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WO2024236032A1 - Thérapies géniques pour la phénylcétonurie - Google Patents

Thérapies géniques pour la phénylcétonurie Download PDF

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Publication number
WO2024236032A1
WO2024236032A1 PCT/EP2024/063362 EP2024063362W WO2024236032A1 WO 2024236032 A1 WO2024236032 A1 WO 2024236032A1 EP 2024063362 W EP2024063362 W EP 2024063362W WO 2024236032 A1 WO2024236032 A1 WO 2024236032A1
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Prior art keywords
aav
pah
polynucleotide
paragraph
cell
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Azadeh Kia
Christopher Davies
Mario Buono
Robert Walmsley
Lisa Chinello
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Evox Therapeutics Ltd
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Evox Therapeutics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/16—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced pteridine as one donor, and incorporation of one atom of oxygen (1.14.16)
    • C12Y114/16001—Phenylalanine 4-monooxygenase (1.14.16.1)
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the disclosure relates to the treatment of diseases and disorders associated with low levels or reduced activity of the phenylalanine hydroxylase (PAH) enzyme, such as Phenylketonuria (PKU).
  • PAH phenylalanine hydroxylase
  • PKU Phenylketonuria
  • the disclosure relates to polynucleotides and polynucleotide sequences encoding PAH and gene therapy vectors comprising the same, which allow for increased expression of PAH in vivo, in particular in the liver.
  • the disclosure further relates to Extracellular Vesicles (EVs) comprising polynucleotide sequences encoding PAH and gene therapy vectors comprising the same and their use in the treatment of diseases and disorders associated with low levels or reduced activity of PAH.
  • EVs Extracellular Vesicles
  • the disclosure further relates to promoter regions, in particular tissue-specific promoter regions that drive expression of the transgenes to which they are operably linked in vivo, in particular in the liver.
  • the disclosure further relates to a bespoke method for assessing the functional activity of PAH in cells or tissues, for example cells in culture, and compositions for use is such methods, for example cell culture media.
  • Phenylketonuria is an inherited genetic disorder caused by mutations in the PAH gene that result in low levels or reduced activity of the phenylalanine hydroxylase (PAH) enzyme. PAH is responsible for the metabolism of the amino acid phenylalanine primarily in the liver.
  • Phenylalanine is present in most foods and accumulates if PKU levels or activity is reduced. The buildup of phenylalanine is associated with a number of harmful effects, which are observed in PKU patients. These include seizures, impaired neurophysiological functioning, behavioural problems and reduced cognitive development. Accumulation of phenylalanine in pregnant women can have further harmful effects and can lead to babies being born with heart problems, microcephaly and low birth weight.
  • Gene therapies involve genetically modifying cells to alter their physiological characteristics.
  • AAV adeno-associated virus
  • a new transgene that encodes for a particular protein can be introduced into a cell, so the cell starts to produce the particular protein that is encoded for.
  • AAV based gene-therapies have had some initial success and an AAV therapeutic that mediates the delivery of a transgene encoding human Factor IX (FIX) is currently in clinical trials for the treatment of Haemophilia B.
  • FIX human Factor IX
  • AAV mediated gene therapy involving the delivery of a transgene that codes for functional PAH by an AAV, is a potential avenue for developing an improved PKU treatment.
  • AAV-mediated gene therapies can induce the onset of dorsal root ganglia toxicity and severe hepatotoxicity. These complications occur most frequently and severely when a high dose of AAV is administered. It has been suggested that such problems could be overcome were it possible to give lower doses of the AAV to patients (Ertl, H.C., 2022. Immunogenicity and toxicity of AAV gene therapy. Frontiers in Immunology, 13).
  • Codon optimisation is a known method in the art that involves changing synonymous codons in the nucleotide sequence in order to enhance expression of a transgene in specific cells, for example in human host cells. Codon optimisation can alter the rate at which a ribosome translates the mRNA that is transcribed from the DNA coding sequence, since there are differences in the availability of complementary tRNAs. If the ribosome is able to use complementary tRNAs that are more readily available, it will translate at a faster rate. However, codon-optimisation does not always lead to an increase in the expression of a polynucleotide.
  • Performing codon optimisation can lead to reduced mRNA stability and disrupt the initiation of translation, thus leading to a decrease in expression at the RNA and/or protein level (Dresios, J., Chappell, S. A., Zhou, W., & Mauro, V. P. (2006). An mRNA-rRNA base-pairing mechanism fortranslation initiation in eukaryotes. Nature structural & molecular biology, 13(1), 30-34; Hausser, J., Syed, A. P., Bilen, B., & Zavolan, M. (2013). Analysis of CDS-located miRNA target sites suggests that they can effectively inhibit translation. Genome research, 23(4), 604-615).
  • codon optimisation can also result in the disruption of protein function, since slowing and pausing of the ribosome at certain times in translation may be necessary for proper protein folding (Stadler, M., & Fire, A. (2011).
  • WO2019/217513 describes a gene therapy construct comprising a codon-optimised transgene encoding PAH for use in an AAV-mediated gene therapy for PKU (BMN307).
  • WO2019/217513 describes a gene therapy construct comprising a codon-optimised transgene encoding PAH for use in an AAV-mediated gene therapy for PKU (BMN307).
  • WO2019/217513 describes a gene therapy construct comprising a codon-optimised transgene encoding PAH for use in an AAV-mediated gene therapy for PKU (BMN307).
  • codon-optimised PAH sequences to provide higher expression, whilst maintaining the functionality of the PAH protein that is produced.
  • such sequences could allow for lower doses
  • AAV therapies could further be improved were it possible to better target specific cell population, since this would allow for lower doses to be administered and would prevent side effects associated with the transduction of other cell-types.
  • a further problem with current AAV-mediated gene therapy is that the AAV can stimulate an antibody- mediated immune response in patients causing the therapeutic effect mediated by the AAV to be repressed. This is thought to be due to neutralising antibodies being produced, which bind to the AAV and prevent it from entering target cells. Up to half of all patients possess neutralising antibodies against AAVs, due to previous exposure to the AAV at some point in their lives. In these patients the effectiveness of AAV mediated gene therapy is severely curtailed. Similarly, any patient that has been previously dosed with the AAV, for the purposes of gene therapy, will have developed immunity against the AAV. Thus, the effectiveness of re-dosing is severely limited.
  • AAV-mediated gene therapies Re-dosing of AAV-mediated gene therapies is highly desirable, since the expression of the transgene delivered by the AAV diminishes over time, especially in children whose organs are still growing. Hence, there is also a need to develop AAV-mediated gene therapies for PKU that can be used in patients with neutralising antibodies against the AAV.
  • the invention provides a polynucleotide comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the polynucleotide preferably encodes phenylalanine hydroxylase (PAH) as defined herein, preferably a PAH protein having 100% sequence identity to SEQ ID NO: 6.
  • PAH phenylalanine hydroxylase
  • the present invention provides an expression cassette comprising the polynucleotide according to the first aspect and a promoter region, wherein the promoter region comprises a promoter sequence, further wherein the polynucleotide sequence is operably linked to the promoter sequence.
  • the present invention provides a gene therapy vector comprising the polynucleotide or the expression cassette as described herein, or a combination thereof.
  • the present invention provides an AAV transfer plasmid, comprising the polynucleotide or the expression cassette as described herein, or a combination thereof.
  • the present invention provides a recombinant AAV vector comprising the polynucleotide or the expression cassette as described herein, or a combination thereof.
  • the present invention provides an AAV viral particle comprising the polynucleotide or the expression cassette as described herein, or a combination thereof.
  • the present invention provides a cell comprising the polynucleotide , the expression cassette , the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector or the AAV viral particle as described herein, or a combination thereof.
  • the present invention provides an in vitro or an in vivo method for inducing PAH expression in a target cell, the method comprising administering to the cell an effective amount of the polynucleotide , the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector or the AAV viral particle as described herein, or a combination thereof.
  • the present invention provides an extracellular vesicle (EV) comprising the polynucleotide , the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector or the AAV viral particle as described herein, or a combination thereof.
  • the EV comprises a liver-targeting moiety.
  • the EV is an exosome.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector, the AAV viral particle or the EV as described herein, or a combination thereof; further comprising a pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical composition comprises an EV, wherein the EV comprises an AAV as described herein; further comprising a pharmaceutically acceptable excipient or carrier.
  • the present invention provides the polynucleotide , the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector, the AAV viral particle, the EV or the pharmaceutical composition as described herein, or a combination thereof, for use as a medicament; preferably for use in a method of treatment or prevention of a disease or disorder in a subject, preferably a disease or disorder associated with low levels or reduced activity of PAH as described herein in a subject in need thereof, preferably Phenylketonuria (PKU) in a subject.
  • PKU Phenylketonuria
  • the invention provides a method of treating a disease or disorder in a subject, preferably a disease or disorder associated with low levels or reduced activity of PAH as described herein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount or a prophylactically effective amount of the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector, the AAV viral particle, the EV or the pharmaceutical composition as described herein, or a combination thereof.
  • the present invention provides a pharmaceutical composition as described herein comprising an EV, wherein the EV comprises an AAV as described herein, further comprising a pharmaceutically acceptable excipient or carrier, for use as a medicament or for treatment or prevention of a disease or disorder as described herein, preferably Phenylketonuria (PKU) in a subject, wherein the subject is administered with two or more doses of the pharmaceutical composition.
  • a pharmaceutically acceptable excipient or carrier for use as a medicament or for treatment or prevention of a disease or disorder as described herein, preferably Phenylketonuria (PKU) in a subject, wherein the subject is administered with two or more doses of the pharmaceutical composition.
  • PKU Phenylketonuria
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an EV, wherein the EV comprises an AAV of the present invention, further comprising a pharmaceutically acceptable excipient or carrier, for use as a medicament for treatment or prevention of Phenylketonuria (PKU) in a subject, wherein the subject has immunity to the AAV comprised in the pharmaceutical composition.
  • PKU Phenylketonuria
  • the present invention provides a promoter region comprising: a liver-specific promoter, a ApoE HCR-1 enhancer, an AMPB enhancer and a SerpinCl enhancer; wherein the ApoE HCR-1 enhancer, the AMPB enhancer and the SerpinCl enhancer are operably linked to the liverspecific promoter; further wherein the liver-specific promoter is a human alpha-1 antitrypsin (hAAT) promoter.
  • hAAT human alpha-1 antitrypsin
  • the present invention provides a cell culture medium for assessing the functional activity of PAH in cells in culture comprising Phenylalanine and L-Sepiapterin.
  • the present invention provides a method for assessing the functional activity of PAH in cells in culture comprising:
  • Figure 1 Figure 1: Figure la is a schematic illustrating the structure of a known ApoE-HCR-l-hAAT promoter region (the LSP1 promoter region).
  • Figure lb and lc (showing the LSP9 promoter and the LSP4 promoter respectively) are schematics illustrating the structures of two newly designed promoter regions.
  • Figure 2a is a graph showing the PAH expression, normalised to a-tubulin, obtained when PAH KO Huh7 cells are transfected with transgene constructs comprising different combinations of promoter regions (LSP1, LSP4 and LSP9) and polynucleotide sequences encoding PAH (WT, CO8, CO9, CO13 and CO21).
  • LSP1, LSP4 and LSP9 promoter regions drive increased expression, with any polynucleotide sequence tested, as compared to LSP1.
  • Figure 2b is an image of a western blot showing the expression of PAH and a-tubulin proteins in Huh7 cells transfected with different PAH transgene constructs (LSP9-CO8, LSP9-CO9 and LSP9-CO21).
  • Figure 2c is a graph quantifying the PAH protein levels, normalised to a-tubulin, in the western blot of Figure 2b. Both Figures 2b and 2c demonstrate that LSP9-CO8, LSP9-CO9 and LSP9-CO21 induce increased expression of PAH, compared with untransfected (UT) Huh7 cells.
  • FIG. 3 A graph showing PAH protein expression levels, normalised to a-tubulin, obtained when PAH KO Huh7 cells were transfected with different PAH transgene constructs (LSP1-BMN 307, LSP9-CO8, LSP9-CO9 and LSP9-CO21).
  • LSP9-CO8, LSP9-CO9 and LSP9-CO21 all show increased expression of PAH, compared with the BMN 307 transgene under the control of an LSP1 promoter.
  • Figure 4 shows a schematic illustrating the steps in the experimental method for testing PAH activity in cell lysates.
  • Figure 4b is an image of a western blot showing that wildtype CAP cells do not express PAH, but that PAH is detected in CAP cells that have been transfected with a construct containing a wildtype PAH transgene.
  • Figure 4c shows tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in CAP cell lysates (WT or untreated; PAH or transfected with the PAH plasmid) incubated with and without phenylalanine (phe) in the Reaction Mix. Phenylalanine is required for tyrosine to be detected.
  • Figure 4d shows tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in CAP cell lysates (WT or untreated; PAH or transfected with the PAH plasmid) treated with and without sonication. Sonication does not affect the tyrosine concentration detected.
  • Figure 4e shows tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in CAP cell lysates (WT or untreated; PAH or transfected with the PAH plasmid) incubated with and without iron in the Reaction Mix. Iron does not affect the tyrosine concentration detected.
  • Figure 4f shows tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in CAP cell lysates (PAH or transfected with the PAH plasmid) with and without catalase in the Reaction Mix. Catalase does not affect the tyrosine concentration detected.
  • a simplified assay without sonication of cells and without iron or catalase in the Reaction Mix can be used to assess PAH activity from cell lysates.
  • Figure 5 Figure 5a and figure 5b are graphs showing tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in mouse liver tissue lysate samples. Endogenous tyrosine background is detected in dose dependent manner.
  • Figure 5a shows the tyrosine production detected over time when different amounts of total protein tissue lysate input were tested.
  • Figure 5b shows the tyrosine production continues to increase over time when 250 pg of tissue lysate is used. Detection of tyrosine is dependent on the presence of phenylalanine in the Reaction Mix.
  • Figure 5c is a graph showing tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in samples obtained from either wildtype mice (WT) or Enu2 mice.
  • the liver tissue obtained from WT mice produces tyrosine over time, but the liver tissue obtained from Enu2 mice does not.
  • the assay can determine PAH activity from liver tissue harvested from a mouse and can detect reduced PAH activity caused by genetic mutations.
  • Figure 6 shows tyrosine production over time, as assessed by measuring tyrosine autofluorescence, in Huh7 cells transfected with constructs containing LSP9 promoter and codon- optimised PAH transgene sequence (LSP9-CO9, LSP9-CO21 and LSP9-CO8).
  • Figure 6b shows the maximum amount of tyrosine detected during the time-course shown in Figure 6a.
  • LSP9-CO8, LSP9- CO9 and LSP9-CO21 all show increased PAH activity, compared with untransfected (UT) Huh7 cells.
  • Figure 7 shows tyrosine could not be detected in conditioned cell culture media (DMEM without phenylalanine and tyrosine supplemented with 400 uM phenylalanine in house) collected from wild-type (WT) or PAH KO Huh7 cells.
  • Figure 7b shows no difference of phenylalanine levels in conditioned media (DMEM without phenylalanine and tyrosine supplemented with 400 uM phenylalanine and BH4 in house) harvested from PAH KO cells compared to WT. Notably, a decrease of phenylalanine was observed with all conditions in the presence of BH4 (48h is here shown).
  • the general decrease in bars 4, 5 and 6 is due to BH4 interference in the assay.
  • the right-hand bar shows background noise detected in media that was not cultured with cells.
  • Figure 7c shows tyrosine is detected in conditioned media samples harvested from WT Huh7 cells cultured in a bespoke media supplemented with 400 pM Phenylalanine, 50 pM (A) or 100 pM (B) of L-Sepiapterin, but not from PAH knocked-out (KO) Huh7 cells.
  • Figure 8a shows a schematic illustrating the steps in the experimental method for testing functional PAH activity in Huh7 cells treated with AAV or ExoAAV.
  • Figure 8b and 8c show the amount of tyrosine detected when PAH knock-out Huh7 cells are infected with AAVs comprising different PAH transgene constructs (BMN 307 or LSP9-CO9) or treated with LSP9-CO9 AAV associated to EVs.
  • Figure 8b shows Tyrosine (pM/h/pg) produced and the relative fold change, compared to BMN 307.
  • Figure 8c shows the normalised tyrosine production compared to BMN 307 (100%, dashed line).
  • Cells infected with LSP9-CO9 AAV show increased PAH activity, compared to cells infected with the BMN 307 AAV.
  • the EVs loaded with LSP9-CO9 AAV also show a further increase in PAH activity, compared to cells infected with a BMN 307 AAV.
  • Figure 8d and 8e show the amount of tyrosine detected when PAH knock-out Huh7 cells are infected with AAVs comprising different PAH transgene constructs (BMN 307or LSP9-CO21) or treated with LSP9-CO21 AAV associated to EVs.
  • Figure 8d shows Tyrosine (pM/h/pg) produced and the relative fold change.
  • Figure 8e shows the normalised tyrosine production compared to BMN 307 (100%, dashed line).
  • Cells infected with LSP9-CO21 AAV show increased PAH activity, compared to cells infected with the BMN 307 AAV.
  • the EVs loaded with LSP9-CO21 AAV also show a further increase in PAH activity, compared to cells infected with a BMN 307 AAV.
  • the present disclosure provides polynucleotides and polynucleotide sequences encoding PAH, expression cassettes, vectors, viral particles and pharmaceutical compositions comprising the same, and their use to treat diseases and disorders associated with PAH deficiency or mutations in the PAH gene that result in low levels or reduced activity of the phenylalanine hydroxylase (PAH) enzyme, such as Phenylketonuria (PKU).
  • PAH phenylalanine hydroxylase
  • PKU Phenylketonuria
  • the inventors have shown that modifications to the gene sequence encoding PAH result in increased transgene expression, in particular in specific tissues, such as liver cells.
  • the polynucleotides and polynucleotide sequences encoding PAH and expression cassettes, vectors and viral particles comprising the same disclosed herein offer advantages for gene therapy as compared to previous gene therapy vectors, including the ability to achieve higher levels of PAH expression in therapeutically relevant tissues, such as liver cells.
  • the PAH protein produced by these newly identified polynucleotide sequences remains functional.
  • the newly identified polynucleotide sequences also have increased liver specificity as compared to the wild-type PAH sequence and BMN307 and are less immunogenic.
  • Phenylalanine hydroxylase is a protein capable of metabolising phenylalanine.
  • PAH as described herein refers to a polypeptide capable of metabolising phenylalanine, preferably a polypeptide capable of converting phenylalanine into tyrosine.
  • the conversion of phenylalanine to tyrosine requires BH4 as a co-factor.
  • Phenylalanine metabolism preferably the conversion of phenylalanine into tyrosine, is also referred to herein as a functional activity or enzyme activity of PAH.
  • the polynucleotide sequence encoding PAH is codon-optimized for expression in a human host cell, preferably a liver cell.
  • the transgene coding sequence is modified, or "codon optimized", to enhance expression by replacing infrequently represented codons with more frequently represented codons.
  • the coding sequence is the portion of the mRNA sequence that encodes the amino acids for translation. During translation, each of 61 trinucleotide codons are translated to one of 20 amino acids, leading to a degeneracy, or redundancy, in the genetic code.
  • tRNAs each bearing an anticodon
  • the disclosure provides a polynucleotide comprising or consisting of a codon optimised PAH sequence.
