WO2022219332A1 - Administration de vecteurs de thérapie génique - Google Patents

Administration de vecteurs de thérapie génique Download PDF

Info

Publication number
WO2022219332A1
WO2022219332A1 PCT/GB2022/050928 GB2022050928W WO2022219332A1 WO 2022219332 A1 WO2022219332 A1 WO 2022219332A1 GB 2022050928 W GB2022050928 W GB 2022050928W WO 2022219332 A1 WO2022219332 A1 WO 2022219332A1
Authority
WO
WIPO (PCT)
Prior art keywords
transgene
pharmaceutical composition
vector
disease
factor
Prior art date
Application number
PCT/GB2022/050928
Other languages
English (en)
Inventor
Uta Griesenbach
Eric Wfw Alton
Robyn BELL
Nikhil FAULKNER
Original Assignee
Imperial College Innovations Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial College Innovations Limited filed Critical Imperial College Innovations Limited
Priority to EP22719987.4A priority Critical patent/EP4323532A1/fr
Publication of WO2022219332A1 publication Critical patent/WO2022219332A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18822—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18841—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18845—Special targeting system for 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates a pharmaceutical compositions comprising a lentiviral vector and a pharmaceutically-acceptable carrier, for intravenous administration, and medical uses and methods of treatment by intravenous administration of said pharmaceutical composition.
  • Retroviruses are a family of RNA viruses (Retroviridae) that encode the enzyme reverse transcriptase. Lentiviruses are a genus of the Retroviridae family, and are characterised by a long incubation period.
  • Retroviruses, and lentiviruses in particular, can deliver a significant amount of viral RNA into the DNA of the host cell and have the unique ability among retroviruses of being able to infect non-dividing cells, so they are one of the most efficient methods of a gene delivery vector.
  • Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins.
  • the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles.
  • pseudotyping allows one to specify the character of the envelope proteins.
  • a frequently used protein to pseudotype retroviral and lentiviral vectors is the glycoprotein G of the Vesicular stomatitis virus (VSV), short VSV-G.
  • VSV Vesicular stomatitis virus
  • Lentiviral vectors especially those derived from HIV-1, are widely studied and frequently used vectors.
  • the evolution of the lentiviral vectors backbone and the ability of viruses to deliver recombinant DNA molecules (transgenes) into target cells have led to their use in many applications.
  • Two possible applications of viral vectors include restoration of functional genes in genetic therapy and in vitro recombinant protein production.
  • the recombinant simian immunodeficiency virus pseudotyped with hemagglutinin- neuraminidase (HN) and fusion (F) proteins from Sendai virus (rSIV.F/HN) is a lentiviral vector that was specifically designed to achieve gene transfer efficiently to the airway epithelium.
  • the method of administration has the potential to enable the use of lower concentrations of viral vector to achieve sufficient levels of therapeutic protein, thus reducing the costs associated with gene therapy and improving safety.
  • SUMMARY OF THE INVENTION The present inventors have now unexpectedly shown that sustained transgene expression can be achieved in a variety of tissue types through intravenous (i.v.) administration of a F/HN pseudotyped lentiviral vector. This is surprising, as this vector was originally developed to facilitate efficient targeting and transduction of airway epithelium, and so would not be predicted to target and transduce other cell types, particularly not with the levels of efficiency observed.
  • the present inventors have identified that a single intravenous dose of transgene-harbouring F/HN pseudotyped lentiviral vector results in markedly higher levels of transgene expression in a range of tissues, including the liver, spleen and kidney when compared with intra-nasal administration of the same vector.
  • the F/HN pseudotyped lentiviral vector can advantageously elicit long-term transgene expression when delivered by intravenous administration according to the invention.
  • the inventors have demonstrated stable high-levels of transgene expression for over six-months, with ongoing experiments expected to maintain these high-levels of expression for at least 12-months.
  • the effective transduction of organs and tissues other than the lungs allows greater flexibility in the choice of transgene, including those which encode proteins which may be more efficiently processed/post-translationally modified in these organs/tissues (for example, the liver).
  • the present invention has the potential to allow for the efficient delivery and expression of transgenes that cannot be efficiently processed/modified when administered by the previous respiratory routes, and hence organ-specific post-translational modifications. Consequently, the present invention makes plausible the effective treatment of diseases beyond those previously considered.
  • the F/HN pseudotyped lentiviral vectors have the potential to achieve clinically relevant levels of therapeutic proteins using lower doses of lentiviral vector, thus improving safety, increasing the likelihood of translating experimental findings into successful therapies and reducing the overall cost of gene therapy (through reduced manufacturing costs).
  • the invention provides a pharmaceutical composition comprising: (i) a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, and which comprises a transgene; and (ii) a pharmaceutically-acceptable carrier; wherein said composition is for intravenous administration.
