of the transcription initiation site. Without wishing to be bound by theory, it is considered that the transcription factor binding site and the transcription initiation site may provide a core promoter region.
  • the promoter may comprise a 5’ untranslated region.
  • the 5’ untranslated region may comprise or consist of a nucleotide sequence having at least about 70%, 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 6 or 19.
  • the 5’ untranslated region may comprise or consist of SEQ ID NO: 6 or 19.
  • the 5’ untranslated region is operably linked to the transcription initiation site.
  • the 5’ untranslated region may be directly downstream of the transcription initiation site.
  • the promoter of the present invention comprises or consists of from 5’ to 3’:
  • nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4 or 17, wherein each of the following elements is present: (a) a RAR binding site at a position corresponding approximately to position 7 to position 13 of SEQ ID NO: 4 or 17; (b) a WT1 binding site at a position corresponding approximately to position 14 to position 30 of SEQ ID NO: 4 or 17; and (c) an enhancer box at a position corresponding approximately to position 49 to position 53 of SEQ ID NO: 4 or 17;
  • nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8, or one or more fragments thereof;
  • the promoter of the present invention comprises or consists of from 5’ to 3’:
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 2, or a variant which is at least 70% identical to SEQ ID NO: 2.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 2, or a variant which is at least 70% identical to SEQ ID NO: 2 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 2, or a variant which is at least 70% identical to SEQ ID NO: 2.
  • Exemplary minimal nephrin promoter - 819 bp (SEQ ID NO: 2)
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 2.
  • the promoter may comprise or consist of a variant of SEQ ID NO: 2 shown as SEQ ID NO: 15 or SEQ ID NO: 46.
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 15, or a variant which is at least 70% identical to SEQ ID NO: 15.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 15, or a variant which is at least 70% identical to SEQ ID NO: 15 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 15, or a variant which is at least 70% identical to SEQ ID NO: 15.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 15.
  • Exemplary minimal nephrin promoter variant - 819 bp (SEQ ID NO: 15)
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 46, or a variant which is at least 70% identical to SEQ ID NO: 46.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 46, or a variant which is at least 70% identical to SEQ ID NO: 46 and wherein the promoter has a length of about 1.1 kb or less. In some embodiments, the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 46, or a variant which is at least 70% identical to SEQ ID NO: 46.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 46.
  • Exemplary minimal nephrin promoter variant - 819 bp (SEQ ID NO: 46)
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 58, or a variant which is at least 70% identical to SEQ ID NO: 58.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 58, or a variant which is at least 70% identical to SEQ ID NO: 58 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 58, or a variant which is at least 70% identical to SEQ ID NO: 58.
  • Exemplary minimal nephrin promoter - 818 bp (SEQ ID NO: 58)
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 58.
  • the promoter may comprise or consist of a variant of SEQ ID NO: 58 shown as SEQ ID NO: 59 or SEQ ID NO: 60.
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 59, or a variant which is at least 70% identical to SEQ ID NO: 59.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 59, or a variant which is at least 70% identical to SEQ ID NO: 59 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 59, or a variant which is at least 70% identical to SEQ ID NO: 59.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 59.
  • Exemplary minimal nephrin promoter variant - 818 bp (SEQ ID NO: 59)
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 60, or a variant which is at least 70% identical to SEQ ID NO: 60.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 60, or a variant which is at least 70% identical to SEQ ID NO: 60 and wherein the promoter has a length of about 1.1 kb or less. In some embodiments, the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 60, or a variant which is at least 70% identical to SEQ ID NO: 60.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 60.
  • Exemplary minimal nephrin promoter variant - 818 bp (SEQ ID NO: 60)
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 3, or a variant which is at least 70% identical to SEQ ID NO: 3.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 3, or a variant which is at least 70% identical to SEQ ID NO: 3 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 3, or a variant which is at least 70% identical to SEQ ID NO: 3.
  • Exemplary minimal nephrin promoter - 265 bp (SEQ ID NO: 3)
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 3.
  • a variant of SEQ ID NO: 3 comprises one or more of the substitutions A63G, C96T, G185C, and C265G.
  • a variant of SEQ ID NO: 3 comprises the substitutions A63G, C96T, G185C, and C265G (cf SEQ ID NOs: 17-20).
  • the promoter may comprise or consist of a variant of SEQ ID NO: 3 shown as SEQ ID NO: 16 or SEQ ID NO: 47.
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 16, or a variant which is at least 70% identical to SEQ ID NO: 16.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 16, or a variant which is at least 70% identical to SEQ ID NO: 16 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 16, or a variant which is at least 70% identical to SEQ ID NO: 16.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 16.
  • Exemplary minimal nephrin promoter variant - 265 bp (SEQ ID NO: 16)
  • the promoter of the present invention comprises or consists of the nucleotide sequence shown as SEQ ID NO: 47, or a variant which is at least 70% identical to SEQ ID NO: 47.
  • the promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO: 47, or a variant which is at least 70% identical to SEQ ID NO: 47 and wherein the promoter has a length of about 1.1 kb or less.
  • the promoter of the present invention consists of the nucleotide sequence shown as SEQ ID NO: 47, or a variant which is at least 70% identical to SEQ ID NO: 47.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 47.
  • Exemplary minimal nephrin promoter variant - 265 bp (SEQ ID NO: 47) GGCCCTGGGGTCACGGAGGCTGGGGAGGCACCGAGGAACGCGCCTGGCATGTGCTGACAGGGGATTTT
  • the present invention provides a polynucleotide comprising the promoter of the present invention.
  • Polynucleotides of the invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotide may be an isolated polynucleotide.
  • Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.
  • polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention.
  • the promoter may be operably linked to one or more protein-coding sequences.
  • the protein-coding sequence may encode any polypeptide of interest.
  • the protein-coding sequence can encode any polypeptide associated with a glomerular disease.
  • the protein-coding sequence may encode a polypeptide involved in with a GBM-associated genetic glomerular disease, such as Alport Syndrome.
  • the protein-coding sequence may encode a polypeptide involved in podocyte-associated genetic glomerular disease.
  • the protein-coding sequence may encode a COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, CFHL1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2,
  • the protein-coding sequence encodes a polypeptide which has a length of 1450 amino acids or more, 1500 amino acids or more, 1550 amino acids or more, 1600 amino acids or more, or 1650 amino acids or more.
  • polypeptide sequence encoded by the polynucleotides of the invention may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.
  • the protein-coding sequence may be codon-optimised. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells (e.g. humans), as well as for a variety of other organisms.
  • the protein coding nucleotide sequences disclosed herein may comprise or lack stop codons at their 3’ end.
  • the present disclosure encompasses the SEQ ID NOs disclosed herein with the stop codons present or absent.
  • the protein-coding sequence may encode a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof.
  • COL4A3, COL4A4 and COL4A5 proteins are approximately 170-185 kDa homologous polypeptides containing collagenous Gly-X-Y repeat sequences frequently interrupted by non- collagenous sequences and forming a triple helix repeat. Each polypeptide also contains a large globular non-collagenous domain at the carboxyl-terminal end.
  • Alport syndrome is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, which result in abnormalities of the collagen IV a345 network of basement membranes.
  • the COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof may be capable of forming a collagen IV a345 network.
  • Approximately 200-300 amino acids may be removed from each of the COL4A3, COL4A4 and COL4A5 polypeptides to produce a truncated transgene suitable for a mini-gene approach.
  • the amino acids may be removed from the triple helix repeat.
  • Preferably the amino acids are not removed from the non-collagenous region.
  • the COL4A3, COL4A4 and COL4A5 polypeptides are full-length polypeptides.
  • the COL4A3, COL4A4 or COL4A5 polypeptide is human.
  • An example human COL4A3 is the COL4A3 having the UniProtKB accession number Q01955.
  • An example human COL4A4 is the COL4A3 having the UniProtKB accession number P53420.
  • An example human COL4A5 is the COL4A5 having the UniProtKB accession number P29400.
  • the COL4A3 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 21 , or a variant which is at least 70% identical to SEQ ID NO: 21.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 21.
  • the COL4A4 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 22, or a variant which is at least 70% identical to SEQ ID NO: 22.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 22.
  • the COL4A5 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 23, or a variant which is at least 70% identical to SEQ ID NO: 23.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 23.
  • the protein-coding sequence may comprise or consist of a COL4A3, COL4A4 or COL4A5 transgene.
  • An example transgene encoding COL4A3 is provided in NM_000091.5.
  • An example transgene encoding COL4A4 is provided in NM_000092.5.
  • An example transgene encoding COL4A5 is provided in NM_000495.5.
  • the COL4A3 transgene may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 24, or a variant which is at least 70% identical to SEQ ID NO: 24.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 24.
  • the COL4A4 transgene may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 25, or a variant which is at least 70% identical to SEQ ID NO: 25.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 25.
  • the COL4A5 transgene may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 26, or a variant which is at least 70% identical to SEQ ID NO: 26.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 26.
  • Exemplary COL4A3 transgene sequence (SEQ ID NO: 24) atgagcgcccggaccgcccccaggccgcaggtgctcctgctgcctgctggtgctcctggcggcggc ggcgcccgcagccagcaagggttgtgtgtctgtaaagacaaaggccagtgcttctgtgacggggccaaag gggagaagggggagaagggctttcctggaccccccggttctctggccagaaaggattcacaggtcct gaaggcttgcctggaccgcagggacccaagggctttccaggacttccaggactcacgggttccaaagg tgtaaggggaccggacccaagggctttccaggacttccaggactcacgggttccaaagg
  • Exemplary COL4A4 transgene sequence (SEQ ID NO: 25) atgtggtctctgcacatagtactaatgaggtgctccttcagattgaccaagtccttggccacaggtcc ctggtcacttatactcattctcttttctgtacaatatgtatatgggagtggaaagaaatacattggtc cttgtggaggaagagattgctctgtttgccactgtgttcctgaaaaggggtctcggggtccaccagga ccaccagggccacagggtccaattggacccctgggagccccaggacccattgggctttcaggagagaa aggaatgagaggggaccgcggccctctggagcagcaggggacaaggagataagggtccaactggtg ttt
  • Exemplary COL4A5 transgene sequence (SEQ ID NO: 26) atgaaactgcgtggagtcagcctggctgccggcttgttcttactggccctgagtctttgggggcagcc tgcagaggctgcggcttgctatgggtgttctccaggatcaaagtgtgactgcagtggcataaaagggg aaaagggagagagagggtttccaggtttggaaggacacccaggattgcctggatttccaggtccagaa gggcctccggggggacaaaagggtgatgatggaattccagggccaccaggaccaaaaggaat cagaggtcctctggacttcctggatttccagggacaccaggaccaaaaggaat cagaggtcctct
  • the COL4A3, COL4A4 or COL4A5 transgene may comprise an intron or intronic sequences, which can be used to improve gene expression.
  • the COL4A3, COL4A4 or COL4A5 transgene may comprise a protein tag, such as a hemagglutinin (HA) tag.
  • HA can be used as an epitope tag and has been shown not to interfere with bioactivity or biodistribution of proteins to which it has been added.
  • the protein tag can facilitate detection, isolation, and purification of the transgene.
  • Other suitable protein tags may include Myc tags, polyhistidine tags and flag tags.
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof.
  • the protein-coding sequence may comprise or consist of a NS-associated transgene.
  • Nephrotic syndrome is a chronic kidney disease characterized by significant proteinuria, hypoalbuminemia, oedema and hyperlipidemia.
  • the NS-associated transgene may be a gene associated with a monogenic form of NS and expressed in podocytes.
  • Suitable NS-associated transgenes include NPHS2, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAFB, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PM M2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAG 12, MY01E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XP05, TNS2 and NLRP3.
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the NS-associated transgene.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the NS-associated transgene.
  • the protein-coding sequence may encode NPHS2, or a fragment and/or variant thereof.
  • NPHS2 is the abbreviated name of the polypeptide encoded by the NPHS2 gene and is also known as podocin.
  • NPHS2 is a 42kDa hairpin like membrane-associated podocyte-specific protein that is a key component of the protein complex at the slit diaphragm; the cell-cell junction between adjacent podocyte foot processes. It localises to lipid rafts and interacts with other important slit diaphragm proteins like nephrin, CD2AP and TRPC6. It is essential in the maintenance of the slit diaphragm, and consequently the integrity of the glomerular filtration barrier.