  • the codon optimisation is based on SerpinAl cDNA codon usage, Fibrin cDNA codon usage or a combination of Factor IX and F2 (prothrombin) cDNA codon usage.
  • the codon optimisation is based on SerpinAl cDNA codon usage or a combination of Factor IX and F2 (prothrombin) cDNA codon usage.
  • the codon optimised PAH sequence does not comprise premature stops, unwanted amino acid substitutions and/or cryptic splicing sites.
  • the codon optimised PAH sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-3.
  • the polynucleotide comprises or consists of a polynucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-3.
  • the polynucleotide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO 1.
  • the polynucleotide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO 3.
  • a "polynucleotide” as described herein refers to a molecule that comprises two or more covalently linked nucleotides.
  • a sequence of the oligonucleotide reference is made to the sequence or order of the covalently linked nucleotides.
  • a polynucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C)
  • this also includes the corresponding RNA sequence (e.g., A, U, G, C) in which "U” replaces "T”.
  • a polynucleotide of the disclosure may comprise one or more modified nucleosides or nucleotides.
  • Polynucleotides include sequences of DNA or RNA or mimetics thereof, which may be isolated from natural sources, recombinantly produced or artificially synthesised.
  • a further example of a polynucleotide as employed in the present invention may be a peptide nucleic acid (PNA; see U.S. Patent No. 6,156,501, which is hereby incorporated by reference in its entirety.)
  • PNA peptide nucleic acid
  • the invention also encompasses situations in which there is a non-traditional base pairing, such as Hoogsteen base pairing, which has been identified in certain tRNA molecules and postulated to exist in a triple helix.
  • polynucleotide includes, for instance, cDNA, RNA, DNA/RNA hybrid, antisense RNA, siRNA, mRNA, ribozyme, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatised, synthetic, or semi-synthetic nucleotide bases.
  • the polynucleotide may be isolated from naturally occurring sources or may be artificially or synthetically produced. Where the polynucleotide is man-made, it may be chemically synthesised, and is typically purified or isolated. Polynucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. In a preferred aspect, the polynucleotide comprises DNA.
  • Percent (%) sequence identity refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical with a reference sequence after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the skill in the art. Standard methods in the include the use of PILEUP and BLAST algorithms to calculate homology or line up sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • sequence identity refers to two or more referenced entities that are the same when they are “aligned” sequences. For example, when two polynucleotide sequences are identical, they have the same nucleic acid base sequence. Similarly, when two amino acid sequences are identical, they have the same amino acid sequence.
  • An “aligned” sequence refers to multiple polynucleotide or amino acid sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence. Sequence identity is interchangeable with the term homology or sequence homology.
  • the polynucleotide of the present disclosure is capable of inducing expression of PAH in a cell.
  • the PAH expression is expression of PAH mRNA and/or PAH protein in the cell.
  • the polynucleotides of the present disclosure are capable of inducing expression of PAH mRNA.
  • PAH mRNA expression may be detected by qPCR.
  • the polynucleotides of the present disclosure are capable of inducing expression of PAH protein. PAH protein expression may be detected by western blot or by immunofluorescence imaging.
  • the polynucleotides described herein are capable of inducing expression of a PAH protein having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6.
  • the increase in PAH expression may be as compared to cells in which PAH is knocked-out, as compared cells of, or cells derived from PKU patients or as compared to wild-type cells.
  • the polynucleotides described herein are capable of inducing an increased in PAH mRNA and/or PAH protein when expressed in a cell, as compared to expression of the wildtype PAH sequence (SEQ ID NO 4).
  • the polynucleotides described herein are capable of inducing an increased in PAH mRNA and/or PAH protein when expressed in a cell, as compared to expression of the BMN307 PAH sequence (SEQ ID NO 5).
  • the polynucleotide of the present disclosure is capable of inducing expression of a PAH protein having a sequence having 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: 6, or a functional fragment or variant thereof; preferably a polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO: 6 or a functional fragment or variant thereof.
  • the polynucleotides described herein are capable of inducing an increase in PAH mRNA and/or PAH protein when expressed in a liver cell.
  • a "liver cell” is defined herein as a cell of the liver or a cell derived from the liver, such as a hepatocyte (HCs), a hepatic stellate cells (HSCs), a Kupffer cells (KCs), a liver sinusoidal endothelial cells (LSECs) or the cell is derived from a HC, HSC, KC or an LSC.
  • the cell may be in vivo, in vitro or ex vivo. In one aspect, the cell is in vitro or ex vivo.
  • the increase in PAH mRNA and/or PAH protein occurs in a hepatocyte or a cell that is derived from a hepatocyte. In a most preferred aspect, the cell is a Huh7 cell.
  • the inventors have shown that modifications to the gene sequence encoding PAH result in increased transgene expression, in particular in specific tissues, such as liver cells. Accordingly, the polynucleotide sequences encoding PAH and expression cassettes, vectors and viral particles comprising the same disclosed herein offer advantages for gene therapy as compared to previous gene therapy vectors, including the ability to achieve higher levels of PAH expression in therapeutically relevant tissues, such as liver cells. Remarkably, despite the modified gene sequences being translated at a faster rate, these sequences still enable the production of functional PAH protein. In addition, the modified gene sequences encoding PAH are less immunogenic than the sequences previously known for gene therapy.
  • the increased expression of PAH occurs specifically in liver cells.
  • a liver-cell specific increase in PAH expression is defined as the polynucleotide inducing a greater fold increase in PAH expression when expressed in a liver cell as compared to when the polynucleotide is expressed in a non-liver cell.
  • a "non-liver cell” is defined herein as a cell of a tissue other than the liver or a cell derived from a tissue other than the liver, such as a cell of, or a cell derived from, the lung, heart, spleen, kidneys, CNS or skin. The cell may be in vivo, in vitro or ex vivo.
  • the increase in expression a non-liver cell is less than 90% 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% of the increase in a liver cell. In a preferred embodiment, there is no detectable increase in PAH expression in a non-liver cell.
  • the PAH protein is capable of metabolising phenylalanine by converting phenylalanine into tyrosine.
  • Phenylalanine metabolism preferably the conversion of phenylalanine into tyrosine, is also referred to herein as a functional activity or enzyme activity of PAH.
  • the polynucleotide of the present disclosure is capable of inducing expression of a PAH protein having functional activity.
  • the polynucleotide of the present disclosure is capable of inducing expression of a PAH protein or a functional fragment or variant thereof having 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 at least 100% of the functional activity of a PAH protein produced by the wildtype PAH sequence (SEQ ID NO 4).
  • the functional activity of PAH can be assessed by methods known in the art. Alternatively, the functional activity of PAH can be assessed by methods and as described herein. In a preferred embodiment, the functional activity of PAH can be assessed by the in vitro PAH assay described herein or the cellular PAH assay described herein. In a further preferred aspect, the functional activity of PAH is validated using both the in vitro PAH assay described herein and the cellular PAH assay described herein.
  • PAH protein or a functional fragment or variant thereof encoded by a polynucleotide of the present disclosure has some detectable level of PAH functional activity, such as at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 80%, 95%, 100% or greater than 100% of the functional activity of a PAH protein encoded by the wildtype PAH sequence (SEQ ID NO 4).
  • the PAH protein or a functional fragment or variant thereof encoded by a polynucleotide as described herein allows at least some reduction in phenylalanine levels to take place in a subject suffering from PKU.
  • the codon optimised sequences described herein encode a PAH protein that remains functional.
  • an expression cassette comprising a polynucleotide as described herein or a polynucleotide sequence or transgene encoding PAH as described herein.
  • expression cassette refers to a DNA segment that is capable in an appropriate setting of driving the expression of a polynucleotide (a "transgene") encoding a therapeutically active polypeptide (e.g., PAH) that is incorporated in said expression cassette.
  • a transgene encoding a therapeutically active polypeptide (e.g., PAH) that is incorporated in said expression cassette.
  • an expression cassette inter alia is capable of directing the cell's machinery to transcribe the transgene into RNA, which is then usually further processed and finally translated into the therapeutically active polypeptide.
  • the expression cassette can be comprised in a gene therapy vector.
  • the term expression cassette excludes polynucleotide sequences 5' to the 5' ITR and 3' to the 3' ITR.
  • transgene refers to a polynucleotide sequence to be inserted into a cell or organism that encodes a polypeptide or a portion of a polypeptide to be expressed in the cell or organism, preferably PAH as described herein.
  • a PAH transgene can include a polynucleotide sequence that is not naturally found in the cell or organism (a heterologous sequence); a nucleic acid sequence that is a variant or mutant form of a sequence naturally found in the cell or organism; or a sequence naturally occurring in the cell or organism.
  • the transgene may be capable of altering the expression of a polypeptide or protein that naturally occurs in the cell or the organism into which it is introduced.
  • the transgene is capable of increasing the expression of a polypeptide and/or protein in the cell or the organism into which it is introduced.
  • the protein is PAH as described herein.
  • the expression cassette of the present disclosure comprises a transgene comprising a polynucleotide sequence encoding PAH as described herein and a promoter sequence, wherein the promoter sequence is operably linked to said polynucleotide sequence encoding PAH.
  • the expression cassette of the present disclosure comprises a polynucleotide as described herein and a promoter region, wherein the promoter region comprises a promoter sequence, wherein the promoter sequence is operably linked to a polynucleotide sequence encoding PAH as described herein.
  • the expression cassette comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-3.
  • the expression cassette comprises a polynucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-3.
  • the expression cassette comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO 1.
  • the expression cassette comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO 3.
  • promoter refers to a DNA-regulatory sequence that is operably linked to a transgene.
  • operably linked means that the transgene (i.e. the polynucleotide sequence encoding PAH as described herein) is controlled or influenced by the promoter such that the promoter affects transcription or expression of the transgene.
  • the promoter may be required for any transcription of the transgene to occur.
  • the promoter may be where relevant proteins, such as RNA polymerase, bind to initiate transcription of transgene.
  • the promoter may be naturally occurring or non-naturally occurring.
  • the promoter is a synthetic promoter that comprises sequences that do not exist in nature. In a preferred aspect, these sequences are designed to regulate the activity of an operably linked gene.
  • the expression cassette described herein comprises a liver-specific promoter.
  • liver-specific promoter refers to a promoter or promoter region that drives gene expression specifically in liver tissue.
  • a liver-specific promoter drives a greater fold increase in the expression of a gene to which it is operably linked in cells of, or cells derived from, the liver, as compared to cells of, or cells derived from, a non-liver tissue.
  • the liverspecific promoter is the human alpha-l-antitrypsin promoter (hAAT) or an active fragments thereof, the mouse thyretin promoter (mTTR), the endogenous human factor VIII promoter (F8), the human albumin minimal promoter or the mouse albumin promoter.
  • the liver specific promoter is the human alpha-l-antitrypsin promoter (hAAT) or an active fragments thereof.
  • the liver-specific promoter consists or comprises a polynucleotide having 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: 12.
  • the expression cassette described herein further comprises an enhancer element, wherein the enhancer element is operably linked to the promoter.
  • enhancer or “enhancer element”, as used herein, refers to a DNA-regulatory element that is operably linked to a promoter.
  • An enhancer is said to be “operably linked to” the enhancer affects the transcription or expression of the transgene that is operably linked to the promoter such that the promoter activates increased transcription as compared to if the enhancer were not present.
  • the operably linked enhancer element is required for any transcription to occur from the promoter to which it is operably linked.
  • the expression cassette described herein comprises a liver-specific enhancer element.
  • liver-specific enhancer element refers to an enhancer element that drives gene expression specifically in liver tissue.
  • a liver-specific enhancer element drives a greater fold increase in the expression from the promoter to which it is operably linked in cells of, or cells derived from, the liver, as compared to cells of, or cells derived from, a non-liver tissue.
  • the liver specific enhancer element is HCR, ApoE, SerpinCl, AMBP, EBP, DBP, HNF1, HNF3, HNF4, HNF6 and Enhl or an active fragment thereof.
  • the expression cassette described herein comprises an ApoE HCR-1 enhancer or an active fragment thereof operably linked to the promoter.
  • the ApoE HCR-1 enhancer consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7.
  • the expression cassette described herein comprises a promoter region comprising a hAAT promoter sequence as described herein operably linked to an ApoE HCR-1 enhancer as described herein.
  • the promoter region consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 15.
  • the expression cassette described herein comprises a AMBP enhancer or an active fragment thereof operably linked to the promoter.
  • AMBP enhancer consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8.
  • the expression cassette described herein comprises a Serpin Cl enhancer or an active fragment thereof operably linked to the promoter.
  • Serpin Cl enhancer consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9.
  • the expression cassette described herein comprises a promoter region comprising a liver specific promoter, as described herein operably linked to an ApoE HCR-1 enhancer as described herein, an AMBP enhancer as described herein and a Serpin Cl enhancer as described herein.
  • the liver specific promoter is a hAAT promoter sequence as described herein.
  • the promoter region allows for increased expression of the transgene to which it is operably linked.
  • the newly identified promoter regions have increased liver specificity as compared to known promoter regions, including liver-specific promoter regions.
  • the expression cassette described herein comprises a promoter region as described herein, wherein the enhancer and promoter elements are structured, in the following order, from 5' to 3' as follows: ApoE HCR-1 enhancer, the SerpinCl enhancer, the AMPB enhancer, HCR enhancer, hAAT promoter sequence.
  • the promoter region further comprises an F9 promoter region to the 5' of the ApoE HCR-1 enhancer.
  • the F9 promoter region has at least 70% 75, 80% 85% 90% 91% 92% 93% 94% 95% 96% 97% 98% 99% or 100% sequence identity to SEQ ID NO 10.
  • the promoter region additionally comprises an Oct-1 transcription factor binding site to the 5' of the F9 promoter region.
  • the Oct-1 transcription factor binding site has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 11.
  • the promoter region consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 13.
  • the expression cassette described herein comprises a promoter region, wherein the enhancer and promoter elements are structured in the following 5' to 3' order: SerpinCl enhancer, AMPB enhancer, ApoE HCR-1 enhancer, HCR enhancer, hAAT promoter.
  • the promoter region additionally comprises an Oct-1 transcription factor binding site to the 5' of the SerpinCl enhancer.
  • the Oct-1 transcription factor binding site has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 11.
  • the promoter region consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 14.
  • the promoter region as described herein is of a size sufficient to allow for high levels of transcription of a transgene to which it is operably linked, whilst still allowing for efficient packaging of the expression cassette, in which it is comprised, into an AAV.
  • the promoter region as described herein is less than 1.5 kb, 1.3 kb, 1.3 kb, 1.1 kb or 1 kb base pairs in length.
  • the expression cassette described herein further comprises a Kozak sequence positioned directly 5' of the PAH transgene and positioned 3' of the promoter region.
  • the Kozak sequence is a DNA sequence having SEQ ID NO: 16.
  • the expression cassette described herein further comprises a polyadenylation sequence.
  • a "polyadenylation sequence” as defined herein is a DNA sequence that is a transcriptional termination region and allows for a poly(A) tail to be added to an RNA transcript.
  • the polyadenylation sequence is positioned to the 3' of the PAH transgene and allows for a poly(A) tail to be added to the RNA transcript that is transcribed from the PAH transgene.
  • the polyadenylation signal sequence is a Bovine growth hormone (bGH) poly(A), SV 40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof.
  • bGH Bovine growth hormone
  • rBG rabbit beta-globin
  • TK thymidine kinase
  • the expression cassette described herein further comprises a polyadenylation sequence.
  • the polyadenylation sequence is downstream of the transgene described herein and allows for a poly(A) tail to be added to the RNA transcript that is transcribed from the transgene sequence.
  • polyadenylation signal sequences include, but are not limited to, Bovine growth hormone (bGH) poly(A), SV 40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof.
  • bGH Bovine growth hormone
  • rBG rabbit beta-globin
  • TK thymidine kinase
  • the polyadenylation sequence is a BgpA sequence, positioned to the 3' of the transgene.
  • the BgpA sequence consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17.
  • the expression cassette described herein further comprises a 3' and a 5' ITR sequence.
  • ITR inverted terminal repeat
  • the term "inverted terminal repeat (ITR)" as used herein is defined as the regions found at the 5' and 3' termini of the AAV genome.
  • the ITRs function in cis as origins of DNA replication and as packaging signals for the viral genome.
  • ITR sequences are well known to a person skilled in the art. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J Viral. (2005) vol. 79, pp. 364-379 which is herein incorporated by reference in its entirety. ITR sequences that find use herein may be full length, wild-type AAV ITRs or fragments thereof that retain functional capability.
  • the ITRs may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication.
  • the AAV ITRs may be derived from any known AAV serotype and, in certain embodiments, derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype.
  • the ITRs used in the present invention are derived AAV2.
  • the 3' ITR sequence consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the 5' ITR sequence consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the expression cassette described herein consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 20-22.
  • the expression cassette described herein consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 20.
  • the expression cassette described herein consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 22.
  • the expression cassette described herein is of a size sufficient that allows for it to be efficiently packaged into an AAV.
  • the expression cassette described herein is less than 5.5 kb, 5 kb, 4.9 kb, 4.8 kb, 4.7 kb, 4.5 kb or 4.4 kb.
  • the transgene is less than 4.4 kb base pairs in length.
  • the present disclosure relates to a gene therapy vector comprising the polynucleotide or expression cassette as described herein, or a combination thereof.
  • the gene therapy vector is an AAV transfer plasmid.
  • AAV transfer plasmid is defined as a plasmid that comprises a transgene and which enables the transgene to be incorporated into the genome of an AAV viral particle.
  • AAV viral particles are produced when the AAV transfer plasmid, comprising the transgene, is expressed in a host cell alongside an AAV rep and cap.
  • the host cell may be an insect cell or a mammalian cell.
  • the host cell is a HEK293 cell.
  • the present disclosure relates to a recombinant AAV vector comprising the polynucleotide encoding PAH as described herein or an expression cassette as described herein, or a combination thereof.
  • the term "recombinant AAV vector" as used herein refers to nucleic acids that are present in the genome of an AAV viral particle.
  • the recombinant AAV vector is a single stranded AAV vector and comprises either the sense strand or the anti-sense strand of the nucleic acid sequences disclosed herein.
  • Recombinant AAV can be produced using any conventional methods known in the art. Such methods are disclosed in WO 2019/217 513 which is herein incorporated by reference in its entirety.
  • the present disclosure relates to a viral particle comprising the polynucleotide or expression cassette as described herein, or a combination thereof.
  • the viral particle is a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus.
  • the viral particle is an AAV viral particle.
  • An "AAV viral particle” is defined herein as a viral particle comprising of at least one AAV capsid protein and an encapsidated recombinant AAV vector. This term is interchangeable with the terms "AAV virion", "AAV virus” and “AAV vector particle” and "AAV”.
  • AAV viral particles can be produced using any conventional methods known in the art. Such methods are disclosed in WO 2019/217 513, which is incorporated herein by reference in its entirety. For instance, AAV comprising a transgene in its genome is produced when an AAV transfer plasmid is expressed in a host cell alongside the AAV rep and cap.
  • the host cell may be an insect cell or a mammalian cell. In a preferred aspect, the host cell is a HEK293 cell.
  • the "AAV capsid” as defined herein is a protein shell that encapsulates the recombinant AAV vector.
  • the AAV capsid is formed of structural VP proteins (a mixture of VP1, VP2 and VP3).