  • Said lentiviral vector may be selected from the group consisting of a Simian immunodeficiency virus (SIV) vector, a Human immunodeficiency virus (HIV) vector, a Feline immunodeficiency virus (FIV) vector, an Equine infectious anaemia virus (EIAV) vector, and a Visna/maedi virus vector.
  • the respiratory paramyxovirus may be a Sendai virus.
  • Said vector may further comprise a promoter, wherein optionally the promoter is selected from the group consisting of a hybrid human cytomegalovirus (CMV) enhancer/elongation factor 1 a (EF1 a) promoter (hCEF), a CMV promoter, an EF1 a promoter and a tissue-specific promoter, preferably a hCEF promoter.
  • CMV human cytomegalovirus
  • EF1 a elongation factor 1 a
  • hCEF tissue-specific promoter
  • the transgene may (a) encode a secreted therapeutic protein selected from the group consisting of Alpha-1 Antitrypsin (AAT), Factor VIII, Surfactant Protein B (SFTPB), Surfactant Protein C (SFTPC) Factor V, Factor VII, Factor IX, Factor X and/or Factor XI, von Willebrand Factor, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), decorin , an anti-inflammatory protein (e.g.
  • IL-10 or TGF ⁇ or monoclonal antibody, an anti-inflammatory decoy, or a monoclonal antibody against an infective agent; or (b) be CFTR, ABCA3, BMPR2 or TRIM72 or a gene associated with an inherited metabolic disorder, lysosomal storage disorder or mucopolysaccharidoses.
  • the promoter may be a hybrid human CMV enhancer/EF1a (hCEF) promoter and the transgene encodes AAT or Factor VIII.
  • the pharmaceutical composition may comprise a concentration of the lentiviral vector of about 1.0 x 10 8 to 1 x 10 9 TU/ml, preferably about 2.5 to 4.5 x 10 8 TU/ml; and/or the composition may be for administration at a dose of about 1.0 x 10 8 to about 1.0 x 10 10 TU/kg, preferably about 5.0 x 10 8 to about 1.0 x 10 10 TU/kg.
  • the pharmaceutical composition may comprise TSSM formulation buffer as a pharmaceutically-acceptable carrier.
  • the invention also provides a pharmaceutical composition of the invention for use in a method of gene therapy, wherein the composition is administered intravenously. Said pharmaceutical composition may be for intravenous administration by bolus administration or infusion administration.
  • the lentiviral vector may integrate into the genome of a target cell; and/or the transgene may exhibit stable expression in a target cell, tissue or organ following a single administration.
  • the transgene may be expressed in a target cell, tissue, or organ for at least 2 weeks, preferably at least 16 weeks, more preferably at least 24 weeks.
  • the target cell may be a liver cell, spleen cell, kidney cell, cardiovascular cell (e.g. an endothelial cell), lung cell, gonad cell, a cell of the central nervous system (CNS) and/or bone marrow cell.
  • the target tissue may be liver, spleen, kidney, cardiovascular tissue (e.g.
  • the pharmaceutical composition may be administered as: (a) a single dose; or (b) repeat doses, optionally wherein the composition is administered (i) at intervals of 3 to 4 weeks, every other month, every three months, every six months, or annually.
  • the gene therapy may treat or prevent a genetic respiratory disease, a haematological disease, an ophthalmological disease, a neurological disease, an autoimmune disease, an infectious disease, a neoplastic disease or an inherited metabolic disorder.
  • the gene therapy may treat or prevent cystic fibrosis (CF); a surfactant deficiency, optionally surfactant protein B (SP-B) deficiency, pulmonary surfactant metabolism dysfunction 2 (SMDP2) or pulmonary surfactant metabolism dysfunction 3 (SMDP3); alpha 1-antitrypsin deficiency (AATD); pulmonary alveolar proteinosis (PAP); chronic obstructive pulmonary disease (COPD); pulmonary hypertension; acute respiratory distress syndrome (ARDS); a pulmonary fibrotic disease, optionally idiopathic pulmonary fibrosis; a pulmonary allergic condition; asthma; a pulmonary bacterial, viral or fungal infection; lung cancer or a dysplastic change in the lungs; a cardiovascular disease; a blood cancer; a blood disorder or blood clotting deficiency, optionally haemophilia; or an inherited metabolic condition, optionally a lysosomal storage disease, mucopolysaccharidosis or mitochondrial disorder.
  • SP-B
  • the genetic respiratory disease may be alpha 1-antitrypsin deficiency and the transgene SERPINA1.
  • the haematological disease may be haemophilia and the transgene may be selected from the group consisting of Factor VIII, Factor V, Factor IX, Factor X and/or Factor XI.