  • a fragment and/or variant of NPHS2 may retain NPHS2 activity.
  • a fragment and/or variant of podocin may regulate glomerular permeability.
  • a fragment and/or variant of NPHS2 may have the same or similar activity to NPHS2, e.g. may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity of NPHS2.
  • a fragment and/or variant of NPHS2 comprises a transmembrane domain, with two cytoplasmic domains at the N- and C-terminus.
  • the NPHS2 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, and rat.
  • the NPHS2 may be a human NPHS2.
  • the NPHS2 may comprise or consist of a polypeptide sequence of UniProtKB accession Q9NP85, or a fragment and/or variant thereof.
  • the NPHS2 comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 55 or a fragment thereof.
  • the NPHS2 comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 55 or a fragment thereof.
  • the NPHS2 comprises or consists of SEQ ID NO: 55 or a fragment thereof.
  • NPHS2amino acid sequence SEQ ID NO: 55
  • the NPHS2 transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 56 or a fragment thereof.
  • the NPHS2 transgene comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 56 or a fragment thereof.
  • the NPHS2 transgene comprises or consists of the nucleotide sequence SEQ ID NO: 56 or a fragment thereof.
  • NPHS2 transgene sequence SEQ ID NO: 56
  • the NPHS2 transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 57 or a fragment thereof.
  • the NPHS2 transgene comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 57 or a fragment thereof.
  • the NPHS2 transgene comprises or consists of the nucleotide sequence SEQ ID NO: 57 or a fragment thereof.
  • ATGGAGAGGAGGGCGCGGAGCTCCTCCAGGGAGTCCCGCGGGCGAGGCGGCAGGACTCCGCACAAGGAG AACAAGAGGGCAAAGGCCGAGAGGAGCGGCGGAGGCCGCGGGCGCCAGGAGGCTGGGCCCGAGCCGTCG GGCTCCGGACGGGCGGGGACCCCGGGGGAGCCCCGAGCGCCCGCCGCCACGGTGGTGGACGTGGATGAG GTCCGAGGCTCCGGCGAGGAGGGCACCGAGGTGGTGGCGCTGTTGGAGAGCGAGCGGCCCGAGGAAGGT ACCAAATCCTCCGGCTTAGGGGCCTGTGAGTGGCTGTCCTCATTTCCCTGCTCTTCATCATCATG ACCTTCCCTTTTTCCATCTGGTTCTGCGTAAAGGTTGTACAAGAGTATGAAAGAGTAATTATATTCCGA CTGGGACATCTGCTTCCTGGAAGGT
  • NPHS2 transgene sequence SEQ ID NO: 57
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding NPHS2, or a fragment and/or variant thereof.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding NPHS2, or a fragment and/or variant thereof.
  • VEGF Vascular endothelial growth factor
  • the protein-coding sequence may comprise or consist of a vascular endothelial growth factor (VEGF)C transgene.
  • VEGF vascular endothelial growth factor
  • VEGFC is a lymphangiogenic growth factor, which is known to signal via two receptors, VEGFR-3 (Flt4) and VEGFR-2 (Flk4).
  • VEGFC is produced by cells in a prepropeptide form, which dimerises before being cleaved into a tetramer.
  • the VEGFC transgene may comprises a polynucleotide encoding any form of VEGFC, such as the prepropeptide form, the tetramer form, the intermediate form, or fully processed mature VEGFC.
  • VEGFC transgene comprises a polynucleotide encoding one or more polypeptides having VEGFC biological activity, i.e., peptides that can bind to and activate VEGFR-2 and/or VEGRF-3. More preferably, the VEGFC transgene comprises a polynucleotide encoding a polypeptide comprising the VEGFC homology domain and having VEGFC biological activity, i.e., a polypeptide that can bind to and activate VEGFR-2 and/or VEGRF-3. Further details of suitable VEGFC polynucleotides and polypeptides include those described in WO 2015/022447 and US 2014/0087002.
  • the VEGFC polynucleotide may comprise the VEGFC open reading frame (ORF) sequence of SEQ ID NO: 36.
  • the VEGFC polynucleotide may comprise a nucleic acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VEGFC ORF sequence of SEC ID NO: 36.
  • the variant sequence may encode a VEGFC polypeptide that has retained the capability to bind and activate VEGFR-2 and VEGFR-3.
  • VEGFC polynucleotide SEQ ID NO: 36
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the VEGF(C) transgene.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the VEGF(C) transgene.
  • the protein-coding sequence may encode a complement protein, or a fragment and/or variant thereof.
  • a “complement protein” is a protein which is part of the complement system.
  • the complement system also known as complement cascade, is a central part of the innate immunity that serves as a first line of defence against foreign and altered host cells.
  • the complement system is composed of plasma proteins produced mainly by the liver or membrane proteins expressed on cell surface. Complement operates in plasma, in tissues, or within cells. Complement proteins collaborate as a cascade to opsonize pathogens and induce a series of inflammatory responses helping immune cells to fight infection and maintain homeostasis (Merle, N.S., et al., 2015. Frontiers in immunology, 6, 262).
  • C5 is cleaved, and the assembly of the membrane attack complex (MAC) is initiated.
  • MAC membrane attack complex
  • the complement protein is selected from the list consisting of CFI, CFH, FHL-1 , C1 INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, and clusterin, or fragments and/or variants thereof.
  • the protein-coding sequence may encode an inhibitor of the complement system, or a fragment and/or variant thereof.
  • an “inhibitor of the complement system” or “complement inhibitor” is a protein which prevents activation of the complement system. Complement is tightly controlled by these inhibitors, which naturally protect self cells and tissues from unwanted complement activation. Complement inhibitors can regulate complement activation in different stages of the classical, lectin, and alternative pathways.
  • the complement inhibitor is a naturally- occurring complement inhibitor, or a fragment and/or variant thereof.
  • the inhibitor of the complement system is an inhibitor of the complement system in humans.
  • Complement inhibitors are grouped into two categories: soluble inhibitors and membrane- bound inhibitors.
  • the inhibitor of the complement system is a soluble complement inhibitor.
  • Soluble complement inhibitors include C1 inhibitor (C1 INH), complement factor I (CFI), complement factor H (CFH), complement factor H-like protein 1 (FHL-1), C4 binding protein (C4BP), clusterin and vitronectin.
  • Membrane-bound regulators include CD46, CD55, CD59, CD35 and CUB and Sushi multiple domain 1 (CSMD1).
  • the inhibitor of the complement system may be selected from: CFI, CFH, FHL-1 , C1INH, C4BP, CD46, CD55, CD59, CD35, vitronectin, clusterin, and CSMD1 , or fragments and/or variants thereof.
  • the inhibitor of the complement system is selected from: CFI, CFH, and FHL-1 , or fragments and/or variants thereof.
  • the protein-coding sequence may encode CFI, or a fragment and/or variant thereof.
  • CFI Complement factor I
  • CFI is a trypsin-like serine protease that inhibits the complement system by cleaving three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b thereby inactivating these proteins.
  • CFI is a glycoprotein heterodimer consisting of a disulfide linked heavy chain and light chain.
  • the heavy chain has four domains: an FI membrane attack complex (FIMAC) domain, CD5 domain, and low density lipoprotein receptor 1 and 2 (LDLrl and LDLr2) domains.
  • FIMAC FI membrane attack complex
  • CD5 CD5 domain
  • LDLrl and LDLr2 low density lipoprotein receptor 1 and 2
  • Factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein Upon binding of the enzyme to the substrate:cofactor complex, the heavy: light chain interface is disrupted, and the enzyme activated by allostery.
  • the light chain contains only the serine protease domain. This domain contains the catalytic triad His-362, Asp-411 , and Ser-507, which is responsible for specific cleavage of C3b and C4b.
  • the CFI or a fragment and/or variant thereof may be capable of cleaving C3b into iC3b and/or may be capable of cleaving iC3b into C3d,g.
  • the fragment and/or variant of CFI may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b-inactivating and iC3b-degradation activity of native CFI.
  • the C3b-inactivating and iC3b-degradation activity of the fragment and/or variant of CFI and native CFI may be determined using any suitable method known to those of skill in the art. For example, using a proteolytic assay.
  • the CFI is a human CFI.
  • An example human CFI is the CFI having the UniProtKB accession number P05156.
  • the CFI may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 49, or a variant which is at least 70% identical to SEQ ID NO: 49.
  • Illustrative CFI polypeptide sequence SEQ ID NO: 49:
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 49.
  • nucleotide sequence encoding CFI is NM_000204.5.
  • a protein-coding sequence encoding CFI may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 50, or a variant which is at least 70% identical to SEQ ID NO: 50.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 50.
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding CFI, or a fragment and/or variant thereof.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding CFI, or a fragment and/or variant thereof.
  • the protein-coding sequence may encode CFH, or a fragment and/or variant thereof.
  • Complement factor H regulates complement activation on self cells and surfaces.
  • CFH competes for binding of complement factor B (CFB) to C3b, acts as a cofactor for CFI- catalysed proteolytic cleavage of C3b, and accelerates the irreversible dissociation of C3bBb and C3b2Bb into their separate components.
  • CFI complement factor B
  • C3bBb complement factor B
  • C3b2Bb C3b2Bb
  • CFH is a large (155 kDa) soluble glycoprotein.
  • CFH is composed from a total of 20 domains, each containing approximately 60 amino acid residues and termed complement control protein modules (CCPs) or short consensus repeats that are joined by short linkers consisting of 3-8 residues.
  • CCP modules are numbered from 1-20 (from the N-terminus of the protein): CCPs 1-4 and CCPs 19-20 engage with C3b while CCPs 7 and CCPs 19-20 bind to GAGs and sialic acid.
  • the CFH or a fragment and/or variant thereof may be capable of binding C3b and/or C3d; and/or acting as a cofactor for the CFI-catalysed proteolytic cleavage of C3b; and/or increasing the irreversible dissociation of C3bBb and C3b2Bb into their separate components.
  • the fragment and/or variant of CFH may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native CFH.
  • the activity of the fragment and/or variant of CFH and native CFH may be determined using any suitable method known to those of skill in the art.
  • the CFH is a human CFH.
  • An example human CFH is the CFH having the UniProtKB accession number P08603.
  • the CFH may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 51 , or a variant which is at least 70% identical to SEQ ID NO: 51.
  • Illustrative CFH polypeptide sequence (SEQ ID NO: 51): MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRK GEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTN DIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPK CVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYI PNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENM RRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGR
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 51.
  • nucleotide sequence encoding CFH is NM_000186.4.
  • a protein-coding sequence encoding CFH may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 52, or a variant which is at least 70% identical to SEQ ID NO: 52.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 52.
  • the CFH fragment may be a splice variant.
  • complement factor H-like protein 1 FHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (CCPs 1-7).
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding CFH, or a fragment and/or variant thereof.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding CFH, or a fragment and/or variant thereof.
  • FHL-1 The protein-coding sequence may encode FHL-1 , or a fragment and/or variant thereof.
  • FHL-1 or a fragment and/or variant thereof may be capable of binding C3b and/or C3d.
  • the fragment and/or variant of FHL-1 may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native FHL-1.
  • the activity of the fragment and/or variant of FHL-1 and native FHL-1 may be determined using any suitable method known to those of skill in the art.
  • the FHL-1 is a human FHL-1.
  • An example human FHL-1 is the FHL-1 having the NCBI Reference Sequence: NP_001014975.1.
  • the FHL-1 may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 53, or a variant which is at least 70% identical to SEQ ID NO: 53.
  • FHL-1 polypeptide sequence SEQ ID NO: 53:
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 53.
  • nucleotide sequence encoding FHL-1 is NM_001014975.2.
  • a proteincoding sequence encoding FHL-1 may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 54, or a variant which is at least 70% identical to SEQ ID NO: 54.
  • FHL-1 polynucleotide sequence SEQ ID NO: 54:
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 54.
  • a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding FHL-1 , or a fragment and/or variant thereof.
  • a promoter consisting of the nucleotide sequence of SEQ ID NO: 47 is operably linked to the protein-coding sequence encoding FHL-1, or a fragment and/or variant thereof.
  • the polynucleotide may comprise one or more further regulatory sequences which may act pre- or post-transcriptionally.
  • the proteincoding sequence may be operably linked to one or more further regulatory sequences.
  • the one or more further regulatory sequences may facilitate expression of the protein in glomerular cells (e.g. podocytes).
  • regulatory sequences are any sequences which facilitate expression of the polypeptides, e.g. act to increase expression of a transcript or to enhance mRNA stability. Suitable further regulatory sequences include for example enhancer elements, post-transcriptional regulatory elements and polyadenylation sites.
  • the polynucleotide of the present invention does not comprise any further regulatory sequences (except the promoter of the invention).
  • the polynucleotide of the invention may comprise an enhancer.
  • the enhancer may be operably linked to the protein-coding sequence.
  • the enhancer may facilitate expression of the protein in glomerular cells (e.g. podocytes).
  • the enhancer is a mammalian enhancer e.g. a human enhancer.