  • the AAV VP proteins are known to determine the cellular tropicity of the AAV virus.
  • the VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes.
  • Cap genes that are derived from different AAV serotypes encode for VP proteins that form capsids which display different tropisms for different cell types.
  • the AAV of the present invention comprises an AAV capsid that has tropism for liver cells.
  • the AAV of the present disclosure comprises an AAV capsid derived from AAV7, AAV8 or AAV9.
  • the AAV capsid is derived from AAV8.
  • the AAV virus is a therapeutic AAV.
  • therapeutic AAV virus or “therapeutic AAV viral particle” refers to an AAV virus that comprises a transgene that encodes a therapeutic protein.
  • the therapeutic protein is PAH as described herein.
  • the present disclosure relates to a cell comprising the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector and/or the AAV viral particle as described herein.
  • the cell is a liver cell or is derived from a cell of the liver.
  • EVs can be loaded with exogenous cargoes, including proteins and polynucleotides of interest, and that the loaded proteins and polynucleotides in this manner results in efficiently delivery of these cargoes into cells.
  • AAVs that are associated with EVs have a number of advantageous effects as compared to AAVs alone.
  • One such advantage is that AAVs that are associated with EVs induce a greater increase in PAH expression, allow for more complete transduction of target cell population and achieve better therapeutic effects when administered at a lower dose. This is due, at least in part, to EVs being able to enter target cells through a variety of different uptake pathways.
  • association with an EV also allows for lower doses of the AAV to be administered, hence has the potential to lesson toxicity in subjects and provides a safer therapeutic.
  • a further advantage of AAVs that are associated with EVs is that AAVs that are associated with EVs overcome problems associated with neutralising antibodies, as the AAV is shielded from neutralising antibodies through its encapsulation by or association with the EV. Moreover, even if neutralising antibodies are still able to bind an AAV that is associated with the EV, the EVs retain the ability to enter target cells, since EVs enter target cells through a variety of different mechanisms. Hence, association of the AAV with EVs can increase the effectiveness of an AAV- mediated gene therapy in patients that have neutralising antibodies against the AAV.
  • the disclosure provides an AAV-mediated gene therapy that is more effective in patients with neutralising antibodies, and which can be re-dosed. Re-dosing in turn facilitates safer administration of the AAV- mediated gene therapy, since the AAV may be given repeatedly at a lower dose, thus further reducing the likelihood of problems such as dorsal root ganglia toxicity and severe hepatotoxicity.
  • AAVs that are associated with EVs are less immunogenic, compared with the AAV alone.
  • AAVs that are associated with EVs can be used to target a particular subset of cells more efficiently than traditional AAVs, which also allows for a lower dose to be administered and has the potential to prevent side-effects caused by the transduction of other cell-types.
  • the present disclosure relates to an extracellular vesicle (EV) comprising the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector and/or the AAV viral particle as described herein.
  • extracellular vesicle or “EV” are used interchangeably herein and can be understood to relate to any type of vesicle that is obtainable from a cell in any form, comprising a membrane that encloses an internal space (i.e., a lumen).
  • the size of EVs may vary considerably, but an EV typically comprises a volume defined by a bi-lipid membrane and having a nano-sized hydrodynamic radius, i.e., a radius below 1000 nm.
  • the volume may comprise the vesicular secretome.
  • the terms 'extracellular vesicle' and/or 'EV' may relate to any type of lipid-based structure (with vesicular morphology or with any other type of suitable morphology) that can act as a delivery or transport vehicle or that has native therapeutic or pharmacological effects.
  • Exosomes, microvesicles and ARMMs are just some examples of the different subtypes that fall under the hereinbefore broader description of EVs and represent particularly preferable EVs, but it will be appreciated that other EVs may also be advantageous in certain circumstances.
  • the different types of EVs are defined by different morphologies, structure, messages and function. EVs can be broadly divided into two categories, (1) ectosomes and (2) exosomes.
  • ectosome or "ectosomes” are used interchangeably herein and can be understood to relate to any type of heterogenous vesicle that is obtainable or derivable from outward budding of the plasma membrane and/or cell membrane of a cell, preferably from neutrophils and monocytes in serum.
  • ectosomes include, but are not limited to, microvesicles, microparticles and large vesicles. Typically, ectosomes range in size from about 50nm to about 1pm.
  • microvesicle and “microvesicles” are used interchangeably herein and can be understood to relate to any type of vesicle that is obtainable or derivable or shed from the plasma membrane or cell membrane of a cell.
  • exosome or “exosomes” are used interchangeably herein and can be understood to relate to any type of vesicle that is obtainable or derivable from the endosomal, lysosomal and/or endo-lysosomal pathway and/or from inward budding of the plasma membrane and/or cell membrane.
  • Exosomes often have a size of from about 30 to about 300 nm, typically in the range of from about 40 to about 250 nm, and sometimes from about 40 to about 160nm, which is a highly suitable size range.
  • the EV is a microvesicle or an exosome.
  • the EV is an exosome.
  • EVs may be derived from any cell type, whether in vivo, ex vivo or in vitro.
  • the "EV Cell Source” as defined herein are the cells from which the EVs are derived.
  • “EV cell source” is used interchangeably with the term “EV producer cells”.
  • EVs may be derived from essentially any cell source, be it a primary cell source or an immortalized cell line.
  • the EV source cells may be any embryonic, fetal, and adult somatic stem cell types, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method, as well as any adult cell source.
  • iPSCs induced pluripotent stem cells
  • the source cells may be select edfrom a wide range of cells and cell lines, for instance mesenchymal stem or stromal cells (obtainable from e.g. bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, chorion, placenta, tooth buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amnion cells and more specifically amnion epithelial cells optionally expressing various early markers, myeloid suppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc.
  • mesenchymal stem or stromal cells obtainable from e.g. bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, chorion, placenta, tooth buds, umbilical cord blood, skin tissue, etc.
  • fibroblasts fibroblasts
  • Cell lines of particular interest include human umbilical cord endothelial cells (HUVECs), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitors, chondrocytes, mesenchymal stromal cells (MSCs) of different origin, amnion cells, amnion epithelial (AE) cells, CEVEC's CAP® cells any cells obtained through amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, etc.
  • HAVECs human umbilical cord endothelial cells
  • HEK human embryonic kidney
  • endothelial cell lines such as microvascular or lymphatic endothelial cells
  • erythrocytes erythroid progenitors
  • chondrocytes chondrocytes
  • the source cells are immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DCs). Essentially any type of cell which is capable of producing EVs is also encompassed herein.
  • the source cell is allogeneic, autologous, or even xenogeneic in nature to the patient to be treated, i.e. the cells may be from the patient himself or from an unrelated, matched or unmatched donor.
  • Preferred producer cells according to the present invention may be a: HEK cell, a HEK293 cell, a HEK293T cell, an MSC, in particular a WJ-MSC cell or a BM-MSC cell, a fibroblast, an amnion cell, an amnion epithelial cell, CEVEC's CAP® cells, a placenta-derived cell, a cord blood cell, an immune system cell, an endothelial cell, an epithelial cell or any other cell type, wherein said cells may be for instance adherent cells, suspension cells, and/or suspension-adapted cells
  • the producer cells are HEK293 cells.
  • HEK293 cells are particularly well-suited to producing viruses, including AAV, which naturally associates with/passively loads into EVs.
  • HEK293 cells have the ability to produce large amounts of virus since they can be transfected with DNA constructs at a high efficiency; since they express Ad5, E1A and E1B genes, which prevent apoptosis; and further since the expression of Ad5 allows for the increased production of recombinant protein from plasmid vectors that contain a CMV promoter.
  • the producer cells are HEK293T cells.
  • HEK 293T cells have all the advantageous characteristics outlined above in relation to HEK293 cells, but in addition also express the SV40 large T antigen, which enables them to produce recombinant proteins within plasmid vectors containing the SV40 promoter.
  • the producer cells are adapted to serum-free cell culture conditions. This is advantageous as it allows for easier purification and downstream processing and reduces the risk of contamination.
  • the producer cells are adapted to suspension culture. Thus, is advantageous as it allows for more efficient scaling of production and an easier production on at a large-scale.
  • the producer cells are HEK293 cells that have been adapted to serum-free, suspension culture conditions.
  • the producer cells are HEK293T cells that have been adapted to serum-free, suspension culture conditions.
  • modified when used in relation to an EV, can mean that the vesicle has been modified either using genetic or chemical approaches, for instance via genetic engineering of the EV-producing cell, preferably an exosome-producing cell or via e.g., chemical conjugation, for instance to attach moieties to the EV, preferably the exosome surface.
  • genetically modified and “genetically engineered” are used interchangeably herein and can mean that the EV, preferably an exosome, is derived from a genetically modified/engineered cell, preferably the cell that has been genetically modified to produce a virus, preferably an AAV.
  • AAVs produced by genetically engineered cells naturally associate with/passively loads the EVs that are also being produced by the genetically engineered cells.
  • the EV may be genetically engineered to express and/or modify the expression of proteins in the lumen, extravesicular membrane and/or displayed on the surface of the EV (e.g., exosome), which is typically incorporated into the EVs, preferably exosomes, produced by those cells. These proteins may directly or indirectly interact with AAVs, thus allowing for the AAVs to associate with/actively load the EV.
  • the AAVs may be produced by the same EV-producer cells and may be loaded endogenously or may be produced by different cells and be loaded exogenously.
  • Such genetically engineered or genetically modified EVs do not occur in nature. Furthermore, the said terms shall be understood to also relate to, in some embodiments, EV mimics, cellular membrane vesicles obtained through membrane extrusion, sonication or other techniques, etc.
  • the present invention normally relates to a plurality of EVs, i.e. a population of EVs which may comprise thousands, millions, billions or even trillions of EVs.
  • EVs may be present in concentrations such as 10 5 , 10 8 , IO 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 ,1O 30 EVs (often termed "particles”) per unit of volume (for instance per ml or per litre), or any other number larger, smaller or anywhere in between.
  • the term "population”, which may e.g. relate to an EV comprising a certain POI shall be understood to encompass a plurality of entities which together constitute such a population.
  • individual EVs when present in a plurality constitute an EV population.
  • the present invention pertains both to individual EVs and populations comprising EVs, as will be clear to the skilled person.
  • the dosages of EVs when applied in vivo may naturally vary considerably depending on the disease to be treated, the administration route, the activity and effects of an associated AAV, the activity and effects of any pharmacokinetic or pharmacodynamic effector moieties, the pharmaceutical formulation, etc.
  • the EV as described herein may comprise a therapeutic cargo.
  • the therapeutic cargo is the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector and/or the AAV viral particle as described herein.
  • the EV of the present invention may comprise more than one of the therapeutic cargo.
  • a single EV comprising more than one therapeutic cargo is advantageous as it increases the therapeutic effect of the EV and the potential for multiplexing of cargos to improve the delivery, therapeutic effect, targeting etc.
  • the EV of the present invention may comprise multiple different therapeutic cargos.
  • the EV may comprise a therapeutic AAV viral particle as described herein and a therapeutic protein cargo, a therapeutic nucleic acid cargo and/or a therapeutic small molecule cargo.
  • the EV described herein comprising an AAV viral particle as described herein which is loaded through interaction with a viral binding protein that the EV comprises. Additionally, or alternatively, the EV described herein comprising an AAV viral particle as described herein is loaded exogenously.
  • the EV of the present disclosure may comprise a therapeutic protein cargo in addition to a therapeutic viral cargo.
  • the therapeutic protein cargo is an immune effector molecule. Immune effector molecules are particularly useful in the case of EVs loaded with a viral (e.g. AAV or lentiviral) cargo but may equally be used where EV is loaded with any cargo according to the invention.
  • the immune effector may act to reduce immunogenicity of the EV.
  • the immune effector functions stimulate immune inhibitors.
  • the immune effector functions inhibit immune stimulating molecules.
  • the EV comprises molecules that stimulate immune inhibitors and molecules that inhibit immune stimulating molecules.
  • the EV therapeutic protein cargo(s) are fused to an EV polypeptide.
  • the EV comprises multimerization domain, a linker and/or a release domain that is fused between the therapeutic protein cargo(s) and the EV polypeptide.
  • the EV as described herein may comprise a cargo that is a nucleic acid cargo described herein.
  • the EV comprises the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid and/or the recombinant AAV vector as described herein.
  • the EV comprises a therapeutic nucleic acid cargo as described herein.
  • the EV of the present invention may comprise the therapeutic nucleic acid molecule in addition to a therapeutic viral cargo and/or a therapeutic protein cargo.
  • the EV described herein comprising a nucleic acid cargo as described herein is loaded through interaction with an RNA/DNA binding protein. Additionally, or alternatively, the EV described herein comprising a nucleic acid cargo as described herein is loaded exogenously.
  • the EV described herein may additionally comprise one or more additional proteins of interest (POI).
  • POIs include:
  • binding proteins for a therapeutic agent such as an RNA binding protein, viral binding protein, small molecule binding proteins, fc-binding proteins
  • the POI is fused to an EV polypeptide.
  • the EV comprises a multimerization domain, a linker and/or a release domain that is fused between the POI and the EV polypeptide.
  • the binding protein may be comprised in a fusion polypeptide with an EV polypeptide.
  • the POI or at least one of the POIs may be a binding protein for a therapeutic cargo.
  • the binding protein may be an RNA or DNA binding protein, a viral binding protein, a small molecule binding protein or an Fc-binding protein.
  • the binding protein is a viral binding protein, such as: proteins capable of binding to adeno-associated virus (AAV) or proteins capable of binding to other types of viruses such as lenti- viruses.
  • the viral binding protein is any protein capable of binding to a viral coat/envelope protein or capable of binding to a viral genome.
  • the viral binding protein is capable of binding to adeno-associated virus (AAV), for instance the AAV-receptor.
  • the viral binding protein is AAVR, GPR108, syndecans or albumin, as well as any domains, parts or derivates, thereof.
  • the binding is a nucleic acid binding protein (NA-binding protein) such as an RNA or DNA binding protein.
  • NA-binding protein such as an RNA or DNA binding protein.
  • the binding protein is a small molecule binding protein, such as any protein, polypeptide, or peptide (i.e. any molecule comprising a sequence of amino acids) to which a small molecule agent can be attached, via non-covalent or covalent attachment, or via a combination of both covalent and non-covalent interactions.
  • a binding protein and a small molecule agent is herein described using terms such as "binding protein-small molecule conjugate” or "binding protein-small molecule drug conjugate” or “binding protein-small molecule agent conjugate", or just “conjugate”.
  • the binding protein is a protein capable of binding to an Fc domain, also known as an Fc-binding protein.
  • Fc binding polypeptide and “Fc binding protein” and “Fc binder” and “Fc-binding protein” and “binder” are used interchangeably herein and shall be understood to relate to any protein, polypeptide, or peptide (i.e. any molecule comprising a sequence of amino acids) which can bind an Fc domain of any POI.
  • the Fc binding polypeptides of the present invention are derived from various sources that are either human or non-human (e.g.
  • mammal sources, bacteria, etc. they have high affinity for Fc domains of various antibody isotypes, subtypes, and species (for instance IgG (as non-limiting examples in the case of IgG, IgGl , lgG2, lgG3, lgG4, lgG2a, lgG2d, and/or lgG2c), IgA, IgM, IgM, IgD, etc.), and they can be fused to EV proteins.
  • the Fc binding polypeptides is Protein A, Protein G, Protein A/G, Z domain, ZZ domain, human FCGRI, human FCGRIIA, human FCGRI IB (as a non-limiting example the accession number 31994), human FCGRIIC (as a nonlimiting example the accession number 31995), human FCGRIIIA (as a non-limiting example the accession number P08637), human FCGR3B (as a non-limiting example the accession number 075015), human FCAMR, human FCERA, human FCAR, mouse FCGRI, mouse FCGRIIB, mouse FCGRIII, mouse, mouse FCGRn, and various combinations, derivatives, or alternatives thereof.
  • the Fc binders are capable of binding an "Fc containing protein".
  • the terms "Fc containing protein” and “protein comprising an Fc domain” and “Fc domain-containing protein” and “Fc domain containing protein” and “Fc domain protein” and similar terms are used interchangeably herein and shall be understood to relate to any protein, polypeptide, or peptide (i.e. any molecule comprising a sequence of amino acids) which comprises an Fc domain, either naturally or as a result of engineering of the protein in question to introduce an Fc domain.
  • the Fc containing protein is a therapeutic cargo or a different POI, for instance a targeting moiety.
  • the EV of the present disclosure comprises a fusion protein comprising an EV polypeptide and an Fc binding polypeptide.
  • the Fc binding polypeptide is bound to an antibody against an AAV capsid, preferably an AAV7, AAV8 or AAV9 capsid, most preferably an AAV8 capsid.
  • the EV described herein comprises a POI that is an endosomal escape domain or endosomal escape moiety.
  • the endosomal escape domain is comprised in a fusion polypeptide also comprising an EV polypeptide.
  • the endosomal escape domain is HA2, VSVG, GALA, B18, HIV TAT PDT (peptide/protein transduction domain), HIV Gp-120, KALA, GALA and INF-7 (derived from the N-terminal domain of influenza virus hemagglutinin HA-2 subunit) or an endosomal escape moiety that acts by causing membrane fusion such as Diphtheria toxin T domain, proton sponge type endosomal escape moieties such as peptides or lipids with histidine or imidazole moieties and cell penetrating peptides (CPPs) and other moieties that enable endosomal escape.
  • HIV TAT PDT peptide/protein transduction domain
  • HIV Gp-120 derived from the N-terminal domain of influenza virus hemagglutinin HA-2 subunit
  • INF-7 derived from the N-terminal domain of influenza virus hemagglutinin HA-2 subunit
  • an endosomal escape moiety that acts by
  • CPPs are typically less than 50 amino acids but may also be longer, are typically highly cationic and rich in arginine and/or lysine amino acids and have the ability to gain access to the interior of virtually any cell type
  • exemplary CPPs may be transportan, transportan 10, penetratin, MTS, VP22, CADY peptides, MAP, KALA, PpTG20, proline-rich peptides, MPG peptides, PepFect peptides, Pep-1, L-oligomers, calcitonin peptides, various arginine-rich CPPs, such as poly-Arg, tat and combinations thereof).
  • endosomal escape domains advantageously assists to drive endosomal escape and thereby enhance the bioactive delivery of the EV per se.
  • Use of endosomal escape strategies is particularly important in the treatment of diseases where the cargo carried within the EV is required to be delivered into the cytosol of the recipient cell or within any other compartment that is outside of the endo-lysosomal system.
  • the EV described herein comprises a POI that is a targeting moiety.
  • a targeting moiety enables the EV to be engineered so as to allow targeted delivery of the EV to a cell, tissue, organ, and/or compartment of interest.
  • the targeting moiety may be the POI comprised in a fusion polypeptide with an EV polypeptide.
  • the targeting moiety is engineered to be displayed on the surface of the EVs.
  • the targeting moiety is a protein, peptide, single chain fragment or any other derivatives of an antibody, obtainable from either humans or from non-human animals, etc.
  • Targeting moieties may be used to target the EVs to cell, subcellular locations, tissues, organs or other bodily compartments.
  • the EV is targeted to the liver.
  • Targeting can be achieved by a variety of means, for instance the use of targeting peptides.