  • the invention also provides a method of gene therapy, said method comprising administering a pharmaceutical composition of the invention to a patient in need thereof, wherein the composition is administered intravenously.
  • the invention further provides the use of a lentiviral vector of the invention for the manufacture of a medicament for use in gene therapy, wherein the medicament is for intravenous administration.
  • the invention further provides a method of expressing a transgene in a target cell, comprising delivering a pharmaceutical composition of the invention into the target cell.
  • Said delivering may comprise integrating the lentiviral vector into said target cell's genome.
  • the invention also provides a method of producing a pharmaceutical composition of the invention, comprising formulating the lentiviral vector with a pharmaceutically-acceptable carrier to produce the pharmaceutical composition in an intravenous formulation.
  • Figure 1 shows the expression levels (expressed as logarithm relative light units- log(RLU)) of gaussia luciferase in peripheral blood sampled repeatedly over a 52 week period following intravenous delivery of increasing doses of SIV-F/HN vector harbouring the gaussia luciferase transgene.
  • Figure 2 shows the expression levels (expressed as relative light units (RLU)) of gaussia luciferase in peripheral blood sampled at 52 weeks following delivery of increasing doses of SIV-F/HN vector harbouring the gaussia luciferase transgene via intravenous and intra-nasal administration.
  • RLU relative light units
  • Figure 3 shows the tissue distribution of luciferase protein one month following a single dose of SIV- F/HN vector harbouring the firefly luciferase transgene (2.6e8 TU/mouse) delivered via either intra- nasal or intravenous administration.
  • Figure 4 shows the concentration of AAT protein in the blood of mice one week after receiving a single dose of SIV-F/HN vector harbouring a human SERPINA1 transgene (vGM109).
  • the term “capable of' when used with a verb, encompasses or means the action of the corresponding verb.
  • “capable of interacting” also means interacting
  • “capable of cleaving” also means cleaves
  • “capable of binding” also means binds and "capable of specifically targeting" also means specifically targets.
  • Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed.
  • the term “consisting essentially of''” refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non-immunogenic ingredients).
  • Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of” and/or “consisting essentially of” such features. Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format.
  • vector retroviral vector
  • retroviral F/HN vector retroviral F/HN vector
  • lentiviral vector and “lentiviral F/HN vector” are used interchangeably to mean a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise stated. All disclosure herein in relation to retroviral vectors of the invention applies equally and without reservation to lentiviral vectors of the invention and to SIV vectors that are pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus (also referred to herein as SIV F/HN or SIV-FHN).
  • the terms “transduced” and “modified” are used interchangeably to describe cells which have been modified to express a transgene of interest. Typically the modification occurs through transduction of the cells.
  • the terms “titre” and “yield” are used interchangeably to mean the amount of lentiviral (e.g. SIV) vector produced by a method of the invention. Titre is the primary benchmark characterising manufacturing efficiency, with higher titres generally indicating that more retroviral/lentiviral (e.g. SIV) vector is manufactured (e.g. using the same amount of reagents).
  • Titre or yield may relate to the number of vector genomes that have integrated into the genome of a target cell (integration titre), which is a measure of “active” virus particles, i.e. the number of particles capable of transducing a cell.
  • Transducing units TU/mL also referred to as TTU/mL
  • TTU/mL Transducing units
  • the total number of (active+inactive) virus particles may also be determined using any appropriate means, such as by measuring either how much Gag is present in the test solution or how many copies of viral RNA are in the test solution.
  • a lentivirus particle contains either 2000 Gag molecules or 2 viral RNA molecules. Once total particle number and a transducing titre/TU have been measured, a particle:infectivity ratio calculated.
  • Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogues, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogues regardless of its size or function.
  • polypeptide proteins and polypeptide
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing.
  • polynucleotides refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analogue thereof.
  • the nucleic acid can be either single-stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA Alternatively, it can be a single-stranded nucleic acid not derived from any double- stranded DNA.
  • the nucleic acid can be DNA.
  • the nucleic acid can be RNA Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides.
  • transgene and “gene” are also used interchangeably and both terms encompass fragments or variants thereof encoding the target protein.
  • transgenes of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. Minor variations in the amino acid sequences of the invention are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to the amino acid sequence of the invention or a fragment thereof as defined anywhere herein.
  • homology is used herein to mean identity.
  • sequence of a variant or analogue sequence of an amino acid sequence of the invention may differ on the basis of substitution (typically conservative substitution) deletion or insertion. Proteins comprising such variations are referred to herein as variants. Proteins of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non- conserved positions. Variants of protein molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure/property-activity relationships [see for example, Wold, et al. Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D.
  • proteins can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of proteins sequence, functional and three-dimensional structures and these properties can be considered individually and in combination.
  • Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation.
  • the term “protein”, as used herein, includes proteins, polypeptides, and peptides.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
  • amino acid sequence is synonymous with the term “peptide”.