  • Enhancers are cis-acting. They can be located up to 1 Mbp (1 ,000,000 bp) away from the gene, upstream or downstream from the start site. Any suitable enhancer may be used, the selection of which may be readily made by the skilled person.
  • tissue specific enhancer is an enhancer which preferentially facilitates expression of a gene in specific cells or tissues.
  • tissue-specific enhancer may facilitate higher expression of a gene in specific cells-types as compared to other cell-types. Higher expression may be measured for example by measuring the expression of a transgene, e.g. GFP, operably linked to the enhancer, wherein expression of the transgene correlates with the ability of the enhancer to facilitate expression of a gene.
  • tissue-specific enhancer may be an enhancer which facilitates gene expression levels at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in a specific cell-type compared to expression levels in other cell types.
  • the enhancer may be a kidney-specific enhancer, preferably a glomerular-specific enhancer, more preferably a podocyte-specific enhancer.
  • the enhancer may be operably linked to the protein-coding sequence.
  • the enhancer may be or may be derived from an enhancer associated with a gene with selective expression in human kidney, glomerular cells, and/or podocytes. Methods to identify the enhancer regions associated with genes will be well known to those of skill in the art.
  • the podocyte-specific enhancer is a NPHS1 or a NPHS2 enhancer, or a fragment or derivative thereof.
  • the podocyte-specific enhancer is a NPHS1 enhancer, or a fragment or derivative thereof.
  • a NPHS1 enhancer has been described in Guo, G., et al. , 2004. Journal of the American Society of Nephrology, 15(11), pp.2851-2856.
  • a 186-bp fragment from the human NPHS1 promoter was capable of directing podocyte-specific expression of a b-galactosidase transgene when placed in front of a heterologous minimal promoter in transgenic mice.
  • the NPHS1 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 27, or a variant which is at least 70% identical to SEQ ID NO: 27.
  • Exemplary NPHS1 enhancer (SEQ ID NO: 21): ctgctgagctgggagaccaccttgatctgacttctcccatcttcccagcctaagccaggccctggggt cacggaggctggggaggcaccgaggaacgcgcctggcatgtgctgacaggggattttatgctccagct gggccagctgggaggagcctgctgggcagaggccagagctgggggctctgg
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 27.
  • the polynucleotide of the present invention does not comprise an enhancer.
  • the polynucleotide of the invention may comprise a Kozak sequence.
  • the Kozak sequence may be operably linked to the protein-coding sequence.
  • a Kozak sequence may be inserted before the start codon of the protein to improve the initiation of translation.
  • the Kozak sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 28, or a variant which is at least 65% identical to SEQ ID NO: 28.
  • the variant may be at least 75%, at least 85%, or at least 90% identical to SEQ ID NO: 28.
  • the polynucleotide of the present invention does not comprise a Kozak sequence.
  • Post-transcriptional regulatory elements are not comprise a Kozak sequence.
  • the polynucleotide of the invention may comprise a post-transcriptional regulatory element.
  • the post-transcriptional regulatory element may be operably linked to the proteincoding sequence.
  • the post-transcriptional regulatory element may improve gene expression.
  • the polynucleotide may comprise a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element
  • the WPRE may be operably linked to the proteincoding sequence.
  • the WPRE sequence may have substitutions, deletions, or insertions within the X-antigen promoter and/or the initiation codon of the X-antigen. This may prevent the production of a functional X-antigen.
  • the WPRE may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 29, or a variant which is at least 70% identical to SEQ ID NO: 29.
  • Exemplary WPRE (SEQ ID NO: 29): aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttac gctatgtggatacgctgctttaatgcctttgtatcatgctattgcttttcccgtatggcttttcattttct cctctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggc gtggc gtggc gtggtgtgcactgtgttttgctgacgcaacccccactggttggggggcattgccaccacctgtcagctccccccctcctctct
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 29.
  • the polynucleotide of the present invention does not comprise a post-transcriptional regulatory element.
  • the polynucleotide of the invention may comprise a polyadenylation signal.
  • the polyadenylation signal may be operably linked to the protein-coding sequence.
  • the polyadenylation signal may improve gene expression.
  • Suitable polyadenylation signals include the early SV40 polyadenylation signal (SV40pA), a chicken beta-globin polyadenylation signal, a bovine growth hormone polyadenylation signal (bGH), or a soluble neuropilin-1 polyadenylation signal.
  • the polyadenylation signal is an early SV40 polyadenylation signal (SV40pA) or a chicken beta- globin polyadenylation signal.
  • the polyadenylation signal is an early SV40 polyadenylation signal (SV40pA).
  • the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 30, or a variant which is at least 70% identical to SEQ ID NO: 30.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 30.
  • the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 31, or a variant which is at least 70% identical to SEQ ID NO: 31.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 31.
  • Exemplary bGH poly(A) signal sequence (SEQ ID NO: 31): ctgtgccttctagttgccagccatctgttgtttgcccctcccgtgccttccttgaccctggaaggt gccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattc tggggggtggggggcaggacaagggggaggattgggaagacaatagcaggcatgctg gggatgcggtgggctctatgg
  • the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 32, or a variant which is at least 70% identical to SEQ ID NO: 32.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 32.
  • Exemplary soluble neuropilin-1 polyadenylation signal (SEQ ID NO: 32): aaataaaatacgaaatg
  • the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 42, or a variant which is at least 70% identical to SEQ ID NO: 42.
  • the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 42.
  • Exemplary chicken beta-globin polyadenylation signal (SEQ ID NO: 42) caataaaagatctttattttcattagatctgtgttggtttttttgtgtgtg
  • the present invention provides a vector comprising the polynucleotide of the present invention.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • the four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • the vector of the present invention is a viral vector.
  • the vector of the invention is preferably an adeno-associated viral (AAV) vector, although it is contemplated that other viral vectors may be used.
  • AAV adeno-associated viral
  • the vector of the present invention may be in the form of a viral vector particle.
  • the viral vector of the present invention is in the form of an AAV vector particle.
  • the vector of the present invention may be capable of transducing kidney cells. In some embodiments, the vector of the present invention is capable of specifically transducing kidney cells.
  • the vector of the present invention is preferably capable of transducing glomerular cells (e.g. podocytes). In some embodiments, the vector of the present invention is capable of specifically transducing glomerular cells (e.g. podocytes). The vector of the present invention is preferably capable of transducing podocytes. In some embodiments, the vector of the present invention is capable of specifically transducing podocytes.
  • AAV Adeno-associated viral
  • the vector of the present invention may be an adeno-associated viral (AAV) vector.
  • the vector of the present invention may be in the form of an AAV vector particle.
  • the AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof.
  • An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replication-deficient.
  • the AAV genome may be in single-stranded form (ssAAV), either positive or negative-sense, or alternatively in double-stranded form (dsAAV).
  • ssAAV single-stranded form
  • dsAAV double-stranded form
  • the use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.
  • the maximum packaging capacity of the single-stranded form is larger than the double-stranded form.
  • the AAV genome is in single-stranded form.
  • AAVs occurring in nature may be classified according to various biological systems.
  • the AAV genome may be from any naturally derived serotype, isolate or clade of AAV.
  • AAV may be referred to in terms of their serotype.
  • a serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies.
  • an AAV vector particle having a particular AAV serotype does not efficiently cross- react with neutralising antibodies specific for any other AAV serotype.
  • AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.
  • the AAV vector of the invention may be an AAV3B, LK03, AAV9, or AAV8 serotype.
  • the AAV vector of the invention may be an AAV3B, LK03, or AAV9 serotype.
  • AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAVs, and typically to a phylogenetic group of AAVs which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAVs which has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognisably distinct population at a genetic level.
  • the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR).
  • ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. ITRs may be the only sequences required in cis next to the therapeutic gene.
  • the AAV genome may also comprise packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV particle.
  • a promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene.
  • the rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof.
  • the cap gene encodes one or more capsid proteins such as VP1 , VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV particle, which determines the AAV serotype.
  • VP1 , VP2, and VP3 may be produced by alternate mRNA splicing (Trempe, J.P. and Carter, B.J., 1988. Journal of virology, 62(9), pp.3356-3363).
  • VP1 , VP2 and VP3 may have identical sequences, but wherein VP2 is truncated at the N-terminus relative to VP1 , and VP3 is truncated at the N-terminus relative to VP2.
  • the AAV genome may be the full genome of a naturally occurring AAV.
  • a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle.
  • the AAV genome is derivatised for the purpose of administration to patients.
  • derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art.
  • the AAV genome may be a derivative of any naturally occurring AAV.
  • the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.
  • the AAV genome is a derivative of AAV2.
  • Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo.
  • a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more.
  • ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR.
  • a preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV (scAAV) genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression. However, the maximum packaging capacity of a scAAV is reduced.
  • the AAV genome is not a scAAV genome.
  • the AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof.
  • the AAV genome may comprise at least one, such as two, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof.
  • the AAV genome may comprise at least one, such as two, AAV2 ITRs.
  • ITRs are preferred to aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases.
  • the formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.
  • ITR elements will be the only sequences retained from the native AAV genome in the derivative.
  • a derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.
  • derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome.
  • Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the AAV vector may be tolerated in a therapeutic setting.
  • the invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome.
  • the invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus.
  • Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.
  • the AAV vector particle may be encapsidated by capsid proteins.
  • the serotype may facilitate the transduction of glomerular cells (e.g. podocytes), for example specific transduction of glomerular cells (e.g. podocytes).
  • the AAV vector particle may be a kidney-specific vector particle.
  • the AAV vector particle is a glomerular-specific (e.g. podocyte-specific) vector particle.
  • the AAV vector particle may be encapsidated by a glomerular-specific (e.g. podocyte-specific) capsid.
  • the AAV vector particle may comprise a glomerular-specific (e.g. podocyte-specific) capsid protein.
  • the AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype.
  • the AAV vector particle also includes mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid.
  • the AAV vector particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface.
  • such ligands may include antibodies for targeting a particular cell surface receptor.
  • a derivative comprises capsid proteins i.e. VP1 , VP2 and/or VP3
  • the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs.
  • the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e. a pseudotyped vector).
  • the AAV vector may be in the form of a pseudotyped AAV vector particle.
  • Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the AAV vector.
  • these derivatives may display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting of podocytes compared to an AAV vector comprising a naturally occurring AAV genome.
  • Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form.
  • Increased efficiency may also relate to an altered tropism range or targeting of podocytes, such that the vector dose is not diluted by administration to tissues where it is not needed.
  • Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are co-transfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties.
  • the capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.
  • Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.
  • Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology.
  • a library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality.
  • error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.
  • capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence.
  • capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.
  • the unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population.
  • the unrelated protein may also be one which assists purification of the viral particle as part of the production process, i.e. an epitope or affinity tag.
  • the site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle.