  • the EV described herein comprises a targeting peptide that may be anywhere from a few amino acids in length to several 100s of amino acids in length, e.g. anywhere in the interval of 3-100 amino acids, 3-30 amino acids, 5-25 amino acids, e.g. 7 amino acids, 12 amino acids, 20 amino acids, etc.
  • Targeting peptides of the as described herein include full length proteins such as receptors, receptor ligands, etc.
  • the targeting peptides described herein may also include antibodies and antibody derivatives, e.g. monoclonal antibodies, single chain variable fragments (scFvs), nanobodies, other antibody domains.
  • the EV described herein comprises a pharmacokinetic or pharmacodynamic effector moiety.
  • Pharmacokinetic Effector Moiety shall be understood to relate to any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacokinetics of the EV.
  • Pharmacodynamic Effector Moiety shall be understood to relate to any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacodynamics of the EV.
  • the pharmacokinetic/pharmacodynamic effector moiety is an albumin binding domain.
  • albumin binding domain (ABD) shall be understood to relate to any protein, peptide, antibody or nanobody, or fragment or domain thereof capable of binding to albumin.
  • ABDs may be derived from any species, preferably the ABD has specific binding affinity for human serum albumin.
  • the ABD is an antibodies or a nanobodies raised against albumin, an ABD derived from PAB protein from Peptostreptococcus magnus or protein G from group C and G streptococci.
  • the albumin binding domain may be an antibody, scFv nanobody, heavy chain antibody (hcAb), single domain antibody (sdAb) such as VHH or VNAR, or a fragment thereof which is capable of binding to albumin.
  • sdAbs and antibody fragments are particularly preferred due to their small size which allows for other additional domains to be introduced into the fusion protein and simple construct generation and expression.
  • the EV described herein comprises both a targeting moiety combined and an ABD.
  • an EV as described herein may comprise an EV polypeptide fused to both a therapeutic protein/a binding protein for a therapeutic cargo (optionally luminally loaded) and a targeting moiety, and on a second construct an ABD fused to an EV polypeptide.
  • the POI-EV polypeptide-targeting moiety and EV polypeptide-ABD constructs are expressed on the same EV.
  • the EV comprises the ABD will become coated in albumin and thus remain in circulation much longer, avoiding uptake by the cells of the immune system, and hence is able to reach the desired target organ.
  • the EV described herein comprises an EV polypeptide as a POL
  • the EV polypeptide is essentially any protein, region, domain, motif, or sequence or stretch of amino acids that is capable of transporting a fusion protein into an EV produced by a given EV-producing cell.
  • the EV polypeptide comprised in a fusion proteins as described herein is a transmembrane EV polypeptide.
  • the EV polypeptides is selected from the group consisting of the following non-limiting examples: CD9, CD53, CD63, CD81, CD54, CDSO, FLOT1 , FLOT2, CD49d, CD71, CD133, CD138, CD235a, AAAT, AT1B3, AT2B4, ALIX, Annexin, BASI, BASP1 , BSG, Syntenin-1 , Syntenin- 2, TSP2, TSP3, Lamp2, Lamp2a, Lamp2b, TSN1 , TSN2, TSN3, TSN4, TSNS TSN6, TSN7, TSPAN8, TSN31, TSN10, TSN11 , TSN12, TSN13, TSN14, TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN4, TSN9, TSN32, TSN33, TNFR, TfRl, syndecan-1 , syndecan-2, syndecan-3, syndecan-4, CD37, CD82,
  • Mutations, truncations, linkers or additions may be introduced into the wildtype sequence of the EV polypeptide to alter its function, for instance a preferred mutant according to the invention is a mutation of the tetraspanin CD63 which replaces the tyrosine in position 235 with alanine (denoted CD63/Y235A).
  • EV proteins has the effect of driving loading of the fusion protein into EVs, such that not only is the therapeutic cargo and/or the POI located in the EV and subsequently secreted by the EV producing cell, but the production of EVs comprising the fusion protein also increased by virtue of the pressure exerted on the EV-producing cell to express and translate the delivered polynucleotide cargo.
  • the EV polypeptide is a tetraspanin such as CD63, CD81, CD9, CD82, CD44, CD47, COSS, TSP2, TSP3, TSP18, LAMP2B, LIMP2, ICAMs, integrins, ARRDC1 , syndecan, syntenin, PTGFRN, BASP1 , MARCKS, MARCKSL 1, TfR, and Alix, as well as derivatives, domains, variants, mutants, or regions thereof.
  • the EV polypeptide may be combined with transmembrane domains from various cytokine receptors, for instance TNFR and gpl30, in order to enhance the loading of the fusion protein into the genetically engineered EVs.
  • the EV described herein further comprises a multimerization domain.
  • the multimerization domain is a homomultimerization domains or a heteromultimerization domains.
  • the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or any higher order of multimerization domain. Multimerization domains enable dimerization, trimerization, or any higher order of multimerization of the fusion polypeptides, which increases the sorting and trafficking of the fusion polypeptides into EVs and may also contribute to increase the yield of vesicles produced by EV-producing cells.
  • Exemplary multimerization domains include: leucine zipper, fold-on domain, fragment X, collagen domain, 2G12 IgG homodimer, mitochondrial antiviral-signaling protein CARD filament, Cardiac phospholamban transmembrane pentamer, parathyroid hormone dimerization domain, Glycophorin A transmembrane, HIV Gp41 trimerisation domain, HPV45 oncoprotein E7 C-terminal dimer domain, and any combination thereof.
  • Linkers according to the invention are useful in providing increased flexibility, improving pharmacokinetics (PK), increasing expression and improving biological activity of the fusion polypeptide constructs, and also to the corresponding polynucleotide constructs, and may also be used to ensure avoidance of steric hindrance and maintained functionality of the fusion polypeptides.
  • PK pharmacokinetics
  • GGGGS glycine or serine linkers which increase stability or flexibility
  • an EV fusion protein as described herein may comprise a suitable release domain.
  • the release domain is cis-cleaving or "self cleaving".
  • self-cleaving protein can mean a naturally occurring protein that excises itself from a native host protein through self-cleavage. It is to be appreciated that certain modifications are desirable to provide a protein that has self-cleaving capability only (i.e., no self-splicing).
  • a protein capable of only self-cleavage (and no splicing) is AI-CM.
  • self-splicing protein can mean a naturally occurring protein that excises itself from a native host protein through self-splicing and ligation of their flanking peptide bonds.
  • the release domain is cis-cleaving or "self cleaving" sequences such as inteins, light induced monomeric or dimeric release domains such as Kaede, KikGR, EosFP, tdEosFP, mEos2, PSmOrange, the GFP-like Dendra proteins Dendra and Dendra2, CRY2-CIBN, etc.
  • inteins light induced monomeric or dimeric release domains
  • KikGR nuclear localization signal
  • EosFP nuclear localization signal
  • tdEosFP tdEosFP
  • mEos2 mEos2, PSmOrange
  • the GFP-like Dendra proteins Dendra and Dendra2, CRY2-CIBN etc.
  • nuclear localization signal (NLS) - nuclear localization signal-binding protein (NLSBP) (NLS-NLSBP) release system may be employed.
  • Protease cleavage sites may also be incorporated into the fusion proteins for spontaneous release, etc.,
  • nucleic acid cargos specific nucleic acid cleaving domains may be included.
  • the nucleic acid cleaving domain is an endonucleases such as Cas6, Casl3, engineered PUF nucleases, site specific RNA nucleases etc.
  • the inclusion of release domains is highly advantageous because they enable release of particular parts or domains from the original fusion polypeptide. This is particularly advantageous when the release of parts of the fusion polypeptide would increase bioactive delivery of the cargo and/or when a particular function of the fusion polypeptide works better when part of a smaller construct.
  • the self-cleaving protein is an intein.
  • mini-intein or “delta-intein” are used interchangeably herein and can be understood to relate to a modified intein, preferably from parent RecA, and lacking the endonuclease domain.
  • the intein may be a slow-cleaving or a fastcleaving intein.
  • Fast-cleaving intein can be understood to relate to an intein or mini-intein that has been modified at the + C-extein position and/or -N-extein position so that the cleavage rate of said intein is quicker/faster than the original RecA, intein, or mini-intein.
  • one may opt to utilize a fast-cleaving cis-cleaving release system such as a fast-cleaving cis-cleaving intein.
  • a fast-cleaving system may be preferable when EVs need to be harvested quickly.
  • the fast-cleaving cis-cleaving release system is based on an intein system, wherein the C- terminal portion of the intein may comprise the amino acid sequences Val-Val-Val-His-Asn or Val-Val- Val-His-Asn-Cys. Certain modifications at the +1 C-extein position, such as the abovementioned example, have been observed to speed up the cleavage rate (i.e., are fast-cleaving).
  • the term "Slow-cleaving intein” can be understood to relate to an intein or mini-intein that has been modified at the + C-extein position and/or -N-extein position so that the cleavage rate of said intein is slower than the original RecA, intein, or mini-intein.
  • An example of a slow-cleaving intein is AI-CM.
  • a slow-cleaving system may be preferable when more time is required to ensure efficient loading of an EV with the desired cargo.
  • the slow-cleaving used herein is selected from the following: mini-inteins, delta inteins, delta-intein-CM and a mini-intein and certain variants, mutations and domains thereof having a desired functionality (such as, but not limited to cleavage action instead of splicing and cleavage rate).
  • the slow-cleaving cis-cleaving release system is based on an intein system, wherein the C-terminal portion of the intein may comprise the amino acid sequences Val-Val-Val-His- Asn, more preferably wherein the C-terminal portion of the intein is modified to comprise Val-Val-Val- His-Asn-Gly. Certain modifications at the +1 C-extein position, have been observed to slow the cleavage rate (i.e., are slow-cleaving).
  • loaded EV is used herein in relation to an EV that is associated with therapeutic cargos and/or POIs as described herein.
  • the EV is considered loaded if the therapeutic cargo/POl remains associated with the EV following one or more EV isolation or purification steps. Affinity purification of EVs is described in WO 2018/153581 Al, WO2019/081474 Al and WO2019/238626 Al, which are incorporated by reference in their entirety.
  • the term "EV loaded with” is used interchangeably with the term “EV associated with” and the term “EV comprising”.
  • the therapeutic cargo(s) carried by the EVs may be present on the inside of the EV lumen, on the outside of the EV and/or in the membrane of the EV.
  • the AAV as described herein is loaded on the outer surface of the EV.
  • the AAV as described herein is loaded within the EV lumen.
  • the EV as described herein comprises AAV as described herein loaded on the outer surface of the EV and loaded within the EV lumen.
  • luminal loading of an AAV viral particle may have the advantage of further shielding from immune cells and neutralising antibodies and providing a less immunogenic therapeutic product
  • surface loading of an AAV viral particle may have the advantage of further increased transgene expression, for instance in the absence of a POI that mediates endosomal escape.
  • the terms "loaded in/on an EV” and “associated with an EV” are used interchangeably.
  • the therapeutic cargo and/or POI described herein is endogenously loaded passively into EVs described herein by the therapeutic cargo being present in the cytosol of the EV producing cells.
  • the fusion of therapeutic cargos and/or POIs to the EV polypeptides described above shall be understood to be endogenously loaded into the EVs.
  • the EV producing cells are transfected with the plasmids required to produce an AAV, including an AAV transfer plasmid as described herein.
  • the EVs produced by the cells naturally have AAVs loaded on/in them.
  • the AAV loaded EVs are then purified in accordance with an EV purification method.
  • the present disclosure relates to cells which have been stably modified to comprise at least one polynucleotide described herein.
  • the cells are stably or transiently transfected with the polynucleotides encoding PAH described herein, the expression cassette described herein, the gene therapy vector described herein or the recombinant AAV vector described herein to render them engineered cells capable of producing EVs described herein.
  • the cells are stably or transiently modified so as to include a construct encoding for a therapeutic protein cargo described herein and/or a POI described herein, which optionally may form part of a fusion protein with an EV polypeptide.
  • the cells are a monoclonal cell or a polyclonal cell line.
  • the cells are HEK293 cells, optionally adapted for suspension.
  • the present disclosure relates to a method of producing EVs as described herein, comprising first introducing into an EV-producing cell polynucleotide constructs to enable the production of an AAV as described herein in the EV-producing cell. Then expressing said constructs to generate EVs comprising AAVs as described herein.
  • the therapeutic cargo and/or POI described herein is exogenously loaded into EVs described herein.
  • the EVs are isolated from the producer cells and then loaded with the therapeutic cargo and/or POI.
  • the EVs are loaded by exogenous active loading, involving the cargo being loaded using any known exogenous loading method.
  • the cargo is loaded by electroporation, by transfection with transfection reagents such as cationic transfection agents such as lipofectamine (RTM), by conjugation of the cargo to a membrane anchoring moiety such as vitamin A, a lipid or cholesterol tail or by means of a cell penetrating peptide (CPP), either in the form of a CPP-cargo conjugate or in the form of a CPP-cargo non-covalent complex or any combination of these methods.
  • this type of active loading results in the therapeutic cargo and/or the POI being located on the inside of the EV, on the outside of the EV or within the membrane of the EV.
  • Any of the cargos described herein may be loaded exogenously.
  • nucleic acid cargoes and/or viral cargos are actively loaded by electroporation, CPP loading or co-incubation with a lipid tagged cargo.
  • the present disclosure also relates to a population of EVs as described herein.
  • the average number of therapeutic cargos as described herein and/or POIs as described herein per EV in the population of EVs as described herein is above one per EV, but it may also be below one per EV.
  • the average number of therapeutic cargos as described herein and/or POIs as described herein per EV is above one per EV.
  • At least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and/or at least 95% of all EVs comprise at least one therapeutic cargos as described herein and/or POIs as described herein.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide sequence encoding PAH as described herein, an expression cassette as described herein, a gene therapy vector as described herein, a recombinant AAV as described herein, an AAV viral particle as described herein, an AAV as described herein, a cell as described herein and a pharmaceutically acceptable excipient and/or carrier.
  • pharmaceutically acceptable is used herein to refer to a material may be administered to a subject without causing any undesirable biological effects.
  • excipient or “carrier” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • the pharmaceutically acceptable carrier or diluent is any substance that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the therapeutic cargo.
  • the excipients and carriers are generally safe and non-toxic.
  • compositions as described herein may be formulated by any known method of formulation.
  • the pharmaceutical compositions of the present disclsoure may be formulated as Oral formulations, including Tablet, Capsule, Sustained release, liquid; Intravenous Formulations; Parenteral Formulations; Topical Formulations; cutaneous administration including cream, ointment, gel, paste, powder; Modified release Formulations including sustained release formulation and Liquid or lyophilized formulations.
  • the pharmaceutical composition as described herein comprises an EV as described herein associated with an AAV as described herein and free AAV as described herein.
  • the EV is associated with an AAV as described herein.
  • the free AAV as opposed to AAV that are associated with EVs, make up more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the AAVs present in the pharmaceutical composition.
  • the free AAV as opposed to AAV that are associated with EVs, make up less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the AAVs present in the pharmaceutical composition.
  • At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the AAV present in the pharmaceutical composition is associated with an EV.
  • the present disclosure relates to an in vivo method for inducing PAH expression, the method comprising administering to a subject an effective amount of a polynucleotide sequence encoding PAH as described herein, an expression cassette as described herein, a gene therapy vector as described herein, a recombinant AAV as described herein, an AAV viral particle as described herein, an AAV as described herein, a cell as described herein and/or a pharmaceutical composition as described herein.
  • PAH expression is induced in a liver cell.
  • PAH expression is induced in a hepatocyte (HCs), a hepatic stellate cells (HSCs), a Kupffer cells (KCs), and/or a liver sinusoidal endothelial cells (LSECs).
  • HCs hepatocyte
  • HSCs hepatic stellate cells
  • KCs Kupffer cells
  • LSECs liver sinusoidal endothelial cells
  • the cell is a hepatocyte.
  • the present disclosure provides a method for treating or preventing a disease or disorder as described herein in a subject comprising administering a therapeutically or prophylactically effective amount of a polynucleotide, an expression cassette, a gene therapy vector, an AAV transfer plasmid, a recombinant AAV vector, an AAV viral particle, a cell, an EV, and/or a pharmaceutical composition as described herein to the subject suffering from or susceptible to the disease.
  • a polynucleotide in a further aspect of the present disclosure provides a polynucleotide, an expression cassette, a gene therapy vector, an AAV transfer plasmid, a recombinant AAV vector , an AAV viral particle, a cell, an EV, and/or a pharmaceutical composition as described herein, for use as a medicament for treatment or prevention of a disease or disorder in a subject as described herein.
  • the present disclosure relates to the use of a polynucleotide, an expression cassette, a gene therapy vector, an AAV transfer plasmid, a recombinant AAV vector, an AAV viral particle, a cell, an EV, and/or a pharmaceutical composition as described herein for the preparation of a medicament for treatment or prevention of a disease in a subject as described herein.
  • an "effective amount” of an AAV, an EV comprising an AAV or a pharmaceutical composition comprising the same may be determined empirically and in a routine manner. In certain embodiments, however, an "effective amount" of recombinant AAV virus ranges from about 1E12 vg/kg body weight to about 1E14 vg/kg body weight, in one embodiment from about 6E12 vg/kg body weight to about 6E13 vg/kg body weight. In another embodiment, a therapeutically effective amount of recombinant AAV virus is about 2E13 vg/kg body weight. In another embodiment, a therapeutically effective amount of recombinant AAV virus is about 6E13 vg/kg body weight.
  • the subject has immunity to the AAV, is capable of producing antibodies against the capsid of the AAV and/or is capable of producing antibodies that bind to a capsid derived from AAV7, AAV8 or AAV9.
  • the subject has immunity to the AAV due to prior natural exposure over the course of their lives.
  • the subject has immunity to the AAV due to having previously been administered with the AAV, or an AAV having a capsid derived from the same serotype, as a therapeutic.
  • Tl assays cell-based in vitro transduction inhibition (Tl) assays, in vivo (e.g., in mice) Tl assays, and ELISA- based detection of total anti-capsid antibodies (TAb) (see, e.g., Masat et al., Discov. Med, vol. 15, pp. 379-389 and Boutin et al., (2010) Hum. Gene Then, vol. 21, pp. 704-712).
  • Tl assays may employ host cells into which an AAV-inducible reporter vector has been previously introduced.
  • the reporter vector may comprise an inducible reporter gene such as GFP, etc.
  • Anti-AAV capsid antibodies present in human serum that are capable of preventing/reducing host cell transduction would thereby reduce overall expression of the reporter gene in the system. Therefore, such assays may be employed to detect the presence of anti-AA V capsid antibodies in human serum that are capable of preventing/reducing cell transduction by the therapeutic AAV PAH virus.
  • TAb assays to detect anti-AAV capsid antibodies may employ solid- phase-bound AAV capsid as a "capture agent" over which human serum is passed, thereby allowing anti-capsid antibodies present in the serum to bind to the solid-phase-bound capsid "capture agent".
  • a "detection agent” may be employed to detect the presence of anti-capsid antibodies bound to the capture agent.
  • the detection agent may be an antibody, an AAV capsid, or the like, and may be detectably-labelled to aid in detection and quantitation of bound anti-capsid antibody.
  • the detection agent is labelled with ruthenium or a ruthenium-complex that may be detected using electrochemiluminescence techniques and equipment. The same above-described methodology may be employed to assess and detect the generation of an anti-AAV capsid immune response in a patient previously treated with a therapeutic AAV virus of interest.
  • contemplated herein are methods that combine techniques for detecting anti-AAV capsid antibodies in human serum and administration of a therapeutic AAV virus for the treatment of PKU, wherein the techniques for detecting anti-AA V capsid antibodies in human serum may be performed either prior to or after administration of the therapeutic AAV virus.
  • Treatment is defined herein as a therapeutic treatment which refers to a treatment administered to a subject who exhibits signs or symptoms of pathology for the purpose of diminishing or eliminating those signs or symptoms.
  • the signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective.
  • “Treat” or “treatment” refers to the reduction or amelioration of the progression, severity, and/or duration of a disease (or symptom related thereto) associated with elevated phenylalanine levels.
  • Ameliorate as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.
  • the treatment is a stable treatment wherein the subject stably expresses the therapeutic protein expressed by the therapeutic AAV virus.
  • Stably expression of a therapeutic protein is defined as the therapeutic protein being expressed for a clinically significant length of time.
  • "Clinically significant length of time” as used herein is defined as expression at therapeutically effective levels for a length of time that has a meaningful impact on the quality of life of the subject.
  • a meaningful impact on the quality of life is demonstrated by the lack of a need to administer alternative therapies.
  • a meaningful impact on the quality of life is demonstrated by an ability to ingest an increased quantity of Phenylalanine, without a worsening of the symptoms associated with PKU.
  • clinically significant length of time is expression for at least two months, for at least six months, for at least eight months, for at least one year, for at least two years, for at least three years, for at least four years, for at least five years, for at least six years, for at least seven years, for at least eight years, for at least nine years, for at least ten years, or for the life of the subject.
  • a treatment can be monitored by measuring levels of phenylalanine in the blood of the treated subject.
  • Precise quantitate assays for determining circulating levels of phenylalanine are well known in the art and include fluorometric assays (see, McCaman, M.W. and Robins, E., (1962) J Lab. Clin. Med, vol. 59, pp. 885-890); thin layer chromatography based assays (see, Tsukerman, G. L. (1985) Laboratornoe delo, vol. 6, pp. 326-327); enzymatic assays (see, La Du, B. N., et al. (1963) Pediatrics, vol. 31, pp.
  • the disease is a disease associated with the in vivo activity of PAH.
  • the disease is associated with reduced expression of PAH and/or reduced activity of PAH in the liver of the subject.
  • the disease is PKU.
  • PKU Phenylketonuria
  • PKU refers to an inherited genetic disorder caused by mutations in the PAH gene, which result in low levels of the enzyme phenylalanine hydroxylase (PAH) or the PAH enzyme having reduced activity. Such mutation results in elevated levels of phenylalanine in PKU patients. This build-up of phenylalanine is associated with a number of harmful effects. These can include seizures, impaired neurophysiologic functioning, behavioural problems and reduced cognitive development. Accumulation of phenylalanine in pregnant women has further harmful effects and can lead to babies being born with heart problems, microcephaly and low birth weight.
  • PAH phenylalanine hydroxylase
  • the polynucleotide sequence encoding PAH as described herein, the expression cassette as described herein, the gene therapy vector as described herein, the recombinant AAV as described herein, the AAV viral particle as described herein, the AAV as described herein, the cell as described herein and/or the pharmaceutical composition as described herein may be administered to a subject, preferably a mammalian subject, most preferably a human subject, through a variety of known administration techniques.
  • auricular otic
  • buccal conjunctival
  • cutaneous dental
  • electro-osmosis endocervical
  • endosinusial endotracheal
  • enteral epidural
  • extra-amniotic extracorporeal
  • hemodialysis infiltration
  • interstitial intra-abdominal
  • intra- amniotic intra-arterial
  • intra-articular intrabiliary
  • intrabronchial intrabursal
  • intracardiac intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracerebroventricular, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intra
  • polynucleotide sequence encoding PAH as described herein, the expression cassette as described herein, the gene therapy vector as described herein, the recombinant AAV as described herein, the AAV viral particle as described herein, the AAV as described herein, the cell as described herein and/or the pharmaceutical composition as described herein is administered to the subject by intravenous (IV) injection.
  • IV intravenous
  • the polynucleotide sequence encoding PAH as described herein, the expression cassette as described herein, the gene therapy vector as described herein, the recombinant AAV as described herein, the AAV viral particle as described herein, the AAV as described herein, the cell as described herein and/or the pharmaceutical composition as described herein is administered to the subject by direct injection into the liver.
  • polynucleotide sequence encoding PAH as described herein, the expression cassette as described herein, the gene therapy vector as described herein, the recombinant AAV as described herein, the AAV viral particle as described herein, the AAV as described herein, the cell as described herein and/or the pharmaceutical composition as described herein is administered to the subject in combination with one or more co-treatments.
  • a "co-treatment is defined" as the use of multiple therapies to treat a single disease.
  • the treatments as described herein may be combined with a phenylalanine restricted diet.
  • the treatments as described herein may be combined a prophylactic and/or therapeutic corticosteroid for the prevention and/or treatment of any hepatotoxicity associated with the treatment as described herein. This may be particularly useful when the treatment comprises an AAV.
  • the medicament comprising a prophylactic or therapeutic corticosteroid treatment may comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg/day of the corticosteroid.
  • the medicament comprising a prophylactic or therapeutic corticosteroid may be administered over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more.
  • the treatments as described herein may additionally or alternatively be combined with tyrosine supplements.
  • the treatments as described herein may additionally or alternatively be combined with KUVAN® and/or PALYNZIQ®.
  • KUVAN® (sapropterin dihydrochloride) is a pharmaceutical formulation of BH4, the natural cofactor for the PAH enzyme, which stimulates activity of the residual PAH enzyme to metabolise Phe into tyrosine.
  • KUVAN® is described in US Patent Nos.
  • PALYNZIQ® pegvaliase-pqpz
  • PKU phenylketonuria
  • PAL YNZIQ® pegvaliase-pqpz
  • US Patent Nos. 7,531,341, 7,534,595, 7,537,923, 7,790,433, 7,560,263, 9,557,34 each of which is incorporated by reference in its entirety.
  • the treatments as described herein may be combined with any number of these additional co-treatments.
  • the present disclosure relates to a dosage regimen, comprising multiple administrations of the EVs.
  • the EVs as described herein are administered to the subject on two occasions.
  • the EVs as described herein are administered to the subject on three occasions.
  • the EVs as described herein are administered to the subject on 4, 5, 6, 7, 8, 9, 10, or more than 10 occasions.
  • the occasions on which the EVs as described herein are administered are hours, days weeks or years apart.
  • a further aspect wherein the treatment described herein relates to the administration of EVs associated with AAVs
  • the present disclosure relates to a dosage regimen, comprising administering to a subject a first dose comprising an AAV and a second dose comprising an EV associated with AAV.
  • the AAV administered in the first dose comprises only free AAVs that are not associated with EVs.
  • the AAVs administered in the first dose are AAVs as described herein.
  • the AAVs administered in the first dose are AAVs comprising a PAH transgene having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 4 or SEQ ID NO 5.
  • the second dose comprises a fewer free AAVs that are not associated with EVs, than were administered in the first dose.
  • the first and second dose as described herein are hours, days weeks or years apart.
  • Synthetic promoter regions are known in the art and comprise known promoters operably linked to known enhancer elements.
  • One such promoter region is the ApoE-HCR-l-hAAT promoter, which comprises the liver specific hAAT promoter sequence operably linked to an ApoE HCR-1 enhancer element.
  • the present disclosure provides promoter regions that enable increased expression of a transgene under its control.
  • the newly identified promoter regions have increased liver specificity as compared to known liver promoter regions.
  • the promoter regions function highly effectively in liver-cells and allows for enhanced expression of PAH transgenes in cells of and derived from the liver, in comparison to the known ApoE-HCR-hAAT promoter (SEQ ID NO 15, referred to as "LSP1" herein).
  • the present disclosure relates to a promoter region comprising an ApoE HCR-1 enhancer or an active fragment thereof operably linked to an hAAT promoter sequence or an active fragment therof.
  • the ApoE HCR-1 enhancer consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7.
  • the promoter region further comprises an AMBP enhancer or an active fragment thereof operably linked to the promoter.
  • AMBP enhancer consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8.
  • the promoter region further comprises a Serpin Cl enhancer or an active fragment thereof operably linked to the promoter.
  • Serpin Cl enhancer consists or comprises a polynucleotide having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9.
  • the promoter region further comprises a hAAT promoter sequence or an active fragment thereof, operably linked to an ApoE HCR-1 enhancer as described herein, an AMBP enhancer as described herein and a Serpin Cl enhancer as described herein.
  • the enhancer and promoter elements of the promoter region are structured in the following order, from 5' to 3': ApoE HCR-1 enhancer, the SerpinCl enhancer, the AMPB enhancer, HCR enhancer, hAAT promoter sequence.
  • the promoter region further comprises an F9 promoter region to the 5' of the ApoE HCR-1 enhancer.
  • the F9 promoter region has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 10.
  • the promoter region additionally comprises an Oct-1 transcription factor binding site to the 5' of the F9 promoter region.
  • the Oct-1 transcription factor binding site has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 11.
  • the promoter region consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 13.
  • the enhancer and promoter elements of the promoter region are structured in the following order, from 5' to 3': SerpinCl enhancer, AMPB enhancer, ApoE HCR-1 enhancer, HCR enhancer, hAAT promoter sequence.
  • the promoter region additionally comprises an Oct-1 transcription factor binding site to the 5' of the SerpinCl enhancer.
  • the Oct-1 transcription factor binding site has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 11.
  • the promoter region consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 14.
  • the promoter regions described herein are of a size sufficient to allow for high levels of transcription of a transgene to which they are operably linked, whilst still allowing for efficient packaging of the expression cassette, in which they are comprised, into an AAV.
  • the promoter regions described herein are less than 1.5 kb, 1.3 kb, 1.3 kb, 1.1 kb or 1 kb base pairs in length.
  • the present disclosure relates to an expression cassette comprising the promoter region as described herein operably linked to a transgene.
  • the transgene is a gene that is usually expressed in the liver.
  • the transgene encodes PAH.
  • the transgene consists or comprises a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1-5.
  • the transgene consists or comprises a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1.
  • the transgene consists or comprises a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3.
  • the expression cassette further comprises a Kozak sequence positioned directly 5' of the transgene and positioned 3' of the promoter region as described herein.
  • the Kozak sequence is a DNA sequence having SEQ ID NO: 16.
  • the expression cassette described herein further comprises a 3' and a 5' ITR sequence.
  • ITR inverted terminal repeat
  • the term "inverted terminal repeat (ITR)" as used herein is defined as the regions found at the 5' and 3' termini of the AAV genome.
  • the ITRs function in cis as origins of DNA replication and as packaging signals for the viral genome.
  • ITR sequences are well known to a person skilled in the art. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J Viral. (2005) vol. 79, pp. 364-379 which is herein incorporated by reference in its entirety. ITR sequences that find use herein may be full length, wild-type AAV ITRs or fragments thereof that retain functional capability.
  • the ITRs may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication.
  • the AAV ITRs may be derived from any known AAV serotype and, in certain embodiments, derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype.
  • the ITRs used in the present invention are derived AAV2.
  • the 3' ITR sequence consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the 5' ITR sequence consists or comprises of a polynucleotide sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the expression cassette described herein is of a size sufficient that allows for it to be efficiently packaged into an AAV.
  • the expression cassette described herein is less than 5.5 kb, 5 kb, 4.9 kb, 4.8 kb, 4.7 kb, 4.5 kb or 4.4 kb.
  • the transgene is less than 4.4 kb base pairs in length.
  • the present disclosure relates to a gene therapy vector comprising a promoter region as described herein or an expression cassette as described herein, or a combination thereof.
  • the gene therapy vector is an AAV transfer plasmid.
  • the present disclosure relates to a recombinant AAV vector comprising a promoter region as described herein or an expression cassette as described herein, or a combination thereof.
  • the present disclosure relates to a viral particle comprising a promoter region as described herein or an expression cassette as described herein, or a combination thereof.
  • the viral particle is a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus.
  • the viral particle is an AAV viral particle.
  • the AAV of the present invention comprises an AAV capsid that has tropism for liver cells.
  • the AAV of the present disclosure comprises an AAV capsid derived from AAV7, AAV8 or AAV9.
  • the AAV capsid is derived from AAV8.
  • the AAV virus is a therapeutic AAV.
  • the present disclosure relates to a cell comprising a promoter region as described herein, the expression cassette as described herein, the gene therapy vector as described herein, the AAV transfer plasmid as described herein, the recombinant AAV vector as described herein and/or an AAV viral particle as described herein.
  • the cell is a liver cell or is derived from a cell of the liver.
  • an extracellular vesicle comprising a promoter region as described herein, an expression cassette as described herein comprising the promoter region as described herein, a gene therapy vector as described herein comprising the promoter region as described herein, a AAV transfer plasmid as described herein comprising the promoter region as described herein, a recombinant AAV vector as described herein comprising the promoter region as described herein and/or a AAV viral particle as described herein comprising the promoter region as described herein.
  • EV extracellular vesicle
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a promoter region as described herein, an expression cassette as described herein comprising the promoter region as described herein, a gene therapy vector as described herein comprising the promoter region as described herein, a recombinant AAV as described herein comprising the promoter region as described herein, an AAV viral particle as described herein comprising the promoter region as described herein, , a cell as described herein comprising the promoter region as described herein and a pharmaceutically acceptable excipient and/or carrier.
  • the present disclosure relates to an in vivo method for inducing PAH expression, the method comprising administering to a subject an effective amount of a promoter region as described herein, an expression cassette as described herein, a gene therapy vector as described herein, a recombinant AAV as described herein, an AAV viral particle as described herein, an AAV as described herein, a cell as described herein and/or a pharmaceutical composition as described herein.
  • PAH expression is induced in a liver cell.
  • PAH expression is induced in a hepatocyte (HCs), a hepatic stellate cells (HSCs), a Kupffer cells (KCs), and/or a liver sinusoidal endothelial cells (LSECs).
  • HCs hepatocyte
  • HSCs hepatic stellate cells
  • KCs Kupffer cells
  • LSECs liver sinusoidal endothelial cells
  • the cell is a hepatocyte.
  • the present disclosure provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a promoter region as described herein, an expression cassette as described herein, a gene therapy vector as described herein, a recombinant AAV as described herein, an AAV viral particle as described herein, an AAV as described herein, a cell as described herein and/or a pharmaceutical composition as described herein to a subject suffering from or susceptible to the disease.
  • the present disclosure relates to the use of a promoter region as described herein, an expression cassette as described herein comprising the promoter region as described herein, a gene therapy vector as described herein comprising the promoter region as described herein, a recombinant AAV as described herein comprising the promoter region as described herein, an AAV viral particle as described herein comprising the promoter region as described herein, a cell as described herein comprising the promoter region as described herein and a pharmaceutically acceptable excipient and/or carrier for the preparation of a medicament for treatment or prevention of a disease in a subject.
  • the present disclosure further provides assays that allow for the efficient and reliable testing of the functional activity of PAH, thus fulfilling a long-felt need in the field of PAH research.
  • a cell assay is provided, which is particularly advantageous since it allows for PAH activity to be monitored in cells in culture, without the need for harvesting the cell.
  • Such an assay allows for PAH activity to be tested, whilst continuing to allow the cells to grow, which may be especially useful during the production of therapeutics.
  • BH4 tetrahydrobiopterin
  • PAH activity as assessed through changes in Tyrosine concentration, could not be detected when cells were cultured in a medium comprising Phenylalanine and BH4.
  • the present inventors found that PAH activity could however be detected if cells were cultured in a medium comprising Phenylalanine and L-Sepiapterin, a BH4 precursor.
  • the present disclosure related to a cell culture medium for assessing the functional activity of a PAH protein in cells in culture comprising Phenylalanine and L-Sepiapterin.
  • the cell culture medium comprises a known amount of Phenylalanine. In one embodiment, the cell culture medium comprises 50 to 1000 pM of Phenylalanine, 100 to 800 pM of Phenylalanine, 200 to 600 pM of Phenylalanine, 300 to 500 pM of Phenylalanine, 350 to 450 pM of Phenylalanine or around 400 pM of Phenylalanine.
  • the cell culture medium comprises at least 50 pM of Phenylalanine, at least 100 pM of Phenylalanine, at least 200 pM of Phenylalanine, at least 300 pM of Phenylalanine, at least 350 pM of Phenylalanine or at least 400 pM of Phenylalanine.
  • the cell culture medium comprises a known amount of L-Sepiapterin.
  • the cell culture medium comprises 5-500 pM of L-Sepiapterin, 10-300 pM of L- Sepiapterin, 25-200 pM of L-Sepiapterin, 50-100 pM of L-Sepiapterin, around 50 pM of L-Sepiapterin or around 100 pM of L-Sepiapterin.
  • the cell culture medium comprises at least 5 pM of L-Sepiapterin, at least 10 pM of L-Sepiapterin, at least 25pM of L-Sepiapterin, at least 40 pM of L-Sepiapterin, at least 50 pM of L-Sepiapterin or at least 100 pM of L-Sepiapterin.
  • L-Sepiapterin as used herein is defined as 2-amino-6-[(2S)-2-hydroxypropanoyl]-7,8-dihydro-lH- pteridin-4-one. L-Sepiapterin can be metabolised into tetrahydrobiopterin via a salvage pathway. L- Sepiapterin is used interchangeably with Sepiapterin.
  • the cell culture medium comprises a known amount of Phenylalanine and L-Sepiapterin. In one embodiment the cell culture medium comprises at least 300 pM of Phenylalanine and at least 25 pM of L-Sepiapterin. In a further preferred embodiment, the cell culture media comprises at least 400 pM of Phenylalanine and at least 50 pM of L-Sepiapterin. In a most preferred embodiment, the cell culture medium comprises around 400 pM of Phenylalanine and around 50-100 pM of L- Sepiapterin.
  • the cell culture medium comprises a known amount of tyrosine.
  • tyrosine is present in the cell culture medium at less than 50 pM, less than 25 pM, less than 10 pM, less than 5 pM, less than 3 pM, less than 2 pM, less than 1 pM, less than 0.5 pM, less than 0.1 pM.
  • the cell culture media contains only trace amounts of tyrosine. In a most preferred embodiment, no detectable tyrosine is present in the cell culture medium.
  • the cell culture medium comprises animal serum.
  • the serum is foetal bovine serum (FBS).
  • the FBS is a dialyzed FBS.
  • animal serum is present at a concentration required to keep the cells alive.
  • animal serum is present at a concentration required for the cells to grow.
  • animal serum is present at a concentration required for optimal cell growth.
  • the animal serum is present in the cell culture medium at a concentration of least 5%, around 5%, at least 10%, around 10%, at least 15%, around 15%, at least 20% or around 20%.
  • the animal serum is dialyzed FBS and is present in the cell culture medium at a concentration of around 10%.
  • the cell culture media comprises L-glutamine, or an equivalent such as L-alanyl-L- glutamine dipeptide.
  • L-glutamine or equivalent is present in the cell culture medium at a concentration required to keep the cells alive.
  • L-glutamine or equivalent is present at a concentration required for the cells to grow.
  • L-glutamine or equivalent is present at a concentration required for optimal cell growth.