  • the capsid protein may be an artificial or mutant capsid protein.
  • artificial capsid as used herein means that the capsid particle comprises an amino acid sequence which does not occur in nature or which comprises an amino acid sequence which has been engineered (e.g. modified) from a naturally occurring capsid amino acid sequence.
  • the artificial capsid protein comprises a mutation or a variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived where the artificial capsid amino acid sequence and the parent capsid amino acid sequences are aligned.
  • the capsid protein may comprise a mutation or modification relative to the wild type capsid protein which improves the ability to transduce podocytes relative to an unmodified or wild type viral particle. Improved ability to transduce podocytes may be measured for example by measuring the expression of a transgene, e.g. GFP, carried by the AAV vector particle, wherein expression of the transgene in podocytes correlates with the ability of the AAV vector particle to transduce podocytes.
  • a transgene e.g. GFP
  • the AAV vector particle may be an AAV3B, LK03, AAV9, or AAV8 vector particle.
  • the present inventors have shown that AAV vector particles with AAV3B, LK03, AAV9 and AAV8 serotypes can transduce podocytes.
  • the AAV vector particle is an AAV3B vector particle or an LK03 vector particle. More preferably, the AAV vector particle is an AAV3B vector particle.
  • the AAV vector particle may comprise an AAV3B, LK03, AAV9, or AAV8 capsid protein.
  • the AAV vector particle comprises an AAV3B capsid protein or an LK03 capsid protein. More preferably, the AAV vector particle comprises an AAV3B capsid protein.
  • the AAV vector particle may comprise AAV3B, LK03, AAV9, or AAV8 capsid proteins VP1, VP2 and VP3.
  • the AAV vector particle comprises AAV3B or LK03 capsid proteins VP1, VP2 and VP3. More preferably, the AAV vector particle comprises AAV3B capsid proteins VP1, VP2 and VP3.
  • the AAV vector particle may comprise one or more AAV2 ITR sequences and AAV3B capsid proteins, LK03 capsid proteins, AAV9 capsid proteins, or AAV8 capsid proteins.
  • the AAV vector particle comprises one or more AAV2 ITR sequences and AAV3B or LK03 capsid proteins. More preferably, the AAV vector particle comprises one or more AAV2 ITR sequences and AAV3B capsid proteins.
  • the AAV vector particle may have an AAV2 genome and AAV3B capsid proteins (AAV2/3B), an AAV2 genome and LK03 capsid proteins, an AAV2 genome and AAV9 capsid proteins (AAV2/9), or an AAV2 genome and AAV8 capsid proteins (AAV2/8).
  • AAV vector particle comprises an AAV2 genome and AAV3B or LK03 capsid proteins. More preferably, the AAV vector particle comprises an AAV2 genome and AAV3B capsid proteins.
  • AAVX/Y may denote a pseudotyped AAV, for example where the ITR sequences are from AAVX and flank a cassette harbouring a payload which is encapsidated into serotype AAVY (i.e. with AAVY capsid proteins).
  • the AAV vector particle may comprise an AAV3B capsid protein.
  • the AAV vector particle may be encapsidated by AAV3B capsid proteins.
  • AAV3A and AAV3B Two distinct AAV3 isolates (AAV3A and AAV3B) have been cloned. In comparison with vectors based on other AAV serotypes, it is thought that AAV3 vectors inefficiently transduce most cell types. However, AAV3B may efficiently transduce podocytes. AA3B has been described in Rutledge, E.A., et al., 1998. Journal of virology, 72(1), pp.309-319.
  • the AAV vector particle may comprise an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and/or an AAV3B VP3 capsid protein.
  • the AAV vector particle may be encapsidated by AAV3B VP1 capsid proteins, AAV3B VP2 capsid proteins, and/or AAV3B VP3 capsid proteins.
  • the AAV vector particle may be encapsidated by AAV3B VP1, VP2, and VP3 capsid proteins.
  • the AAV3B VP1 capsid protein may comprise or consist of the amino acid sequence shown as SEQ ID NO: 33, or a variant which is at least 90% identical to SEQ ID NO: 33.
  • Exemplary AAV3B VP1 capsid protein (SEQ ID NO: 33):
  • the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 33.
  • the AAV3B VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 33, or N-terminal truncations of a variant which is at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 33.
  • the AAV vector particle may comprise an LK03 capsid protein.
  • the AAV vector particle may be encapsidated by LK03 capsid proteins.
  • the AAV-LK03 cap sequence consists of fragments from seven different wild-type serotypes (AAV1, 2, 3B, 4, 6, 8, 9) and is described in Lisowski, L, et al., 2014. Nature, 506(7488), pp.382-386.
  • the present inventors have demonstrated that AAV-LK03 vectors can achieve high transduction of close to 100% in human podocytes in vitro.
  • the AAV vector particle may comprise an LK03 VP1 capsid protein, an LK03 VP2 capsid protein, and/or an LK03 VP3 capsid protein.
  • the AAV vector particle may be encapsidated by LK03 VP1 capsid proteins, LK03 VP2 capsid proteins, and/or LK03 VP3 capsid proteins.
  • the AAV vector particle may be encapsidated by LK03 VP1, VP2, and VP3 capsid proteins.
  • the LK03 VP1 capsid protein may comprise or consist of the amino acid sequence shown as SEQ ID NO: 34, or a variant which is at least 90% identical to SEQ ID NO: 34.
  • Exemplary LK03 VP1 capsid protein (SEQ ID NO: 34):
  • the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 34.
  • the LK03 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 34, or N-terminal truncations of a variant which is at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 34.
  • the AAV vector particle may comprise an AAV9 capsid protein.
  • the AAV vector particle may be encapsidated by AAV9 capsid proteins.
  • AAV9 vectors can achieve high transduction in human podocytes in vitro.
  • the AAV vector particle may comprise an AAV9 VP1 capsid protein, an AAV9 VP2 capsid protein, and/or an AAV9 VP3 capsid protein.
  • the AAV vector particle may be encapsidated by AAV9 VP1 capsid proteins, AAV9 VP2 capsid proteins, and/or AAV9 VP3 capsid proteins.
  • the AAV vector particle may be encapsidated by AAV9 VP1, VP2, and VP3 capsid proteins.
  • the AAV9 VP1 capsid protein may comprise or consist of the amino acid sequence shown as SEQ ID NO: 35, or a variant which is at least 90% identical to SEQ ID NO: 35.
  • Exemplary AAV9 VP1 capsid protein (SEQ ID NO: 35):
  • the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 35.
  • the AAV9 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 35, or N-terminal truncations of a variant which is at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 35.
  • the vector of the present invention may be a retroviral vector or a lentiviral vector.
  • the vector of the present invention may be a retroviral vector particle or a lentiviral vector particle.
  • a retroviral vector may be derived from or may be derivable from any suitable retrovirus.
  • retroviruses include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV).
  • MMV murine leukaemia virus
  • HTLV human T-cell leukaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • Fujinami sarcoma virus FuSV
  • Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.
  • LTR long terminal repeats
  • the LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.
  • the LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5.
  • U3 is derived from the sequence unique to the 3’ end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA.
  • U5 is derived from the sequence unique to the 5’ end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • gag, pol and env may be absent or not functional.
  • a retroviral vector In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and/or integrating its genome into a host genome.
  • Lentivirus vectors are part of the larger group of retroviral vectors.
  • lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV).
  • non-primate lentiviruses examples include the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anaemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • the lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • other retroviruses such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • a lentiviral vector is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
  • the lentiviral vector may be a “primate” vector.
  • the lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans).
  • non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.
  • HIV-1- and HIV-2-based vectors are described below.
  • the HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of Rev and/or RRE, full-length HIV-1 RNAs accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.
  • HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs.
  • the viral vector used in the present invention has a minimal viral genome.
  • minimal viral genome it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell.
  • the vector lacks a functional gag-pol and/or env gene and/or other genes essential for replication.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell.
  • transcriptional regulatory control sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).
  • the vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted.
  • SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors.
  • the transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.
  • LTR long terminal repeat
  • the vectors may be integration-defective.
  • Integration defective lentiviral vectors can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.
  • the vector of the present invention may be an adenoviral vector.
  • the vector of the present invention may be an adenoviral vector particle.
  • the adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate.
  • adenovirus There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology.
  • the natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms.
  • Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.
  • Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes.
  • the large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 10 12 .
  • Adenovirus is thus one of the best systems to study the expression of genes in primary non- replicative cells.
  • Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence, the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome.
  • the vector of the present invention may be a herpes simplex viral vector.
  • the vector of the present invention may be a herpes simplex viral vector particle.
  • Herpes simplex virus is a neurotropic DNA virus with favorable properties as a gene delivery vector.
  • HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells.
  • Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans enabling straightforward production of high-titre pure preparations of non-pathogenic vector.
  • the genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes.
  • Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime.
  • the vectors are non-pathogenic, unable to reactivate and persist long-term.
  • HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed targeting the tropism of HSV vectors.
  • Suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p.42.
  • the vector of the present invention may be an alphaviral vector.
  • the vector of the present invention may be an alphaviral vector particle.
  • the vector of the present invention may be a flaviviral vector.
  • the vector of the present invention may be a flaviviral vector particle.
  • Self-amplifying ssRNA viruses comprise of alphaviruses (e.g. Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1) and flaviviruses (e.g. Kunjin virus, West Nile virus, and Dengue virus) possessing a genome of positive polarity.
  • alphaviruses e.g. Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1
  • flaviviruses e.g. Kunjin virus, West Nile virus, and Dengue virus possessing a genome of positive polarity.
  • Alphaviruses have been mainly applied in preclinical gene therapy studies for cancer treatment.
  • Alphavirus vectors can be delivered in the form of naked RNA, layered plasmid DNA vectors and recombinant replication-deficient or -proficient particles.
  • the vector of the present invention may be a rhabdoviral vector.
  • the vector of the present invention may be a rhabdoviral vector particle.
  • the vector of the present invention may be a measles viral vector.
  • the vector of the present invention may be a measles viral vector particle.
  • Rhabdoviruses e.g. rabies and vesicular stomatitis virus
  • measles viruses carry negative strand genomes.
  • recombinant vesicular stomatitis virus (VSV) has been applied for preclinical gene therapy studies.
  • Measles viruses e.g. MV-Edm
  • the vector of the present invention may be a Newcastle disease viral vector.
  • the vector of the present invention may be a Newcastle disease viral vector particle.
  • NDV Newcastle disease virus
  • the vector of the present invention may be a poxviral vector.
  • the vector of the present invention may be a poxviral vector particle.
  • poxviruses have found several applications as gene therapy vectors. For instance, vaccinia virus vectors have demonstrated potential for treatment of cancer. Vaccinia virus is large enveloped poxvirus that has an approximately 190 kb linear, double-stranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it useful for the delivery of large genes. A number of attenuated vaccinia virus strains are known in the art that are suitable for gene therapy applications, for example the MVA and NYVAC strains.
  • the vector of the present invention may be a picornaviral vector.
  • the vector of the present invention may be a picornaviral vector particle.
  • Picornoviruses are non-enveloped ssRNA viruses. Coxsackieviruses belonging to Picornaviridae, have been applied as oncolytic vectors.
  • the invention also encompasses variants, derivatives, homologues and fragments thereof.
  • a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions.
  • a variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.
  • a variant promoter sequence retains at least some level of the activity and specificity of the promoter sequence from which it is obtained.
  • derivative as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.
  • amino acid substitutions may be made, for example from 1, 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogues.
  • Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
  • homologue as used herein means a variant having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence.
  • homology can be equated with “identity”.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence.
  • homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology or identity between two or more sequences.
  • Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs).
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • percent homology preferably percent sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.
  • “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.
  • Such variants, derivatives, homologues and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis.
  • synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made.
  • the flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut.
  • the DNA is then expressed in accordance with the invention to make the encoded protein.
  • the present invention provides a cell comprising the polynucleotide or the vector of the invention.
  • the cell may be an isolated cell.
  • the cell may be a human cell, suitably an isolated human cell.
  • Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation.
  • the vector of the present invention is introduced into the cell by transfection or transduction.
  • the cell may be any cell type known in the prior art.
  • the cell may be a producer cell.
  • the term “producer cell” includes a cell that produces viral particles, after transient transfection, stable transfection or vector transduction of all the elements necessary to produce the viral particles or any cell engineered to stably comprise the elements necessary to produce the viral particles.
  • Suitable producer cells will be well known to those of skill in the art.
  • Suitable producer cell lines include HEK293 (e.g. HEK293T), HeLa, and A549 cell lines.
  • the cell may be a packaging cell.
  • packaging cell includes a cell which contains some or all of the elements necessary for packaging an infectious recombinant virus.
  • the packaging cell may lack a recombinant viral vector genome.
  • packaging cells contain one or more vectors which are capable of expressing viral structural proteins. Cells comprising only some of the elements required for the production of enveloped viral particles are useful as intermediate reagents in the generation of viral particle producer cell lines, through subsequent steps of transient transfection, transduction or stable integration of each additional required element. These intermediate reagents are encompassed by the term “packaging cell”. Suitable packaging cells will be well known to those of skill in the art.
  • the cell may be a kidney cell or glomerular cell, for example a podocyte.
  • the cell may be an immortalized kidney cell or glomerular cell, for example an immortalized podocyte.
  • Suitable podocyte cell lines will be well known to those of skill in the art, for example CIHP-1. Methods to generate immortalized podocytes will be well known to those of skill in the art. Suitable methods are described in Ni, L, et al., 2012. Nephrology, 17(6), pp.525-531.
  • the present invention provides pharmaceutical composition comprising the polynucleotide or vector of the invention or the cell of the invention.
  • a pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent i.e. the vector. It preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
  • the formulation is sterile and pyrogen free.
  • the carrier, diluent, and/or excipient must be “acceptable” in the sense of being compatible with the vector and not deleterious to the recipients thereof.
  • the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used.
  • compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).
  • Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
  • the vector, cell, or pharmaceutical composition according to the present invention may be administered in a manner appropriate for treating and/or preventing the diseases described herein.
  • the quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials.
  • the pharmaceutical composition may be formulated accordingly.
  • the vector, cell or pharmaceutical composition according to the present invention may be administered parenterally, for example, intravenously, or by infusion techniques.
  • the vector, cell or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9).
  • the pharmaceutical composition may be formulated accordingly.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the vector, cell or pharmaceutical composition according to the present invention may be administered systemically, for example by intravenous injection.
  • the vector, cell or pharmaceutical composition according to the present invention may be administered locally, for example by targeting administration to the kidney.
  • the vector, cell or pharmaceutical composition may be administered by injection into the renal artery or by ureteral or subcapsular injection.
  • compositions may comprise vectors or cells of the invention in infusion media, for example sterile isotonic solution.
  • the pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the vector, cell or pharmaceutical composition may be administered in a single or in multiple doses. Particularly, the vector, cell or pharmaceutical composition may be administered in a single, one off dose.
  • the pharmaceutical composition may be formulated accordingly.
  • the vector, cell or pharmaceutical composition may be administered at varying doses (e.g. measured in vector genomes (vg) per kg).
  • doses e.g. measured in vector genomes (vg) per kg.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual subject and it will vary with the age, weight and response of the particular subject.
  • doses of 10 10 to 10 14 vg/kg, or 10 11 to 10 13 vg/kg may be administered.
  • the pharmaceutical composition may further comprise one or more other therapeutic agents.
  • kits comprising the vector, cells and/or pharmaceutical composition of the present invention.
  • kits are for use in the methods and used as described herein, e.g., the therapeutic methods as described herein.
  • kits comprise instructions for use of the kit components.
  • the present invention provides the vector, cell or pharmaceutical composition according to the present invention for use as a medicament.
  • the present invention provides use of the vector, cell or pharmaceutical composition according to the present invention in the manufacture of a medicament.
  • the present invention provides a method of administering the vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.
  • the vector, cell or pharmaceutical composition according to the present invention may be used to treat glomerular diseases in a subject.
  • the subject is a human subject.
  • the present invention provides the vector, cell or pharmaceutical composition according to the present invention for use in preventing or treating a glomerular disease.
  • the present invention provides use of the vector, cell or pharmaceutical composition according to the present invention for the manufacture of a medicament for preventing or treating a glomerular disease.
  • the present invention provides a method of preventing or treating a glomerular disease comprising administering the vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.
  • Glomerular diseases can be classified as either nephrotic or nephritic.
  • Nephrotic syndrome often involves factors that affect the integrity of podocyte-podocyte or podocyte-GBM interactions.
  • factors involved in the etiology of nephritic syndrome can vary, but may include circulating platelets and white blood cells, the GBM, resident glomerular endothelial cells, and mesangial cells. (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).
  • the glomerular disease is a genetic glomerular disease, i.e. a glomerular disease which is inherited.
  • Genetic glomerular diseases include podocyte-associated genetic glomerular diseases, such as nephrotic syndrome, and GBM-associated glomerular diseases, such as Alport Syndrome.
  • the glomerular disease is a podocyte-associated genetic glomerular disease.
  • Podocyte-associated genetic glomerular diseases include Congenital nephrotic syndrome of the Finnish type, Congenital nephrotic syndrome type 2, Familial nephrotic syndrome type 3, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, Nephrotic syndrome caused by mutations in CD2AP, Nephrotic syndrome caused by mutations in actinin-4, Nephrotic syndrome caused by mutations in TRPC6, and Epstein and Fechtner syndrome.
  • the glomerular disease is nephrotic syndrome.
  • the glomerular disease is a GBM-associated genetic glomerular disease.
  • Podocyte-associated genetic glomerular diseases include X-linked Alport syndrome, Autosomal recessive Alport syndrome, Autosomal dominant Alport syndrome, Thin basement membrane diseases, Pierson syndrome, and Nail-patella syndrome.
  • the glomerular disease is Alport syndrome (AS).
  • AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease and thin basement membrane nephropathy.
  • Example 1 design, construction, and testing of minimal nephrin promoters
  • NPHS1 promoter A human NPHS1 promoter has been described in Moeller et al. 2002 J Am Soc Nephrol, 13(6): 1561-7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6): F1027-32.
  • This NPHS1 promoter is a 1.2kb fragment and appears to be podocyte-specific. This is referred to hereafter as a “FL” nephrin promoter and is shown in Figure 1A.
  • the FL nephrin promoter was initially cut to 822 bp (819 bp excluding start codon) by deleting the N-terminal sequence. This is referred to hereafter as a “midi” nephrin promoter and is shown in Figure 1B.
  • the midi nephrin promoter was further cut to 268 bp (265 bp excluding start codon) by removing putative general transcription domains from the central region. This is referred to hereafter as a “mini” nephrin promoter and is shown in Figure 1C.
  • Midi nephrin promoter pACE_hNPHS1 promoter was used as a template to introduce BamHI and Clal restriction sites as shown in Figure 2A. Fragments were then gel extracted and digested with Clal and BamHI for 1h at 37°C prior to ligation into pLenti GFP Blast vector. Ligations were further transformed into stable competent E.Coli cells, DNA was extracted and sequenced (Midi Promoter). The final lentiviral vector is shown in Figure 2B.
  • Mini nephrin promoter pACE_hNPHS1 promoter was used to PCR the overhangs (OHs) as shown in Figure 3A. Two sections of the promoter containing OHs were gel extracted for the NEBuilder HiFi Assembly reaction into pLenti GFP Blast vector. The ligation reaction was then cleaned using the DNA clean up kit, prior to its transformation into stable competent E.Coli cells. DNA was extracted and sequenced. The final lentiviral vector is shown in Figure 3B.
  • the minimal nephrin promotors were used to express GFP in in vitro cell models to check efficacy and podocyte-specificity.
  • pLenti GFP Blast Nephrin Promoter constructs (Full Length, Midi and Mini) were used to transfect HEK293T cells for 48h to make virus, which was further used to create human conditionally immortalized podocytes stably expressing either GFP-tagged FL NPHS1, midi or mini promoters.
  • ciPodocytes Conditionally immortalised human podocytes (ciPodocytes) were transfected with the lentiviral vectors to determine whether the minimal promoters were able to drive GFP expression. Both the midi and mini nephrin promoters were shown to drive GFP expression.
  • Figure 4 shows a representative fluorescence microscopy image showing GFP expression from the mini nephrin promoter.
  • Figure 5 shows a representative western blot showing GFP expression from the mini nephrin promoter.
  • the lentiviral vectors were also used to transduce human glomerular cells.
  • ciPodocytes and glomerular endothelial cells were transduced with a lentivirus comprising GFP coupled to the mini nephrin reporter.
  • Figure 6A-C shows FACS analysis displaying median GFP fluorescence (AFU) of all live singlets of conditionally immortalised human podocytes (LY) and glomerular endothelial cells (GEnC) using a Novocyte Analyser.
  • AFU median GFP fluorescence
  • Untransduced cells were compared with those transduced with lentivirus constructs harbouring a GFP expression cassette controlled by the full length human nephrin promoter (hNPHSlGFP) or the mini human nephrin promoter (265. GFP). All cells were differentiated for 10 days, trypsinised (100 uL) and diluted in PBS, 2%FBS, 1:1000 DRAQ7 (150uL). Data and error bars represent 3 technical repeats (100 uL, >2500 cells) ⁇ SEM. These results show podocyte specificity for a minimal nephrin promoter when compared to glomerular endothelial cells.
  • Example 2 design, construction, and testing of minimal nephrin promoter coupled to COL4A3, COL4A4, and COL4A5
  • AAV transfer plasmids comprising COL4A3, COL4A4, and COL4A5 coupled to the mini nephrin promoter (“265”) were designed and constructed:
  • AAV viral vectors were prepared using standard methods:
  • Figure 8A shows immunoprecipitation experiments of the full-length FLAG-tagged Col4a3 (LK03) or Col4a5 (LK03) in human differentiated ciPodocytes pulled down with the anti-FLAG antibody. Anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human-FLAG IgG was used as a control.
  • Figure 8B shows western blots of protein lysates showing the expression levels of Col4a3 (LK03 capsid serotype), Col4a5 (LK03) and Col4a5 (2/9 capsid serotype) in human or mouse differentiated ciPodocytes. Non-infected human and mouse cipodocytes were used as controls.
  • Figure 8C shows confocal images showing immunofluorescence staining of transduced Col4a5 in Human wild-type CiPodocytes/Col4a5 3xFlag AAV CiPodocytes with F-Actin.
  • Col4a5 is present at the cytosolic level in the human differentiated podocytes infected with Col4a5 3xFlag AAV virus in comparison to the wild-type counterpart.
  • HGTI1 Human Embryonic Kidney cells grown in DMEM supplemented with 10% FBS were triple transfected with pHelper (HGTI1), one of the two pAAV Rep-Cap (LK03 or AAV9) and one of the three ITR-expression plasmids containing 1) CFH under the 265bp minimal nephrin promoter of SEQ ID NO: 47 (pAAV-265-CFH), 2) CFI under the full-length minimal nephrin promoter of SEQ ID NO: 14 (pAAV-FL-CFI) or 3) CFHL1 under the full-length minimal nephrin promoter of SEQ ID NO: 14 (pAAV-FL-CFHL1). All constructs were tagged with MYC and FLAG.
  • Transfection was carried out on a 150mm culture dish in serum-free media in the presence of polyethylenimine (PEI). Media was changed to DMEM with FBS the following day after transfection. On Day 4 post-transfection, media and cells were collected and processed separately. Media was frozen and stored at -80°C. Cells were lysed with RIPA buffer supplemented with proteinase inhibitors and stored at -80°C.