  • the L-glutamine or equivalent is present in the cell culture media at a concentration of l-3mM.
  • the L-glutamine or equivalent is present in the cell culture media at around 2 mM.
  • the cell culture media is a DMEM media comprising around 400 pM of Phenylalanine, around 50-100 pM of L-Sepiapterin, around 2 pM of L-glutamine or an equivalent, around 10% dialyzed FBS and does not contain any detectable tyrosine.
  • the present disclosure relates to provides a method for assessing the functional activity of PAH in cells in culture comprising first culturing the cells in the cell culture medium as described herein and then harvesting a sample of the cell culture medium and assessing the level of tyrosine present.
  • the cells are cultured in the medium as described herein for at least 24 hrs, at least 36 hrs, at least 48 hours at least 60 hours or at least 72 hours before the harvesting of a sample of the cell culture medium.
  • the cells are cultured in the medium of the present invention for 48-96 hours, 60-84 hours or, in a preferred aspect, around 72 hours before the harvesting of a sample of the cell culture medium.
  • the level of tyrosine is assessed using a quantitative colorimetric detection assay.
  • the level of tyrosine is detected by enzymatically oxidating the tyrosine and detecting the signal at OD 492 nm.
  • the present disclosure provides a method for assessing the functional activity of a PAH transgene in cells in culture, the method comprising first expressing the PAH transgene in a cell in culture, second culturing the cells in the cell culture medium of the present invention and third harvesting a sample of the cell culture medium and assessing the level of tyrosine present.
  • the cells are cultured in the medium of the present invention for at least 24 hrs, at least 36 hrs, at least 48 hours at least 60 hours or at least 72 hours before the harvesting of a sample of the cell culture medium. In a one aspect of the present invention the cells are cultured in the medium of the present invention for 48-96 hours, 60-84 hours or, in a preferred aspect, around 72 hours before the harvesting of a sample of the cell culture medium.
  • the level of tyrosine is assessed using a quantitative colorimetric detection assay.
  • the level of tyrosine is detected by enzymatically oxidating the tyrosine and detecting the signal at OD 492 nm.
  • the cells are deficient in PAH.
  • the cells are Huh7 cells in which PAH has been knocked down or knocked out.
  • the cells are treated with the PAH transgene for at least 6 hours, 12 hours, 18 hours or 24 hours before the cell culture medium as described herein is added. In a preferred aspect, the cells are treated with the PAH transgene for around 24 hours before the cell culture medium as described herein is added. In one aspect the cells are washed before the cell culture medium as described herein is added. In a further preferred aspect, the cells are washed with PBS.
  • the PAH transgene is delivered to cells by a virus comprising a PAH transgene in its viral genome.
  • the AAV may be associated with an EV.
  • the PAH transgene is delivered to cells by transfection of a plasmid comprising a sequence encoding PAH.
  • the present disclosure provides a method of manufacturing a gene therapy product for use in the treatment of PKU, comprising the steps of first manufacturing a gene therapy product for the treatment of PKU and second, assessing the functional PAH activity that can be induced in cells in culture by the gene therapy product for the treatment of PKU using a cell assay as described herein.
  • the method of manufacturing a gene therapy product for use in the treatment of PKU further comprises a final step of producing a pharmaceutical composition comprising a gene therapy product for use in the treatment of PKU that has been validated as capable of inducing functional PAH activity in cells.
  • the cells are liver cells.
  • the gene therapy product for the treatment of PKU is a virus comprising a PAH transgene in its viral genome.
  • the AAV may be associated with an EV.
  • the present disclosure provides a method of manufacturing a gene therapy product for use in the treatment of PKU, comprising the steps of manufacturing a gene therapy product for the treatment of PKU in cells in culture and assessing the functional PAH activity in the cells used to produce the gene therapy product for use in the treatment of PKU using a cell assay as describe herein.
  • the method of manufacturing a gene therapy product for use in the treatment of PKU further comprises a final step of producing a pharmaceutical composition comprising a gene therapy product for use in the treatment of PKU that has been validated as capable of inducing functional PAH activity in the cells in which the gene therapy product is produced.
  • the cells are AAV or EV producer cells.
  • the cells are HEK293 cells.
  • the gene therapy product for the treatment of PKU is a virus comprising a PAH transgene in its viral genome.
  • the AAV may be associated with an EV.
  • the present disclosure further provides assays that allow for the efficient and reliable testing of the functional activity of PAH, thus fulfilling a long-felt need in the field of PAH research. Whilst assays for assessing PAH activity from cell lysates are known, such assays are highly complex.
  • the present disclosure provides a highly simplified Reaction Mix and a simplified methodology that can be used to assess PAH activity in both cell lysates and tissue samples.
  • the simplified Reaction Mix disclosed herein is particularly advantageous since it does not require the addition of ferrous ammonium iron (II) sulphate, which must be prepared fresh on the day of use, thus reduces waste and saves time.
  • the simplifies Reaction Mix does not require a catalase, which is a highly toxic, thus is safer to use.
  • the present disclosure provides a Reaction Mix for assessing PAH activity comprising Phenylalanine, Tetrahydrobiopterin (BH4), a Reducing Agent, a salt and a pH buffer.
  • a Reaction Mix for assessing PAH activity comprising Phenylalanine, Tetrahydrobiopterin (BH4), a Reducing Agent, a salt and a pH buffer.
  • the Reaction Mix does not comprise any detectable iron. In a most preferred aspect, the Reaction Mix does not comprise any detectable ferrous ammonium iron (II) sulphate. In a further preferred aspect, the Reaction Mix does not comprise any detectable catalase.
  • the Reaction Mix does not comprise any detectable ferrous ammonium iron II sulphate or any detectable catalase.
  • the Reaction Mix comprises phenylalanine at a concentration of more than 0.1 mM, more than 0.5 mM, more than 0.7 mM, more than 0.8 mM, more than 0.9 mM or more than ImM. In a preferred embodiment the Reaction Mix comprises phenylalanine at a concentration of around 1 mM.
  • the Reducing Agent is Dithiothreitol (DTT).
  • DTT Dithiothreitol
  • the Reaction Mix comprises DTT at a concentration of more than 0.5 mM, more than 1 mM, more than 1.5 mM, more than 1.7 mM, more than 1.8 mM, more than 1.9 mM or more than 2 mM.
  • the Reaction Mix comprises DTT at a concentration of around 2 mM.
  • the salt is NaCI or KCI.
  • the Reaction Mix comprises NaCI at a concentration of 5-500 mM, 100-300 mM or 150-250 mM. In a preferred aspect, the Reaction Mix comprises NaCI at a concentration of around 200 mM.
  • the pH buffer is Na-HEPES.
  • the Reaction Mix comprises Na-HEPES at a concentration of 5-50 mM, 10-30 mM or 15-25 mM. In a preferred aspect, the Reaction Mix comprises Na-HEPES at a concentration of around 20 mM.
  • the Reaction Mix comprises less than BH4 in a concentration of 25-190 pM, 40-150 pM, 50-100 pM, 60-90 pM or 70-80 pM.
  • the Reaction Mix comprises BH4 in a concentration around 75 pM.
  • the Reaction Mix is made up in water.
  • the Reaction Mix comprises around ImM phenylalanine, around 75 pM BH4, around 2 mM DTT, around 200mM NaCI, around 20mM NA-HEPES and is made up in water.
  • this Reaction Mix does not comprise any detectable ferrous ammonium iron II sulphate or any detectable catalase.
  • the present disclosure relates to a method for assessing the functional activity of PAH in cells in culture, comprising first obtaining a cell lysate by lysing the cells, then combining the cell lysate with the Reaction Mix of the present invention and finally assessing the level of tyrosine in the mixture.
  • the cell lysates are obtained by lysing cells in a PBS-based lysis buffer.
  • lysis buffer as used herein is defined as a buffer solution used for the purpose of breaking open cells.
  • the lysis buffer may comprise buffering salts and ionic salts to regulate the pH and osmolarity of the lysate.
  • the lysis buffer may comprise one or more detergents to break up membrane structures.
  • the lysis buffer additionally comprises a protease inhibitor.
  • lysate comprising at least 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg of protein is mixed with the Reaction Mix. In a preferred aspect lysate comprising around 100 pg of protein is mixed with the Reaction Mix.
  • the level of tyrosine is assessed by detecting tyrosine autofluorescence.
  • the level of tyrosine is assessed by detecting the level of fluorescence at or around 274/304nm excitation/emission.
  • Assessing PAH activity by quantifying tyrosine autofluorescence is particularly advantageous, since this provides a direct measurement of the tyrosine that has been produced and is not dependent on a further enzymatic step, which is a further step in which inaccuracies may be introduced.
  • it allows for the amount of tyrosine produced to be measured over time, rather than having to choose one particular time point at which to measure the tyrosine concentration in a sample, thus providing more information.
  • the level of tyrosine is assessed at least 2 mins, 5mins, 10 mins, 15 mins, 20 mins, 30 mins, 40 mins, 50 mins, 60 mins, 90 mins or 120 mins, 180 mins, 210 mins after the cell lysate and the Reaction Mix are combined.
  • the assessment of the level of tyrosine making a standard curve using different known amounts of tyrosine.
  • the tyrosine samples used in the standard curve are mixed with the same amount of BH4 as is used in the Reaction Mix that is used in the assay of the present invention. This overcomes the issues uncovered by the present inventors relating to the presence of BH4 lowering the detectable tyrosine autofluorescence.
  • the cell is a cell in which PAH has been overexpressed.
  • the present disclosure provides a method for assessing the functional activity of PAH in a tissue sample comprising first processing the tissue sample by digestion and/or homogenisation, then combining the processed tissue sample with the Reaction Mix of the present invention and finally assessing the level of tyrosine in the mixture.
  • tissue sample as used herein is defined as multiple cells that have been taken from an animal.
  • the tissue sample comprises tens of cells, hundreds of cells, thousands of cells, tens of thousands of cells or more.
  • the tissue sample may include the entire organ or may comprise part any part of an organ/tissue.
  • the tissue sample may be from any tissue in the body including a liver sample, a lung sample, a heart sample, a spleen sample, a kidneys sample, a brain sample and/or a skin sample.
  • the tissue sample may comprise multiple cell types, for instance a liver tissue sample may comprise hepatocytes (HCs), a hepatic stellate cells (HSCs), a Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), immune cells and/or blood cells.
  • the tissue sample may be taken from any animal.
  • the animal is a mammal, such as a mouse or a non-human primate.
  • the animal is a human.
  • the tissue sample may be taken from an animal that is dead or alive.
  • the tissue sample is taken as a biopsy from a living human.
  • the digestion is a mechanical digestion.
  • the homogenisation is performed with a bead homogeniser.
  • the tissue sample is both mechanically digested and homogenised.
  • the tissue sample is both mechanically digested and homogenised with a bead homogeniser.
  • the assessment of the level of tyrosine making a standard curve using different known amounts of tyrosine is a preferred aspect, the tyrosine samples used in the standard curve are mixed with the same amount of BH4 as is used in the Reaction Mix that is used in the assay of the present invention. This overcomes the issues uncovered by the present inventors relating to the presence of BH4 lowering the detectable tyrosine autofluorescence.
  • the sample is a liver tissue sample.
  • the tissue sample is a murine tissue sample.
  • the sample is a murine liver tissue sample.
  • the cell lysates are obtained by lysing cells in a PBS-based lysis buffer.
  • lysate comprising at least 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg of protein is mixed with the Reaction Mix. In a preferred aspect lysate comprising around 100 pg of protein is mixed with the Reaction Mix.
  • the level of tyrosine is assessed by detecting tyrosine autofluorescence. In a preferred embodiment the level of tyrosine is assessed by detecting the level of fluorescence at or around 274/304nm excitation/emission.
  • the level of tyrosine is assessed at least 2 mins, 5mins, 10 mins, 15 mins, 20 mins, 30 mins, 40 mins, 50 mins, 60 mins, 90 mins or 120 mins, 180 mins, 210 mins after the cell lysate and the Reaction Mix are combined.
  • the cells are Huh7 cells. In a further embodiment the cells are deficient in PAH. In a preferred aspect, the cells in which PAH has been knocked down or knocked out.
  • the cells are treated with the PAH transgene for at least 6 hours, 12 hours, 18 hours or 24 hours, 36 hours 48 hours, 60 hours or 72 hours before the cells are lysed. In a preferred aspect, the cells are treated with the PAH transgene for around 72 hours before the cells are lysed.
  • the PAH transgene is delivered to the cells in culture by a virus comprising a PAH transgene in its viral genome.
  • the AAV may be loaded in/on an EV.
  • the transgene is delivered to cells by transfection of a plasmid comprising the PAH transgene sequence.
  • the present disclosure provides a method for assessing the functional activity of a PAH transgene in an in vivo tissue, the method comprising first expressing the PAH transgene in an animal; then obtaining a tissue sample from the animal; then assessing the functional activity of PAH in the tissue sample obtained by a method of the present invention.
  • tissue sample is harvested from the left lateral lobe of a murine liver and the tissue sample is snap frozen.
  • the processed tissue sample mixed with the Reaction Mix comprises at least 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 150 pg, 175 pg, 200 pg, 230 pg or 250 pg of protein.
  • lysate comprising around 100-150 pg of protein is mixed with the Reaction Mix.
  • lysate comprising around 150 pg of protein is mixed with the Reaction Mix.
  • the digestion is a mechanical digestion.
  • the homogenisation is performed with a bead homogeniser.
  • the tissue sample is both mechanically digested and homogenised.
  • the tissue sample is both mechanically digested and homogenised with a bead homogeniser.
  • the level of tyrosine is assessed by detecting tyrosine autofluorescence. In a preferred aspect the level of tyrosine is assessed by detecting the level of fluorescence at or around 274/304nm excitation/emission.
  • the level of tyrosine is assessed at least 1 min, 2 mins, 5mins, 10 mins, 15 mins, 20 mins, 30 mins, 40 mins, 50 mins, 60 mins after the processed tissue sample and the Reaction Mix are combined.
  • the assessment of the level of tyrosine making a standard curve using different known amounts of tyrosine.
  • the tyrosine samples used in the standard curve are mixed with the same amount of BH4 as is used in the Reaction Mix that is used in the assay as described herein. This overcomes the issues uncovered by the present inventors relating to the presence of BH4 lowering the detectable tyrosine autofluorescence.
  • the tissue sample is a liver tissue sample.
  • the tissue sample is a murine tissue sample.
  • the tissue sample is a murine liver tissue sample.
  • the PAH transgene is expressed in an animal by administering to the animal a viral particle comprising a PAH transgene in its viral genome.
  • the AAV may be associated with an EV.
  • the present disclosure relates to a method for diagnosing a disorder associated with reduced PAH activity and/or expression comprising taking a biopsy from the liver and assessing the functional activity of PAH in the tissue sample obtained by the biopsy using a method of the present invention.
  • the disorder associated with reduced PAH activity and/or expression is PKU.
  • the present disclosure relates to a method for monitoring a PKU therapeutic comprising taking a biopsy from the liver of a patient that has been previously treated with a therapeutic for PKU and assessing the functional activity of PAH in the tissue sample obtained by the biopsy using a method of the present invention.
  • therapeutic is a gene therapy therapeutic.
  • the therapeutic is an AAV comprising a PAH transgene in its single stranded viral vector.
  • the AAV may be loaded on/in an EV.
  • the PAH transgene is a PAH transgene of the present invention
  • the present disclosure relates to an AAV comprising a PAH transgene in its single stranded viral vector, optionally wherein the AAV is loaded on/in an EV, for use in the treatment or prophylaxis of PKU, wherein the subject has been found to have deficient PKU activity using the assay of the present invention.
  • the PAH transgene is a polynucleotide sequence encoding PAH as described herein
  • the present invention related to a method of treating or preventing PKU in a subject, comprising administering an effective amount of an AAV comprising a PAH transgene in its viral genome, optionally wherein the AAV is associated with an EV, wherein the subject has been found to have deficient PKU activity using the assay of the present invention.
  • the PAH transgene is a PAH transgene of the present invention
  • a polynucleotide sequence comprising or consisting of a codon optimised PAH sequence.
  • the polynucleotide of paragraph 1 to 7 wherein the codon optimised PAH sequence comprises less than 8, less than 7 , less than 6, less than 5, less than A, less than 3, less than 2 or less than 1 CG motifs.
  • the polynucleotide of paragraph 10, wherein the codon optimised PAH sequence has at least 94% sequence identity to SEQ ID NO: 3.
  • the polynucleotide of paragraph 20 wherein the PAH protein has 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6.
  • the polynucleotide of paragraph 23, wherein the increase in expression of a PAH protein is an increase in expression of a functional PAH protein.
  • the polynucleotide of any one of paragraphs 18 to 24, wherein the cell is a liver cell.
  • An expression cassette comprising the polynucleotide of any of paragraphs 1 to 25 and a promoter region comprising or consisting of a promoter sequence, wherein the promoter sequence is operably linked to the polynucleotide.
  • the expression cassette of paragraph 26 wherein the promoter sequence is a liver specific promoter sequence.
  • the expression cassette of paragraph 27, wherein the liver specific promoter sequence is a hAAT promoter sequence.
  • the expression cassette of paragraph 28 wherein the liver specific promoter sequence is a hAAT promoter sequence.
  • the expression cassette of paragraph 31 wherein the ApoE HCR-1 enhancer has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7.
  • the expression cassette of paragraph 33 wherein the SerpinCl enhancer has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9.
  • the expression cassette of paragraph 29 to 34 wherein the promoter sequence is operably linked to a AMBP enhancer.
  • the expression cassette of paragraph 35 wherein the AMBP enhancer has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 8.
  • the expression cassette of paragraph 29 to 36 wherein the promoter sequence is operably linked to an F9 promoter region.
  • the expression cassette of paragraph 37 wherein the F9 promoter region has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10.
  • a promoter region comprising:
  • an AMPB enhancer further wherein the ApoE HCR-1 enhancer, the SerpinCl enhancer and the AMPB enhancer are operably linked to the liver-specific promoter.
  • the promoter region of paragraph 42 to 51 wherein the enhancer and promoter elements are structured, in the following order, from 5' to 3' as follows: SerpinCl enhancer, AMPB enhancer, ApoE HCR-1 enhancer, HCR enhancer, hAAT promoter.
  • the promoter region of paragraph 53, wherein the promoter region further comprises a F9 promoter region positioned 5' of the ApoE HCR-1 enhancer.
  • the promoter region of paragraph 42 to 56 wherein the promoter region has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 14.
  • the promoter region of paragraph 40 to 58 wherein the promoter region is capable of inducing expression of a transgene to which it is operably linked.
  • An expression cassette comprising the promoter region of paragraph 42 to 62 and a transgene, wherein the transgene is operably linked to the promoter region.
  • the expression cassette of paragraph 63, wherein the promoter region is capable of inducing expression of the transgene in a cell.
  • the expression cassette of paragraph 63 or paragraph 64 wherein the promoter region is capable of inducing increased expression of the transgene in a cell, as compared to when the transgene is operably linked to a promoter region having SEQ ID NO. 15.
  • the expression cassette of paragraphs 63 to paragraph 65 wherein the transgene consists or comprises of a polynucleotide encoding PAH.
  • the expression cassette of paragraph 66 wherein the polynucleotide encoding PAH is a polynucleotide of paragraph 1 to paragraph 25.
  • the expression cassette of paragraphs 63 to 67 further comprising a Kozak sequence positioned between the promoter region and the transgene.
  • the expression cassette of paragraph 68 wherein the Kozak sequence positioned immediately 5' of the start codon of the transgene.
  • the expression cassette of paragraph 68 or 69, wherein the Kozak sequence has at least 50%, 62.5%, 75%, 87.5% or 100% sequence identity to SEQ ID NO. 16.
  • the expression cassette of paragraph 63 to 70 comprising a polyadenylation sequence positioned to the 3' of the transgene.
  • the expression cassette of paragraph 71 wherein the polyadenylation sequence is a BgpA sequence having at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 17.
  • the expression cassette of paragraph 63 to 70 comprising a 3' ITR sequence positioned to the 3' of the transgene.
  • the expression cassette of paragraph 71 or 72 comprising a 3' ITR sequence positioned to the 3' of the BgpA sequence.
  • the expression cassette of paragraph 73 or 74 wherein the 3' ITR sequence is derived from AAV2.
  • the expression cassette of paragraph 73 to 75 wherein the 3' ITR sequence has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 18.
  • the expression cassette of paragraph 63 to 76 comprising a 5' ITR sequence positioned to the 5' of the promoter region.
  • the expression cassette of paragraph 77 wherein the 5' ITR sequence is derived from AAV2.
  • the expression cassette of paragraph 77 or 78, wherein the 5' ITR sequence has at least 70%, 75, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 19.
  • the expression cassette of paragraph 63 to 79 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 20.
  • the expression cassette of paragraph 63 to 79 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 21.
  • the expression cassette of paragraph 63 to 79 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 22.
  • the expression cassette of paragraph 63 to 82 wherein the expression cassette is capable of inducing expression of PAH in a cell.
  • the expression cassette of paragraph 83, wherein the expression of a PAH is expression of a PAH protein.
  • the expression cassette of paragraph 85 wherein the PAH protein has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6
  • the expression cassette of paragraph 88 wherein the increased expression of a PAH is increased expression of a PAH protein.
  • the expression cassette of paragraph 89 or paragraph 90, wherein the PAH protein has functional PAH activity.
  • the expression cassette of paragraphs 88 to 91, wherein the increase in expression of PAH is liver-cell specific.
  • a plasmid comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92.
  • a gene therapy vector comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92.
  • An AAV transfer plasmid comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92.
  • a recombinant AAV vector comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92.
  • the recombinant AAV vector of paragraph 96 wherein the AAV vector is a single stranded AAV vector
  • a viral particle comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92.
  • the viral particle of paragraph 98 wherein the viral particle is an AAV.
  • the viral particle of paragraph 99 wherein the viral particle comprises a capsid derived from AAV8.
  • An EV comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97 and/or the viral particle of paragraph 98 to 100.
  • the EV of paragraph 101 wherein the EV is a microvesicle.
  • the EV of paragraph 101 to 103 wherein the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97 and/or the viral particle of paragraph 98 to 100 is loaded on the surface of the EV.
  • the EV of paragraph 101 to 104 wherein the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97 and/or the viral particle of paragraph 98 to 100 is loaded in the lumen of the EV.
  • a cell comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100 and/or the EV of paragraph 101 to 105.
  • the cell of paragraph 106 wherein the cell is a liver cell or is a cell derived from the liver.
  • An in vitro or in vivo method for inducing PAH expression in a cell comprising administering to the cell an effective amount of the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100 and/or the EV of paragraph 101 to 105.
  • the /n vitro or in vivo method of paragraph 108 wherein the cell is a liver cell or is a cell derived from the liver.
  • a pharmaceutical composition comprising the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100, the EV of paragraph 101 to 105 and/or the cell of paragraph 106 or 107, and a pharmaceutically acceptable excipient and/or carrier.
  • a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100, the EV of paragraph 101 to 105, the cell of paragraph 106 or 107 and/or the pharmaceutical composition of paragraph 110, to a subject suffering from or susceptible to the disease.
  • paragraphs 103 to 118 wherein the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100, the EV of paragraph 101 to 105, the cell of paragraph 106 or 107 and/or the pharmaceutical composition of paragraph 110 is administered to the subject by intravenous (IV) injection.
  • IV intravenous
  • paragraphs 103 to 118 wherein the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100, the EV of paragraph 101 to 105, the cell of paragraph 106 or 107 and/or the pharmaceutical composition of paragraph 110 is administered to the subject by direct injection into the liver.
  • paragraphs 103 to 120 wherein the polynucleotide of paragraph 1 to 25, the promoter region of paragraph 42 to 62 or the expression cassette of paragraph 26 to 41 or 63 to 92, the plasmid of paragraph 93, the gene therapy vector of paragraph 94, the AAV transfer plasmid of paragraph 95, the recombinant AAV vector of paragraph 96 or 97, the viral particle of paragraph 98 to 100, the EV of paragraph 101 to 105, the cell of paragraph 106 or 107 and/or the pharmaceutical composition of paragraph 110 is administered to the subject in combination with one or more co-treatments.
  • An EV comprising a AAV viral particle of paragraph 99 or 100.
  • the EV of paragraph 122 wherein the AAV viral particle is loaded on the surface of the EV.
  • An EV comprising multiple AAV viral particles of 99 or 100.
  • the EV of paragraphs 125 or 126, wherein the AAV viral particles are loaded in the lumen of the EV.
  • the EV of paragraphs 125 wherein a proportion of the AAV viral particles are loaded on the surface of the EV and a proportion of the AAV viral particles are loaded in the lumen of the EV.
  • An in vitro method for inducing PAH expression in a cell comprising administering to the cell an effective amount of an EV of paragraphs 122 to 130 or a population of EVs of paragraph 131.
  • the in vitro method of paragraph 132, wherein the cell is a liver cell or a cell derived from the liver.
  • a pharmaceutical composition comprising an EV of paragraphs 122 to 130 or a population of EVs of paragraph 131 and a pharmaceutically acceptable excipient and/or carrier.
  • a pharmaceutical composition comprising an EV of paragraphs 122 to 130 or a population of EVs of paragraph 131 and free AAV that is not associated with the EV, and a pharmaceutically acceptable excipient and/or carrier, wherein the free AAV is an AVV of paragraph 99 or 100.
  • An in vivo method for inducing PAH expression comprising administering to a subject an effective amount of an EV of paragraphs 122 to 130, a population of EVs of paragraph 131 and/or a pharmaceutical composition of paragraph 134 to 136.
  • a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an EV of paragraphs 122 to 130, a population of EVs of paragraph 131 and/or a pharmaceutical composition of paragraph 134 to 136, to a subject suffering from or susceptible to the disease.
  • An EV of paragraphs 122 to 130, a population of EVs of paragraph 131 and/or a pharmaceutical composition of paragraph 134 to 136 for use as a medicament for treatment or prevention of a disease in a subject.
  • the use or method of paragraphs 138 to 142, wherein the disease is PKU.
  • the use or method of paragraphs 138 to 147, the EV, population of EVs and/or pharmaceutical composition is administered to the subject by direct injection into the liver.
  • the use or method of paragraphs 138 to 149, the EV, population of EVs and/or pharmaceutical composition is administered to the subject in combination with one or more co-treatments.
  • the use or method of paragraphs 138 to 150, wherein two or more doses of the EV, population of EVs and/or pharmaceutical composition is administered are administered to the subject.
  • the use or method of paragraph 151, wherein the different doses of the EV, population of EVs and/or pharmaceutical composition are administered to the subject hours, days weeks or years apart.
  • the use or method of paragraph 151 or paragraph 152, wherein three doses are administered to the subject.
  • paragraph 151 or paragraph 152 wherein 4, 5, 6, 7, 8, 9, 10 or more than 10 doses are administered to the subject.
  • the use or method of paragraph 138 to 154 wherein the subject is first administered with an AAV comprising a transgene encoding PAH and then administered with the EV, population of EVs and/or pharmaceutical composition.
  • the use or method of paragraph 155 wherein the first dose of the AAV is administered days, weeks or years apart before the subsequent dose of the EV, EVs or pharmaceutical composition is administered.
  • the use or method of paragraph 155 or 156, wherein the first dose of the AAV consists of free AAVs that are not associated with EVs.
  • the use or method of paragraph 155 to 157 wherein the first dose of the AAV comprises an AAV comprising a transgene sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 4 or SEQ ID NO 5.
  • the use or method of paragraph 155 to 157, wherein the first dose of the AAV comprises an AAV of paragraph 99 or paragraph 100.
  • the use or method of paragraph 155 to 159, wherein the subsequent dose of the EV, population of EVs and/or pharmaceutical composition comprises a fewer free AAVs that are not associated with EVs than were administered in the first dose.
  • a cell culture medium for assessing the functional activity of a PAH protein in cells in culture comprising Phenylalanine and L-Sepiapterin.
  • the cell culture media of paragraph 161 comprising comprises at least 50 pM of Phenylalanine, at least 100 pM of Phenylalanine, at least 200 pM of Phenylalanine, at least 300 pM of Phenylalanine, at least 350 pM of Phenylalanine.
  • the cell culture media of paragraph 161 or 162 comprising at least 5 pM of L-Sepiapterin, at least 10 pM of L-Sepiapterin, at least 25pM of L-Sepiapterin, at least 40 pM of L-Sepiapterin, at least 50 pM of L-Sepiapterin or at least 100 pM of L-Sepiapterin.
  • the cell culture media of paragraph 161 to 163 comprising at least 200 pM of Phenylalanine and at least 25pM of L-Sepiapterin.
  • the cell culture media of paragraph 161 to 164 comprising around 400 pM of Phenylalanine and around 50-100 pM of L-Sepiapterin.
  • the cell culture media of paragraph 161 to 165 further comprising a known amount of tyrosine.
  • the cell culture media of paragraph 161 to 166 comprising less than 0.5 pM.
  • the cell culture media of paragraph 161 to 167 comprising only trace amounts of tyrosine.
  • the cell culture media of paragraph 161 to 168 wherein no detectable tyrosine is present in the cell culture medium.
  • a method for assessing the functional activity of PAH in cells in culture comprising the following steps:
  • the method of paragraph 177 to 179 wherein the cells are cultured in the medium of paragraphs 161 to 171 for around 72 hours before the harvesting of a sample of the cell culture medium.
  • the method of paragraphs 177 to 180 wherein the level of tyrosine present in the sample is assessed by adding an enzyme that oxidates the tyrosine and then measuring the signal at OD 492 nm.
  • the method of paragraphs 177 to 181 wherein the cells are liver cells or ells derived from the liver.
  • the method of paragraph 182 wherein the cells are Huh7 cells.
  • the method of paragraph 177 to 183 wherein the cells are deficient in PAH.
  • the method of paragraph 177 to 184 wherein the cells have no detectable functional PAH activity.
  • the method of paragraph 177 to 185 wherein the cells are treated with the PAH transgene for 6 hours, 12 hours, 18 hours or 24 hours before the cells are cultured in the cell culture medium of 161 to 171.
  • the method of paragraph 177 to 185 wherein the cells are treated with the PAH transgene for around 24 hours before the cells are cultured in the cell culture medium of 161 to 171.
  • the method of paragraph 177 to 187 wherein the PAH transgene is delivered to cells by a viral particle comprising a PAH transgene in its viral genome.
  • a method of manufacturing a gene therapy product for use in the treatment of PKU comprising the following steps:
  • a method of manufacturing a gene therapy product for use in the treatment of PKU comprising the following steps: (i) manufacturing a gene therapy product for the treatment of PKU in cells in culture; and
  • a Reaction Mix for assessing PAH activity comprising Phenylalanine, Tetrahydrobiopterin (BH4), a Reducing Agent, a salt and a pH buffer.
  • the Reaction Mix of paragraph 198, wherein the Reaction Mix does not comprise any detectable catalase.
  • the Reaction Mix of paragraph 198 to 201 comprising more than 0.5 mM phenylalanine.
  • the Reaction Mix of paragraph 198 to 202 comprising around 1 mM phenylalanine.
  • the Reaction Mix of paragraph 198 to 203 wherein the Reducing Agent is Dithiothreitol (DTT).
  • the Reaction Mix of paragraph 198 to 204 comprising more than 1 mM of DTT.
  • the Reaction Mix of paragraph 198 to 205 comprising around 2 mM of DTT.
  • the Reaction Mix of paragraph 198 to 206 wherein the salt is NaCI.
  • the Reaction Mix of paragraph 198 to 207 comprising 100-300 mM of NaCI.
  • the Reaction Mix of paragraph 198 to 208 comprising around 200 mM of NaCI.
  • the Reaction Mix of paragraph 198 to 209 wherein the pH buffer is Na-HEPES.
  • the Reaction Mix of paragraph 198 to 210 comprising 10-30 mM of Na-HEPES..
  • the Reaction Mix of paragraph 198 to 211 comprising around 20 mM of Na-HEPES.
  • the Reaction Mix of paragraph 199 to 212 comprising 50-190 pM of BH4.
  • the Reaction Mix of paragraph 198 to 214 wherein the components are dissolved in water.
  • the Reaction Mix of paragraph 198 to 215 comprising around ImM phenylalanine, around 75 pM BH4, around 2 mM DTT, around 200mM NaCI, around 20mM NA-HEPE; wherein the components are dissolved in water; further wherein the Reaction Mix does not comprise any detectable ferrous ammonium iron II sulphate or any detectable catalase.
  • a method for assessing the functional activity of PAH in cell in culture comprising the following steps
  • step (ii) combining the cell lysate of step (i) with a Reaction Mix of paragraphs 198 to 216;
  • step (iii) assessing the level of tyrosine in the mixture obtained in step (ii).
  • the method of paragraph 217 wherein the cell lysates is obtained by lysing cells in a PBS- based lysis buffer.
  • the method of paragraph 217 to 220 wherein the level of tyrosine is assessed by detecting tyrosine autofluorescence.
  • the method of paragraph 221, wherein the level of tyrosine is assessed by detecting the level of fluorescence at or around 274/304nm excitation/emission The method of paragraph 217 to 222, wherein the level of tyrosine is assessed at least 10 minutes after the cell lysate and the Reaction Mix are combined.
  • the method of paragraph 217 to 223, the cell lysed in step (i) is a cell in which PAH has been overexpressed.
  • a method for assessing the functional activity of PAH in a tissue sample comprising the following steps:
  • step (iii) assessing the level of tyrosine in the mixture obtained in step (ii).
  • the method of paragraph 225 wherein in step (ii) processed tissue sample comprising at least 150 pg of is combined with the Reaction Mix of paragraphs 198 to 216.
  • the method of paragraph 225 wherein in step (ii) processed tissue sample comprising around 250 pg of is combined with the Reaction Mix of paragraphs 198 to 216.
  • the method of paragraph 224 to 227 wherein in step (i), the digestion is a mechanical digestion.
  • step (i) the homogenisation is performed with a bead homogeniser.
  • a method of monitoring a subject that has been treated with a therapeutic for PKU comprising the following steps:
  • PAH phenylalanine hydroxylase
  • An expression cassette comprising the polynucleotide of any one of embodiments 1 to 3 and a promoter region, wherein the promoter region comprises a promoter sequence, further wherein the polynucleotide sequence is operably linked to the promoter sequence.
  • the expression cassette of embodiment 4, wherein the promoter sequence is a liver-specific promoter sequence.
  • the expression cassette of embodiment 4 or embodiment 5, wherein the promoter sequence comprises a human alpha-1 antitrypsin (hAAT) promoter sequence; wherein the promoter region further comprises a ApoE HCR-1 enhancer, an AMPB enhancer and a SerpinCl enhancer; wherein the ApoE HCR-1 enhancer, the AMPB enhancer and the SerpinCl enhancer are operably linked to the human alpha-1 antitrypsin (hAAT) promoter sequence.
  • hAAT human alpha-1 antitrypsin
  • the expression cassette of embodiment 6, wherein the promoter region is structured, from 5' to 3' as follows: the ApoE HCR-1 enhancer, the SerpinCl enhancer, the AMPB enhancer, the HCR enhancer, the hAAT promoter sequence.
  • the expression cassette of any one of embodiments embodiment 4 to 7, wherein the promoter region comprises or consists of a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO: 13.
  • the expression cassette of any one of embodiments 4 to 8 comprising or consisting of a polynucleotide sequence having at least 90% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.
  • a gene therapy vector comprising the polynucleotide of any one of embodiments 1-3, or the expression cassette of any one of embodiments 4 to 9, or a combination thereof.
  • An AAV transfer plasmid comprising the polynucleotide of any one of embodiments 1-3, or the expression cassette of any one of embodiments 4 to 9, or a combination thereof.
  • a recombinant AAV vector comprising the polynucleotide of any one of embodiments 1-3, or the expression cassette of any one of embodiments 4 to 9, or a combination thereof.
  • An AAV viral particle comprising the polynucleotide of any one of embodiments 1-3, or the expression cassette of any one of embodiments 4 to 9, or a combination thereof.
  • a cell comprising the polynucleotide of any one of embodiments 1 to 3, the expression cassette of any one of embodiments 4 to 9, the gene therapy vector of embodiment 10, the AAV transfer plasmid of embodiment 11, the recombinant AAV vector of embodiment 12 and/or the AAV viral particle of embodiment 13.
  • An in vitro or an in vivo method for inducing PAH expression in a target cell comprising administering to the cell an effective amount of the polynucleotide of any one of embodiments 1 to 3, the expression cassette of any one of embodiments 4 to 9, the gene therapy vector of embodiment 10, the AAV transfer plasmid of embodiment 11, the recombinant AAV vector of embodiment 12 and/or the AAV viral particle of embodiment 13.
  • An extracellular vesicle comprising the polynucleotide of any one of embodiments 1 to 3, the expression cassette of any one of embodiments 4 to 9, the gene therapy vector of embodiment 10, the AAV transfer plasmid of embodiment 11, the recombinant AAV vector of embodiment 12 and/or the AAV viral particle of embodiment 13.
  • a pharmaceutical composition comprising the polynucleotide of any one of embodiments 1 to 3, the expression cassette of any one of embodiments 4 to 9, the gene therapy vector of embodiment 10, the AAV transfer plasmid of embodiment 11, the recombinant AAV vector of embodiment 12, the AAV viral particle of embodiment 13 and/or the EV of embodiment 16 or embodiment 17; further comprising a pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical composition according to embodiment 18, comprising an EV wherein the EV comprises an AAV viral particle according to embodiment 13; further comprising a pharmaceutically acceptable excipient or carrier.
  • PKU Phenylketonuria
  • the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector, the AAV viral particle, the EV or the pharmaceutical composition for the use according to embodiment 20, wherein the subject is administered with two or more doses of the pharmaceutical composition.
  • the polynucleotide, the expression cassette, the gene therapy vector, the AAV transfer plasmid, the recombinant AAV vector, the AAV viral particle, the EV or the pharmaceutical composition for the use according to embodiment 20, wherein the subject has immunity to the AAV comprised in the pharmaceutical composition.
  • a promoter region comprising: a liver-specific promoter sequence, an ApoE HCR-1 enhancer, an AMPB enhancer and a SerpinCl enhancer; wherein the ApoE HCR-1 enhancer, the AMPB enhancer and the SerpinCl enhancer are operably linked to the liver-specific promoter; further wherein the liver-specific promoter is a human alpha-1 antitrypsin (hAAT) promoter.