  • PEI polyethylenimine
  • Protein concentration in the cell lysates was measured using Pierce BCA protein assay and 10ug of total protein from each sample was loaded onto a 4-15% polyacrylamide Tris-Glycine gel. A total of 2.6ul of media from each sample was loaded onto the gel. Protein was transferred to a nitrocellulose membrane using the iBlot2 dry blotting system.
  • the following primary antibodies were used for protein detection: anti-Factor H (Abeam, cat. ab124769), anti-Factor I (Abeam, ab278524), anti-MYC-tag (CST, cat.2276S), anti-FLAG-tag (CST, cat. 14793S) and anti-GAPDH (Millipore, cat. MAB374).
  • Factor H was expressed in both the cell lysates and the media from 293T HEK cells transfected with the CFH expression plasmid ( Figure 9, lanes 2, 3, 8, 9). Expression was detected using a Factor H-specific antibody. Factor H was not detected in the cell lysates or media of untransfected cells or cells transfected with the CFI or CFHL1 expression plasmids ( Figure 9, lanes 1, 4-7, 10-13). These results demonstrate that a 265bp minimal nephrin promoter allows expression of the transgene in target cells.
  • Conditionally immortalised human podocytes with a mutation in endogenous Factor H (Muehlig et al, 2020) grown in RPMI supplemented with 10% FBS and 1% ITS were seeded in 6-well culture plates and grown at 33°C until 70-80% confluency.
  • Cells were incubated with AAV2/9 containing a CFH transgene under the control of the 265bp minimal nephrin promoter (SEQ ID NO: 47). Cells incubated without the virus were used as a non-transduced (NT) control.
  • NT non-transduced
  • cells were transferred to a non- permissive temperature of 37°C to allow for cell differentiation and transgene expression. Media was changed twice on the subsequent days following transduction.
  • cell media was collected and the concentration of human Factor H was measured by ELISA using an anti-Factor H antibody (Abeam, cat. ab252359).
  • Human podocytes with a mutation in Factor H were transduced with AAV2/9 virus containing a CFH transgene under the control of the 265 bp minimal nephrin promoter demonstrated higher concentrations of human Factor H in the culture media than untransduced cells ( Figures 10A and 10B). This demonstrates that a Factor H transgene delivered by AAV and under the control of a 265bp minimal nephrin promoter can be expressed in human podocytes.
  • Conditionally immortalised human podocytes with a mutation in endogenous Factor H (Muehlig et al, 2020) grown in RPMI supplemented with 10% FBS and 1% ITS were seeded in 6-well culture plates and grown at 33°C until 70-80% confluency.
  • plasmid transfection cells were incubated with 1.5ug of expression plasmid in serum-free media in the presence of polyethylenimine. Cells where no plasmid was added were used as a non-transfected (NT) control. The media was changed the following day to media containing FBS. On Day 3 posttransfection, media was collected and analysed by ELISA (Abeam, cat. ab252359).
  • AAV2/9 gene therapy product pAAV.NPHS1(265).hCFH.WPRE.bGH
  • saline 100 mI of AAV2/9 gene therapy product (pAAV.NPHS1(265).hCFH.WPRE.bGH) or saline was administered to wild-type C57BL6 mice by IV tail vein injection.
  • AAV expressed tagged wild- type human CFH transgene was under control of the 265bp minimal nephrin promoter (SEQ ID NO: 47).
  • AAV was harvested and purified by ultracentrifugation 3 days later and titrated ( ⁇ 1.5x10e13/ml) in PBS. All animals completed the study on Day 21 and culled. Kidneys were snap frozen in liquid nitrogen and used for RNA extraction by RNeasy Micro Kit (Qiagen Cat. No. / ID: 74004) as per the manufacturers' protocol.
  • RNA was then converted into cDNA using High-Capacity RNA-to-
  • Quantitative qPCR was performed on DNA samples from kidneys of pAAV_CFH injected mice. Standard curve qPCR with SYBR green reagents and passive ROX method was used to detect ITR presence in the kidney DNA samples. Viral genomes per pg of DNA were calculated using a standard curve of known quantities of an ITR amplicon. Finally, viral genomes per cell were calculated based on the assumption that diploid mouse cells have 6pg of DNA.
  • Immunofluorescence staining was performed on the frozen kidney sections 5 microns thick using an anti-nephrin antibody (PROGEN) and an anti-CFH antibody (ab124767).
  • Tissue was embedded in OCT compound (VWR, cat. number 361603E) and snap frozen in liquid nitrogen. 10uM sections were cut using a cryostat (Thermo, Cryostar NX270). Tissue sections were fixed with 4% Paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.3% Triton-X for 15 minutes at room temperature and blocked with 5% BSA for 30 minutes at room temperature.
  • Plasmids were constructed comprising eGFP under the control of: (i) the full-length (FL) minimal nephrin promoter of SEQ ID NO: 14 (PS0281); (ii) the 818bp minimal nephrin promoter of SEQ ID NO: 59 (PS0301); or (iii) the 265bp minimal nephrin promoter of SEQ ID NO: 47 (P20282).
  • Podocytes, glomerular endothelial cells (GENCs), and proximal tube epithelial cells (PTECs) were seeded at a density of 1.5e5 cells per well of a six well plate and allowed to attach overnight. For HEK293T cells, 5e5 cells per well were used. The following day 3ug of plasmid DNA in 150ul of DMEM was mixed with 9ul PEI (also in 150ul DMEM), incubated for 15 minutes at room temperature, and analysed 48 hours later by flow cytometry for eGFP expression. Non transfected cells were used as a negative control for gating of negative and positive populations.
  • Example 8 Expression of GFP using 265bp v FL minimal nephrin promoters (AAV transduction) in kidney cells
  • AAV Production 60-80% confluent 293T Human Embryonic Kidney cells grown in DMEM supplemented with 10% FBS were triple transfected with pHelper (HGTI1, PS0150), a Rep- Cap plasmid (LK03, PS0240) and one of the two ITR-expression plasmids containing the Full Length (FL) minimal nephrin promoter or the 265bp minimal nephrin promoter driving eGFP expression (PS0281 and PS0282, respectively). Transfection was carried out on a 150mm culture dish in the presence of polyethylenimine (PEI). Media was changed to DMEM with FBS the following day after transfection.
  • PEI polyethylenimine
  • AAV QC AAV samples were titred via qPCR to determine the number of viral genomes per ml of viral supernatant. Primers designed to target the eGFP sequence were used. SDS-PAGE analysis was performed on samples to determine purity. Alkaline gel electrophoresis was used to demonstrate incorporation of full transgene cassettes into the viral particles.
  • AAV Transduction Cells were seeded into wells of a 6-well plate at a density of 1.5e5 cells/well and allowed to adhere overnight. The following day viral particles were added a multiplicity of infection (MOI) of 5e5 particles per cell. Cells were placed at 37 degrees to initiate differentiation and cultured for 10 days. After 10 days cells were analysed for eGFP expression by flow cytometry.
  • MOI multiplicity of infection
  • 265bp minimal nephrin promoter is capable of driving eGFP expression in target cells. Additionally, that this promoter may be incorporated into an AAV vector for use in gene therapy.
  • the use of a 265bp minimal nephrin promoter, in place of the full length minimal nephrin promoter, may allow the incorporation of more genetic material into a transgene cassette for AAV purposes.
  • Example 9 Expression of human podocin using 265bp v FL minimal nephrin promoters (plasmid transfection) in HEK Plasmids were constructed comprising HA-tagged podocin under the control of: (i) the full- length (FL) minimal nephrin promoter of SEQ ID NO: 14 (FL.NPSH1-Podocin-HA); or (ii) the 265bp minimal nephrin promoter of SEQ ID NO: 47 (265.hNPSH1-Podocin-HA).
  • Example 10 Expression of human podocin using 265bp v FL minimal nephrin promoters (AAV transduction) in human podocytes
  • AAV Production 60-80% confluent 293T Human Embryonic Kidney cells grown in DMEM supplemented with 10% FBS,1% Penicillin/Streptomycin and 1% sodium pyruvate were triple transfected with pHelper (HGTI1, PS0150), pAAV Rep-Cap (LK03, PS0240) and one of the two ITR-expression plasmids containing the Full Length (FL) minimal nephrin promoter or the 265bp minimal nephrin promoter driving Podocin-HA expression (FL.NPSH1-Podocin-HA and 265.hNPSH1-Podocin-HA, respectively).
  • Transfection was carried out on a 150mm culture dish in the presence of polyethylenimine (PEI)at a ratio of 1 :2. Media was changed to DMEM with FBS the following day after transfection. On Day 4 post-transfection, media and cells were collected and processed separately. PEG was added to filtered supernatants to precipitate viral particles which were subsequently centrifuged at 1500g for 20 minutes. Cell pellets were resuspended in TD buffer and frozen at -80 degrees, followed by thawing at 37 degrees and vortexed. Freeze-thaw cycles were repeated 5 times. Pellets from the PEG treated supernatant were then added to the lysed cells.
  • PEI polyethylenimine
  • Benzonase and Sodium deoxycholate was further added and incubated at 37 degrees for 30 minutes. Samples were then centrifuged and the supernatant filtered and subsequently purified via iodixanol gradient ultracentrifugation.
  • AAV QC AAV samples were titred via qPCR to determine the number of viral genomes per ml of viral supernatant. Primers designed to target the Podocin sequence were used.
  • AAV Transduction Cells were seeded into wells of a 12-well plate at a density of 0.5e5 cells/well and allowed to adhere overnight. The following day viral particles were added a multiplicity of infection (MOI) of 5e5 particles per cell. Cells were placed at 37 degrees to initiate differentiation and cultured for 9 days. After 9 days cells were harvested and stained with anti-HA antibody for HA expression by flow cytometry as a measure of Podocin expression.
  • MOI multiplicity of infection
  • Example 11 In vivo expression of GFP using 265bp v FL promoters via AAV transduction
  • the 265- and FL- nephrin promoters were evaluated in vivo in WT mice (C57/BI6).
  • qPCR analysis performed on the kidney and GFP copy number normalised to 18S (housekeeping gene). 18S rRNA is a reliable normalisation gene for real time PCR.
  • a promoter comprising (i) a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 and wherein the promoter has a length of about 1.1 kb or less.
  • a promoter which comprises or consists of a nucleotide sequence having at least 70% identity to a nucleotide sequence according to SEQ ID NO: 1 but wherein:
  • the promoter according to para 2 wherein the promoter comprises (i) a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 and/or wherein the promoter has a length of about 1.1 kb or less.
  • the promoter further comprises (ii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 5, a nucleotide sequence having at least 70% identity to SEQ ID NO: 6, and/or a nucleotide sequence having at least 70% identity to SEQ ID NO: 7.
  • the promoter further comprises (iii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 8, or one or more fragments thereof.
  • the promoter comprises from 5’ to 3’: (i) a nucleotide sequence having at least 70% identity to SEQ ID NO: 4; (iii) optionally a nucleotide sequence having at least 70% identity to SEQ ID NO: 8, or one or more fragments thereof; and (ii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 5, a nucleotide sequence having at least 70% identity to SEQ ID NO: 6, and/or a nucleotide sequence having at least 70% identity to SEQ ID NO: 7.
  • the promoter has a length of about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less.
  • the promoter has a length of from 0.265 kb to 1.0 kb, from 0.265 kb to 0.9 kb, from 0.265 kb to 0.8 kb, from 0.265 kb to 0.7 kb, from 0.265 kb to 0.6 kb, from 0.265 kb to 0.5 kb, from 0.265 kb to 0.4 kb, or from 0.265 kb to 0.3 kb.
  • retinoic acid receptor binding site comprises or consists of a nucleotide sequence shown as SEQ ID NO: 10 or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 10.
  • the promoter according to para 10 or 11 wherein the WT1 binding site comprises or consists of a nucleotide sequence shown as SEQ ID NO: 11 or a nucleotide sequence having one, two or three substitutions, deletions, or insertions compared to SEQ ID NO: 11.
  • the promoter according to any one of paras 10 to 12 wherein the enhancer box comprises or consists of a nucleotide sequence shown as SEQ ID NO: 12 or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 12.
  • nucleotide sequence having at least 70% identity to SEQ ID NO: 4 comprises one or more of: (a) a retinoic acid receptor binding site; (b) a WT 1 binding site; and (c) an enhancer box.
  • nucleotide sequence having at least 70% identity to SEQ ID NO: 4 comprises one or more of: (a) a retinoic acid receptor binding site; (b) a WT 1 binding site; and (c) an enhancer box.
  • the transcription factor binding region comprises or consists of a nucleotide sequence shown as SEQ ID NO: 13 or a nucleotide sequence having one, two, three, four or five substitutions, deletions, or insertions compared to SEQ ID NO: 13.
  • the promoter comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 2 or SEQ ID NO: 3.
  • a promoter which consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 2.
  • a promoter which consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 3.
  • a polynucleotide comprising the promoter according to any one of paras 1 to 21.
  • the protein coding sequence is operably linked to one or more further regulatory elements, such as a post-transcriptional regulatory element and/or a polyadenylation sequence.
  • a vector comprising the polynucleotide according to any one of paras 22 to 24.
  • the vector is a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an alphaviral vector, a flaviviral vector, a rhabdoviral vector, a measles viral vector, a Newcastle disease viral vector, a poxviral vector, and a picornaviral vector, preferably wherein the vector is an AAV vector.
  • AAV adeno-associated virus
  • a cell comprising the polynucleotide according to any one of paras 22 to 24, or the vector according to any one of paras 25 to 29.
  • a pharmaceutical composition comprising the polynucleotide according to any one of paras 22 to 24, the vector according to any one of paras 25 to 29, or the cell according to para 30.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/GB2022/050649 2021-03-12 2022-03-14 Minimal nephrin promoter Ceased WO2022189811A1 (en)

Priority Applications (16)

Application Number Priority Date Filing Date Title
AU2022235209A AU2022235209A1 (en) 2021-03-12 2022-03-14 Minimal nephrin promoter
JP2023555709A JP2024509316A (ja) 2021-03-12 2022-03-14 最小ネフリンプロモーター
BR112023018362A BR112023018362A2 (pt) 2021-03-12 2022-03-14 Promotor mínimo de nefrina
EP22710714.1A EP4305181B1 (en) 2021-03-12 2022-03-14 Minimal nephrin promoter
DK22710714.1T DK4305181T3 (en) 2021-03-12 2022-03-14 Minimal nefrin-promotor
CN202280034399.8A CN117377770A (zh) 2021-03-12 2022-03-14 最小肾病蛋白启动子
MX2023010573A MX2023010573A (es) 2021-03-12 2022-03-14 Promotor minimo de nefrina.
US18/280,699 US20250283072A1 (en) 2021-03-12 2022-03-14 Minimal nephrin promoter
PL22710714.1T PL4305181T3 (pl) 2021-03-12 2022-03-14 Minimalny promotor nefryny
KR1020237034581A KR20230161459A (ko) 2021-03-12 2022-03-14 최소 네프린 프로모터
ES22710714T ES3056304T3 (en) 2021-03-12 2022-03-14 Minimal nephrin promoter
CA3211566A CA3211566A1 (en) 2021-03-12 2022-03-14 Minimal nephrin promoter
EP25181596.5A EP4722371A2 (en) 2021-03-12 2022-03-14 Promoter
HRP20251561TT HRP20251561T1 (hr) 2021-03-12 2022-03-14 Minimalni promotor nefrina
IL305714A IL305714A (en) 2021-03-12 2022-03-14 Minimal nephrin factor
FIEP22710714.1T FI4305181T3 (fi) 2021-03-12 2022-03-14 Minimaalinen nefriinipromoottori