  • hAAT human alpha-1 antitrypsin
  • a cell culture medium for assessing the functional activity of PAH in cells in culture comprising Phenylalanine and L-Sepiapterin.
  • a method for assessing the functional activity of PAH in cells in culture comprising:
  • any of the features described herein may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
  • any of the polynucleotides, expression cassettes, gene therapy vectors, the AAV transfer plasmids, recombinant AAV vectors, AAV viral particles, EVs or pharmaceutical compositions described herein can be used in any of the described methods, such as the methods of treatment. Any and all such combinations are explicitly envisaged as forming part of the invention. 6.
  • the present inventors sought to design constructs that allow for increased expression of PAH.
  • codon optimised sequences were generated based on cDNA sequence of proteins or mRNAs highly expressed from liver.
  • the codon usage table was used as input to the online application OPTIMISER to obtain 18 different codon optimised sequences.
  • 3 sequences codon optimised based on human codon usage were further manipulated to remove CG motifs, premature stops, unwanted amino acid substitutions and to verify cryptic splicing.
  • Codon-optimised nucleotide sequences encoding the PAH variants were gene- synthesised and cloned into AAV2-LSPl-BgpA backbone by GenScript.
  • CO8 is codon optimised based on Fibrin cDNA codon usage
  • CO9 is codon optimised based on SerpinAl cDNA codon usage
  • CO21 is codon optimised based on FIX and F2 (prothrombin) combined cDNA codon usage.
  • CO9, CO8 and CO21 have SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 respectively.
  • Codon-optimisation does not always lead to an increase in the expression of a polynucleotide, as codon-optimisation can lead to reduced mRNA stability and disrupt the initiation of translation, thus leading to a decrease in expression at the RNA and/or protein level.
  • the present inventors then went on to evaluate the level of expression induced by the 44 novel constructs, to determine which constructs lead to an increase in PAH expression.
  • CO9 is codon optimised based on SerpinAl cDNA codon usage and has SEQ ID NO 1.
  • CO8 is codon optimised based on Fibrin cDNA codon usage and has SEQ ID NO 2.
  • CO21 is codon optimised based on FIX and F2 (prothrombin) combined cDNA codon usage and has SEQ ID NO 3.
  • the inventors also designed 8 new promoter.
  • the new promoters comprise different liver enhancer elements, as single elements and/or in combination. These liver specific enhancers regions were selected based on conserved DNA regions amongst vertebrates.
  • the plasmids comprising the new designed promoters operably linked to PAH sequences were transfected into human Huh7 liver cells.
  • the 8 new promoters designed and tested only 2 showed an improved level of expression, as compared to LSP1, following plasmid transfection into HuH7 liver cells (data not shown).
  • These promoters were LSP9 and LSP4, having SEQ ID NO 12 and 13 respectively and shown schematically in Figure lb and lc respectively.
  • the present inventors selected CO9, CO8 and CO21 to be closed under the LSP4 promoter as well as LSP9 promoter to further evaluate the PAH expression.
  • the present inventors investigated the PAH expression levels induced by constructs comprising C08, C09 and C021 and the LSP4 and LSP9, in Huh7 cells in which PAH had been knocked-out, as compared to a wild-type PAH sequence under the control of the LSP1 promoter.
  • the inventors seeded PAH KO Huh7 cells onto 6-well tissue culture plates and then returned to the incubator for between 16-24 hours to allow them to adhere to the plastic.
  • PAH KO Huh7 cells were seeded onto 6-well tissue culture plates and returned to the incubator for between 16-24 hours to allow them to adhere to the plastic.
  • Cells were harvested by first washing with PBS then adding lOOpI of RIPA buffer, including a Protease Inhibitor, and incubating the cells on ice for 15 minutes. Cells lysates were transferred to an Eppendorf tube and centrifuged for at 15,000 g for 15 minutes.
  • Total protein content of the cell lysates was assessed using a BCA assay kit (ThermoFisher Scientific, #23225), in accordance with the manufacturer's protocol.
  • membranes were washed in PBS + 0.1% Tween and the membrane was then incubated with secondary antibodies (Donkey anti-mouse IR680 Antibody (Licor ,#926-68022) or Donkey anti-rabbit IR800 Antibody (Licor, #926-32213)), diluted in PBS blocking buffer, for 1 hour at room temperature with gentle agitation. Finally, membranes were washed again and imaged using the Licor Odyssey instrument (Licor, #EQ-0120) (2 minutes acquisition time per channel).
  • the average normalised value of PAH is shown in Table 2 below (column 2).
  • Table 2 and Figure 2a show that the PAH sequences CO8, CO9 and CO21 have increased averaged normalised PAH expression levels, as compared to the wildtype PAH sequence (compare rows 2, 5 and
  • Table 1 and Figure 2a show new promoters LSP4 and LSP9 drive an increase in the average normalised expression of the operably linked PAH transgene PAH, as compared to the known LSP1 promoter sequence (compare row 3 and 4 to row 2; row 6 and 7 to row 5; and row
  • plasmid DNA (LSP9-CO09, LSP9-CO021 or LSP9-CO08) was added to 1.2mL of serum-free DMEM media and 96 pL of Fugene HD. The mixture incubated at room temperature for 15 minutes and the complex was added directly to Huh7 cells.
  • the medium was changed for fresh DMEM supplemented with 10% FBS.
  • the cells were left for a further 48 hours, then washed in PBS, trypsinised and spun at 300g for 5 minutes. The pellet was then washed in PBS and spun again at 300g for 5 minutes.
  • Figure 2b shows that LSP9-CO9, LSP9-CO21 and LSP9-CO8 all induce an increase in the expression of PAH protein in wildtype Huh7 cells, as compared with an untransfected control.
  • Figure 2c shows the quantification of the western blot image of Figure 2b, carried out in accordance with the method described above.
  • the present inventors then went on to investigate whether the three constructs that had induced the largest increase in PAH expression (LSP9-CO9; LSP9-CO21 and LSP9-CO8) could provide an improvement over a known construct for PAH that that is currently in clinical trials, BMN 307.
  • the BMN307 construct comprises a codon optimised PAH sequence that is operably linked to the LSP1 promoter.
  • the present inventors were aware that codon optimisation can also result in the disruption of protein function, since slowing and pausing of the ribosome at certain times in translation may be necessary for proper protein folding. Hence, the present inventors developed an in vitro assay for assessing the functional activity of PAH in cell lysates.
  • Figure 4a shows the methodology followed by the present inventors to assess PAH activity in cell culture lysates.
  • the CAP cells were lysed in a PBS-based lysis buffer and the lysates were sonicated.
  • a BCA was then performed and 100 pg of protein from the cell lysate was added to a well of a 96-well plate.
  • Known amounts of tyrosine are also added into the 96 well plate in order to generate a standard curve.
  • the initial Reaction Mix used by the inventors comprised 1 mM phenylalanine, 0.04 mg/mL catalase, 100 pM ferrous ammonium iron II sulphate, 200 pM BH4, 5 mM DTT, 200mM NaCI and 20mM NA-HEPES made up in water.
  • the inventors also included a well with 100 pg of protein from the PAH transfected CAP cell lysate where a Reaction Mix that lacked Phenylalanine was added.
  • the inventors then detect tyrosine autofluorescence over time and determine the concentration of tyrosine using the standard curve generated from the wells containing known amounts of tyrosine.
  • Figure 4c shows that this methodology can be used to assess functional PAH activity, since increased tyrosine was detected over time in the lysate from the CAP cells that were transfected with a PAH construct.
  • This methodology allows for PAH activity to be assessed by direct measurement of the tyrosine concentration, thus is highly accurate and reliable.
  • Tissue samples were taken from the livers of wildtype mice and were mechanically digested then homogenised with a bead homogeniser.
  • a BCA was then performed and either 20 pg, 50 pg, 100 pg or 150 pg of protein from the tissue sample is added to a well of a 96 well plate. Known amounts of tyrosine are also added into wells of the 96 well plate in order to generate a standard curve.
  • Figure 5a shows that tyrosine is produced by the wildtype liver samples over time, thus functional PAH activity is being detected. In line with this, a higher amount of protein input results in a greater amount of Tyrosine being generated. This methodology allows for PAH activity to be assessed by direct measurement of the tyrosine concentration, thus is highly accurate and reliable.
  • Enu2 mice are described by Shedlovsky A, et al. (Mouse models of human phenylketonuria, (1993), Genetics, vol. 134, pages 1205-1210). These mice carry a T835C missense mutation in exon 7 of their phenylalanine hydroxy lase gene that results in a phenylalanine to serine substitution at amino acid 263 (F263 S) of the enzyme. Homozygous mutant Enu2 mice show severe hyperphenylalanemia.
  • Tissue samples from the livers of wildtype mice and Enu2 mice were snap frozen tissue from the left lateral lobe of the liver. 100-150 mg of the left lateral lobe tissue was digested with a scalpel on dry ice. The digested tissue was placed in a precellys soft tissue homogeniser tube and 500 pL of PBS was added and the precellys homogeniser machine was then used to produce a homogenous lysate. After homogenisation, the samples were centrifuged for 10 mins and the supernatants were added to 500 pL of PBS supplemented with proteinase inhibitors.
  • a BCA (Thermo, cat no. 23227) was performed and 250pg of tissue lysate was added into a well of a black 96 well plate. Known amount of tyrosine were also added into the plate for generation of a tyrosine standard curve.
  • Tyrosine production was read using fluorescence kinetic readout on the spectramax (274/304 nm ex/em). 1 read was taken every 2.5 mins for 3.5 hours.
  • Tyrosine production was then calculated by subtracting the without Phe control from the samples with Phe, to remove background from endogenous tyrosine, then using the tyrosine standard curve to extrapolate the tyrosine concentration.
  • Figure 5c shows that Enu2 mice had inhibited PAH activity, with no difference in tyrosine production over time and a maximum tyrosine production of 50pM.
  • the in vitro assay could be used to differentiate between liver tissue samples from wildtype and Enu2 mice.
  • EXAMPLE 6 Transfection with the new PAH transgene constructs induces functional PAH activity in cells Codon optimisation can also result in the disruption of protein function, since slowing and pausing of the ribosome at certain times in translation may be necessary for proper protein folding.
  • the present inventors went on to assess whether the newly designed constructs (LSP9-CO9; LSP9-CO8 and LSP9-CO21), which had been demonstrated to increase the expression of PAH, have the ability to induce the expression of functional PAH.
  • the inventors transfected wildtype Huh7 cells with the LSP9-CO9, LSP9-CO8 or LSP9-CO21 and used the in vitro assay, outlined in example 4, to assess functional PAH activity by direct measurement of the tyrosine concentration in cell lysates.
  • the cells were lysed in a PBS-based lysis buffer, a BCA was performed and 100 pg of protein from each cell lysates was added to a well in a 96 well plate. Known amounts of tyrosine are also added into wells of the 96 well plate in order to generate a standard curve. 150 pL of the Reaction Mix (ImM phenylalanine, 75 pM BH4, 2 mM DTT, 200mM NaCI, 20mM NA-HEPES and is made up in water) was added to each well. The inventors then measured tyrosine autofluorescence over time and determined the concentration of tyrosine using a standard curve generated from the readings of the wells containing known amounts of tyrosine.
  • Reaction Mix ImM phenylalanine, 75 pM BH4, 2 mM DTT, 200mM NaCI, 20mM NA-HEPES and is made up in water
  • Figure 6a shows that the new transgenes LSP9-CO9, LSP-C08 and LSP9-CO21 all induce expression of functional PAH, since an increase in tyrosine expression is observed over time, which is not detected in the untransfected control.
  • Figure 6b shows the maximum tyrosine concentration detected in each of the cell lysates and again demonstrates that LSP9-CO9, LSP-C08 and LSP9-CO21 all induce an increase in tyrosine concentration, as compared to the untransfected control, thus showing that the transgene constructs induce expression of functional PAH.
  • the inventors initially tried using a bespoke media of DMEM without tyrosine and phenylalanine with 10 % dialyzed FBS, glutamax and 400 pM Phenylalanine, however no tyrosine was detected in the supernatant sample collected from wildtype Huh7 (see Figure 7a). This demonstrates that the phenylalanine present in this first bespoke media was not being metabolised into tyrosine by the PAH present in the wildtype Huh7 cells, thus this media cannot be used to assess whether cells have functional PAH activity.
  • the inventors then tried a new bespoke media comprising 500 pM of BH4, a co-factor required for PAH activity.
  • the second bespoke media is composed of DMEM without amino acids supplemented which was then supplemented with 400 pM L-Arginine monohydrochloride, 200 pM L-Cystine dihydrochloride, 200 pM L-H istidine monohydrochloride monohydrate, 800 pM L-Threonine, 40 pM Glycine, 800 pM L-Lysine monohydrochloride, 400 pM L-Serine, 0.78 pM L-Tryptophan, 8 pM L- Valine, 8 pM L-lsoleucine, 2 pM L-Methionine, 8 pM L-Leucine.
  • the second bespoke media was further supplemented with 10 % dialyzed FBS, GlutaMAX and 50 pM Phenylalanine.
  • PAH activity was then assessed by measuring whether there was any reduction in the provided phenylalanine (50 uM), in the supernatant samples collected from Huh7 cells.
  • phenylalanine 50 uM
  • this decrease was also seen in the control media sample that has not been added to cells (bar 6 of Figure 7b).
  • Figure 7b demonstrates that the phenylalanine present in this second bespoke media was not being metabolised by the PAH present in the wildtype Huh7 cells, thus this media also cannot be used to assess whether cells have functional PAH activity.
  • This third bespoke media is composed of DMEM without tyrosine and phenylalanine, 10 % dialyzed FBS, glutamax, 400pM Phenylalanine, Antibiotic-Antimycotic and either 50 pM or 100 pM of L- Sepiapterin.
  • This bespoke media was added to Huh7 cells, as outlined above, and a sample of the supernatant was harvested 72 hrs or 96 hrs after the bespoke media was. PAH activity was then assessed by measuring the Tyrosine concentration present in supernatant that was collected from each of these cell lines, using a colorimetric tyrosine kit (Abeam, #abl8375).
  • the inventors Having developed the bespoke cell culture medium, which allows for PAH activity to be monitored in cells in culture, without the need for harvesting the cells, and given that codon optimisation can also result in the disruption of protein function, the inventors then used this assay to confirm whether the AAVs comprising the two constructs that induce the largest increase in PAH expression (LSP9-CO9 and LSP9-CO21) are able to transduce cells and induce expression of functional PAH.
  • the present inventors investigated how delivery of the constructs in a product wherein the AAVs are associated with EVs compares with delivery of the constructs in a standard AAV product.
  • AAVs comprising either the new LSP9-CO9 construct, the new LSP9-CO21 construct, or an LSP1- BMN307 construct were produced alongside EVs loaded with AAVs comprising either the new LSP9- CO9 construct, the new LSP9-CO21 construct, or an LSP1-BMN307 construct.
  • HEK293 cells were seeded in 10-layer cell factories and cultured in DMEM media supplemented with 10% FBS. The next day, the cells were transfected with the relevant transgene construct (comprising SEQ. ID NO 20, 21, 22 or 24) alongside a helper plasmid (pALD-X80) and a Rep-Cap plasmid (Rep2-Cap8-neo VI). 24 hours post-transfection, the media was changed from 10% FBS in DMEM to serum free DMEM.
  • conditioned media was collected from the cells. Sequential centrifugations were preformed, and cells were separated from the conditioned media. The conditioned media was treated with denerase and 4 mM MgCL for 1 hour at 37°C shaking at rate of 95 rpm, to eliminate host cell DNA and RNA.
  • the cells were also harvested 96 hours post-transfection. A tube containing ⁇ 45 mL of resuspended cell pellet was frozen and thawed three times by alternating between dry ice and a 37°C bead bath or water bath and then stored at -80°C.
  • TFF Tangential Flow Filtration
  • the pooled peak containing fractions (1.5 mL) were added to the upper compartment of the pre-equilibrated amicon filter.
  • the compartment was topped up with PBS to approximately 13 mL, before centrifuging at 2000 x g for ⁇ 1 min at room temperature. This concentration was repeated for a total of three rounds and all three permeates were collected into a separate falcon tube (approximately 35 mL).
  • the concentrated retentate - 1.5 mL - was mixed up and down by pipetting gently over the membrane surface, avoiding creating bubbles and the retentate was then transferred to a 50 mL falcon tube.
  • the AAV drug product was then passed through a 0.22 PES syringe filter and formulated to a 0.2% concentration of HSA.
  • Figure 8a shows the methodology developed by the present inventors for assessing PAH activity in PAH KO Huh7 cells in culture.
  • Cells were seeded in cell culture plates and grown in DMEM media supplemented with 10% Heat Inactivated FBS and GlutaMAX. After 6 hrs this medium was removed and the cells were treated with either AAV or exoAAV material.
  • Each of the AAV or ExoAAV samples added to cells were at a titre of le5 MOL 24 hrs later, the medium was removed, cells were washed with PBS and a bespoke cell culture medium (of DMEM without tyrosine and phenylalanine supplemented with 10 % dialyzed FBS, glutamax, 400 pM, Antibiotic-Antimycotic and 100 pM L- Sepiapterin) was then added to the cells. The cells were then cultured in the bespoke medium for a further 72 hours. PAH activity was assessed by measuring the Tyrosine concentration present in the collected supernatant, using a colorimetric tyrosine kit (Abeam, #abl8375).
  • Figure 8b and 8c show that AAVs comprising the new LSP9-CO9 construct induce expression of functional PAH. Moreover, in line with the LSP9-CO9 construct inducing increased PAH expression compared with the known BMN307 construct ( Figure 3), cells transduced with AAVs comprising the new LSP9-CO9 transgene show increased PAH activity as compared with cells transduced with AAVs comprising the BMN 307 construct.
  • Figure 8d and 8e show that AAVs comprising the new LSP9-CO21 construct induce expression of functional PAH. Moreover, in line with the new LSP9-CO21 construct inducing increased PAH expression compared with the known BMN307 construct ( Figure 3), cells transduced with AAVs comprising the new LSP9-CO21 construct show increased PAH activity as compared with cells transduced with AAVs comprising the BMN307 construct.
  • Figures 8b-8e show that the delivery of the new constructs in a product wherein the AAVs are associated with EVs also results in the induction of functional PAH activity.
  • SEQ ID NO 20 - LSP9-CO9 SEQUENCE (ITR to ITR) TTGGCCACTCCCTCTCTGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT
  • SEQ ID NO 24 - LSP1-BMN 307 SEQUENCE (ITR to ITR) cctgcaggcagctgcgcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcga gcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcGGCCGCACGCGTAGGCTCAGAGGCACACAGGAG TTTCTGGGCTCACCCTGCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGTGCTGCCTCTGAA GTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAAC ACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGCTGACC

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Abstract

L'invention concerne des polynucléotides et des séquences polynucléotidiques codant pour la phénylalanine hydroxylase (PAH), des cassettes d'expression, des vecteurs, des particules virales, des vésicules extracellulaires (VE) et des compositions pharmaceutiques les comprenant, et leurs utilisations, par exemple pour traiter des maladies et des troubles associés à de faibles niveaux ou à une activité réduite de PAH, telle que la phénylcétonurie (PKU).
PCT/EP2024/063362 2023-05-15 2024-05-15 Thérapies géniques pour la phénylcétonurie Pending WO2024236032A1 (fr)

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