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB2103470.7 2021-03-12
GBPCT/GB2021/050633 2021-03-12
PCT/GB2021/050633 WO2021181118A1 (en) 2020-03-12 2021-03-12 Gene therapy
GBGB2103470.7A GB202103470D0 (en) 2021-03-12 2021-03-12 Promoter
PCT/GB2021/051668 WO2022003357A1 (en) 2020-06-30 2021-06-30 Vector
GBPCT/GB2021/051668 2021-06-30

Publications (1)

Publication Number Publication Date
WO2022189811A1 true WO2022189811A1 (en) 2022-09-15

Family

ID=75623235

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2022/050649 Ceased WO2022189811A1 (en) 2021-03-12 2022-03-14 Minimal nephrin promoter

Country Status (18)

Country Link
US (1) US20250283072A1 (https=)
EP (2) EP4722371A2 (https=)
JP (1) JP2024509316A (https=)
KR (1) KR20230161459A (https=)
CN (1) CN117377770A (https=)
AU (1) AU2022235209A1 (https=)
BR (1) BR112023018362A2 (https=)
CA (1) CA3211566A1 (https=)
DK (1) DK4305181T3 (https=)
ES (1) ES3056304T3 (https=)
FI (1) FI4305181T3 (https=)
GB (1) GB202103470D0 (https=)
HR (1) HRP20251561T1 (https=)
IL (1) IL305714A (https=)
MX (1) MX2023010573A (https=)
PL (1) PL4305181T3 (https=)
PT (1) PT4305181T (https=)
WO (1) WO2022189811A1 (https=)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024105415A1 (en) * 2022-11-18 2024-05-23 The University Of Bristol Methods of delivering a viral vector to a kidney
EP4397976A1 (en) * 2023-01-09 2024-07-10 x-kidney diagnostics GmbH Biomarkers for the pre-clinical and/or early-stage detection and/or diagnosis of kidney diseases
WO2025003703A1 (en) * 2023-06-29 2025-01-02 Purespring Therapeutics Limited Promoters
WO2025003710A1 (en) * 2023-06-29 2025-01-02 Purespring Therapeutics Limited Polynucleotides, aav vectors, and pharmaceutical compositions for treating nephrotic syndrome
WO2025003709A1 (en) * 2023-06-29 2025-01-02 Purespring Therapeutics Limited Regulatory elements
WO2025003704A3 (en) * 2023-06-29 2025-02-13 Purespring Therapeutics Limited Polynucleotides, aav vectors, and pharmaceutical compositions for treating complement-mediated kidney diseases
US12589167B2 (en) 2020-03-12 2026-03-31 The University Of Bristol AAV gene therapy vector with podocyte-specific promoter

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202003109D0 (en) * 2020-03-04 2020-04-15 Univ Bristol Gene therapy

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017815A1 (en) 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Retroviral vectors
US20140087002A1 (en) 2012-09-21 2014-03-27 Laurantis Pharma Oy Autologous Lymph Node Transfer In Combination With VEGF-C Or VEGF-D Growth Factor Therapy to Treat Lymphedema And To Improve Reconstructive Surgery
WO2015022447A1 (en) 2013-08-14 2015-02-19 Laurantis Pharma Oy Therapeutic use of vegf-c and ccbe1
WO2020148548A1 (en) * 2019-01-18 2020-07-23 The University Of Bristol Aav gene therapy for treating nephrotic syndrome

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017815A1 (en) 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Retroviral vectors
US20140087002A1 (en) 2012-09-21 2014-03-27 Laurantis Pharma Oy Autologous Lymph Node Transfer In Combination With VEGF-C Or VEGF-D Growth Factor Therapy to Treat Lymphedema And To Improve Reconstructive Surgery
WO2015022447A1 (en) 2013-08-14 2015-02-19 Laurantis Pharma Oy Therapeutic use of vegf-c and ccbe1
WO2020148548A1 (en) * 2019-01-18 2020-07-23 The University Of Bristol Aav gene therapy for treating nephrotic syndrome

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
"NCBI", Database accession no. NP_001014975.1
"Uniprot", Database accession no. Q01955
"UniProtKB", Database accession no. P08603
ATSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
AUSUBEL, F.M. ET AL.: "Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
AYUSO, E. ET AL., CURRENT GENE THERAPY, vol. 10, no. 6, 2010, pages 423 - 436
CHIANG, C.K.INAGI, R., NATURE REVIEWS NEPHROLOGY, vol. 6, no. 9, 2010, pages 539
DEVEREUX ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 387
FEMS MICROBIOL. LETT., vol. 177, no. 1, 1999, pages 187 - 50
GUO G ET AL: "WT1 Activates a Glomerular-Specific Enhancer Identified from the Human Nephrin Gene", JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, vol. 15, no. 11, 1 November 2004 (2004-11-01), US, pages 2851 - 2856, XP055929896, ISSN: 1046-6673, Retrieved from the Internet <URL:https://jasn.asnjournals.org/content/jnephrol/15/11/2851.full.pdf> DOI: 10.1097/01.ASN.0000143474.91362.C4 *
GUO, G. ET AL., JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, vol. 15, no. 11, 2004, pages 2851 - 2856
LILLEY, D.M.DAHLBERG, J.E.: "Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA", 1992, ACADEMIC PRESS
LISOWSKI, L. ET AL., NATURE, vol. 506, no. 7488, 2014, pages 382 - 386
MADEIRA, F. ET AL., NUCLEIC ACIDS RESEARCH, vol. 47, no. W1, 2019, pages W636 - W641
MERLE, N.S. ET AL., FRONTIERS IN IMMUNOLOGY, vol. 6, 2015, pages 262
MERTEN, O.W. ET AL., MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT, vol. 3, 2016, pages 16017
MOELLER ET AL., J AM SOC NEPHROL, vol. 13, no. 6, 2002, pages 1561 - 7
NADEAU, I.KAMEN, A., BIOTECHNOLOGY ADVANCES, vol. 20, no. 7-8, 2003, pages 475 - 489
NI, L. ET AL., NEPHROLOGY, vol. 17, no. 6, 2012, pages 525 - 531
PICCONI ET AL., MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT, vol. 1, 2014, pages 14014
PIEKAROWICZ, K. ET AL., METHODS & CLINICAL DEVELOPMENT, vol. 15, 2019, pages 157 - 169
RISTOLA M ET AL: "Regulation of nephrin gene by the Ets transcription factor, GA-binding protein", NEPHROLOGY DIALYSIS TRANSPLANTATION, vol. 28, no. 4, 1 April 2013 (2013-04-01), GB, pages 846 - 855, XP055929781, ISSN: 0931-0509, Retrieved from the Internet <URL:https://academic.oup.com/ndt/article-pdf/28/4/846/12928529/gfs482.pdf> DOI: 10.1093/ndt/gfs482 *
RISTOLA M ET AL: "Transcription of nephrin-Neph3 gene pair is synergistically activated by WT1 and NF- B and silenced by DNA methylation", NEPHROLOGY DIALYSIS TRANSPLANTATION, vol. 27, no. 5, 6 October 2011 (2011-10-06), GB, pages 1737 - 1745, XP055929853, ISSN: 0931-0509, DOI: 10.1093/ndt/gfr576 *
ROE, B.CRABTREE, J.KAHN, A.: "DNA Isolation and Sequencing: Essential Techniques", 1996, JOHN WILEY & SONS
RUTLEDGE, E.A. ET AL., JOURNAL OF VIROLOGY, vol. 72, no. 1, 1998, pages 309 - 319
TABASSUM, A. ET AL., INTERDISCIPLINARY SCIENCES: COMPUTATIONAL LIFE SCIENCES, vol. 6, no. 1, 2014, pages 32 - 39
TREMPE, J.P.CARTER, B.J., JOURNAL OF VIROLOGY, vol. 62, no. 9, 1988, pages 3356 - 3363
VAUGHAN, M.R.QUAGGIN, S.E., JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, vol. 19, no. 1, 2008, pages 24 - 33
WONG ET AL., AMERICAN JOURNAL OF PHYSIOLOGY RENAL PHYSIOLOGY, vol. 279, no. 6, 2000, pages F1027 - 32
WONG MA ET AL., AM J PHYSIOL RENAL PHYSIOL, vol. 279, no. 6, 2000, pages F1027 - 32

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12589167B2 (en) 2020-03-12 2026-03-31 The University Of Bristol AAV gene therapy vector with podocyte-specific promoter
WO2024105415A1 (en) * 2022-11-18 2024-05-23 The University Of Bristol Methods of delivering a viral vector to a kidney
EP4397976A1 (en) * 2023-01-09 2024-07-10 x-kidney diagnostics GmbH Biomarkers for the pre-clinical and/or early-stage detection and/or diagnosis of kidney diseases
WO2025003703A1 (en) * 2023-06-29 2025-01-02 Purespring Therapeutics Limited Promoters
WO2025003710A1 (en) * 2023-06-29 2025-01-02 Purespring Therapeutics Limited Polynucleotides, aav vectors, and pharmaceutical compositions for treating nephrotic syndrome
WO2025003709A1 (en) * 2023-06-29 2025-01-02 Purespring Therapeutics Limited Regulatory elements
WO2025003704A3 (en) * 2023-06-29 2025-02-13 Purespring Therapeutics Limited Polynucleotides, aav vectors, and pharmaceutical compositions for treating complement-mediated kidney diseases

Also Published As

Publication number Publication date
US20250283072A1 (en) 2025-09-11
CN117377770A (zh) 2024-01-09
MX2023010573A (es) 2023-11-03
AU2022235209A1 (en) 2023-10-05
CA3211566A1 (en) 2022-09-15
IL305714A (en) 2023-11-01
KR20230161459A (ko) 2023-11-27
PL4305181T3 (pl) 2026-01-26
BR112023018362A2 (pt) 2023-12-05
ES3056304T3 (en) 2026-02-19
EP4722371A2 (en) 2026-04-08
PT4305181T (pt) 2025-12-04
HRP20251561T1 (hr) 2026-02-13
JP2024509316A (ja) 2024-02-29
FI4305181T3 (fi) 2025-12-12
GB202103470D0 (en) 2021-04-28
EP4305181B1 (en) 2025-11-05
DK4305181T3 (en) 2025-12-15
EP4305181A1 (en) 2024-01-17

Similar Documents

Publication Publication Date Title
EP4305181B1 (en) Minimal nephrin promoter
JP7340471B2 (ja) ウイルスベクター産生系
US20230175016A1 (en) Vector
US20260053952A1 (en) Methods of delivering a viral vector to a kidney
EP4735053A1 (en) Regulatory elements
WO2025003703A1 (en) Promoters
RU2854890C1 (ru) Вектор
WO2024201058A1 (en) Promoter
WO2025003704A2 (en) Polynucleotides, aav vectors, and pharmaceutical compositions for treating complement-mediated kidney diseases
WO2026074284A1 (en) Methods of transducing podocytes
EP4683677A1 (en) Gene therapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22710714

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 305714

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: MX/A/2023/010573

Country of ref document: MX

Ref document number: 3211566

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2023555709

Country of ref document: JP

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023018362

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 803864

Country of ref document: NZ

Ref document number: 2022235209

Country of ref document: AU

Ref document number: AU2022235209

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2022235209

Country of ref document: AU

Date of ref document: 20220314

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 202347067512

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 20237034581

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020237034581

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2023126025

Country of ref document: RU

Ref document number: 2022710714

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 11202306723P

Country of ref document: SG

ENP Entry into the national phase

Ref document number: 2022710714

Country of ref document: EP

Effective date: 20231012

WWE Wipo information: entry into national phase

Ref document number: 202280034399.8

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 112023018362

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20230911

WWE Wipo information: entry into national phase

Ref document number: 523450663

Country of ref document: SA

WWE Wipo information: entry into national phase

Ref document number: 523450663

Country of ref document: SA

WWP Wipo information: published in national office

Ref document number: 18280699

Country of ref document: US

WWG Wipo information: grant in national office

Ref document number: 2022710714

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 523450663

Country of ref document: SA