  • the terms "protein” and "polypeptide” are used interchangeably herein.
  • the conventional one-letter and three- letter codes for amino acid residues may be used.
  • the 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Amino acid residues at non-conserved positions may be substituted with conservative or non- conservative residues. In particular, conservative amino acid replacements are contemplated.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine).
  • basic side chains e.g., lysine, arginine, or histidine
  • acidic side chains e.g.
  • conservatively modified variants in a protein of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.
  • Non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).
  • an electropositive side chain e.g., Arg, His or Lys
  • an electronegative residue e.g., Glu or As
  • “Insertions” or “deletions” are typically in the range of about 1, 2, or 3 amino acids. The variation allowed may be experimentally determined by systematically introducing insertions or deletions of amino acids in a protein using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more than routine experiments for a skilled person.
  • a “fragment” of a polypeptide comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or more of the original polypeptide.
  • the polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell.
  • the natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.
  • the polynucleotides of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method or the tri-ester method, and may be performed on commercial automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • isolated in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.
  • Degenerate codons encompassing all possible codons for a given amino acid are set forth below:
  • a “variant” nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof).
  • a nucleic acid sequence or fragment thereof is “substantially homologous” (or “substantially identical”) to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more% of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.
  • a “variant” nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the “variant” and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions.
  • Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM.
  • the pH is typically between 7.0 and 8.3.
  • Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention.
  • Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST (as described below).
  • preferential codon usage refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid.
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different codons may be preferential.
  • Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • any nucleic acid sequence may be codon-optimised for expression in a host or target cell.
  • the vector genome or corresponding plasmid
  • the REV gene or corresponding plasmid
  • the fusion protein (F) gene or correspond plasmid
  • the hemagglutinin-neuraminidase (HN) gene or corresponding plasmid, or any combination thereof may be codon-optimised.
  • a “fragment” of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide.
  • a “fragment” of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of said polynucleotide (e.g. at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950 or 1000 consecutive nucleic acid residues of said polynucleotide).
  • a fragment may include at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest.
  • a fragment as defined herein retains the same function as the full-length polynucleotide.
  • the terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • the terms “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g.
  • “reduction” or “inhibition” encompasses a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition (i.e. abrogation) as compared to a reference level.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250% or more compared with a reference level, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level.
  • an "increase” is an observable or statistically significant increase in such level.
  • the terms “high expression” as used herein encompasses the potential for therapeutic expression.
  • the terms “individual”, “subject”, and “patient”, are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and/or therapy optimisation is desired.
  • the mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
  • the individual, subject, or patient is a human.
  • An “individual” may be an adult, juvenile or infant.
  • An “individual” may be male or female.
  • a "subject in need" of treatment for a particular condition can be an individual having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications or symptoms related to such a condition, and optionally, have already undergone treatment for a condition as defined herein or the one or more complications or symptoms related to said condition.
  • a subject can also be one who has not been previously diagnosed as having a condition as defined herein or one or more or symptoms or complications related to said condition.
  • a subject can be one who exhibits one or more risk factors for a condition, or one or more or symptoms or complications related to said condition or a subject who does not exhibit risk factors.
  • the term “healthy individual” refers to an individual or group of individuals who are in a healthy state, e.g. individuals who have not shown any symptoms of the disease, have not been diagnosed with the disease and/or are not likely to develop the disease e.g. cystic fibrosis (CF) or any other disease described herein).
  • CF cystic fibrosis
  • Preferably said healthy individual(s) is not on medication affecting CF and has not been diagnosed with any other disease.
  • the one or more healthy individuals may have a similar sex, age, and/or body mass index (BMI) as compared with the test individual.
  • BMI body mass index
  • Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.
  • control and “reference population” are used interchangeably.
  • Lentiviral vector The invention relates to the intravenous delivery of a pharmaceutical composition
  • a pharmaceutical composition comprising (i) a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins and which comprises a transgene; and (ii) a pharmaceutically-acceptable carrier.
  • HN hemagglutinin-neuraminidase
  • F fusion proteins and which comprises a transgene
  • lentivirus refers to a family of retroviruses, which are themselves members of the Retroviridae family of RNA viruses that encode the enzyme reverse transcriptase.
  • lentiviruses suitable for use in the present invention include Simian immunodeficiency virus (SIV), Human immunodeficiency virus (HIV), Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV), and Visna/maedi virus.
  • SIV Simian immunodeficiency virus
  • HV Human immunodeficiency virus
  • FV Feline immunodeficiency virus
  • EIAV Equine infectious anaemia virus
  • Visna/maedi virus is an SIV vector (including all strains and subtypes), such as a SIV-AGM (originally isolated from African green monkeys, Cercopithecus aethiops).
  • SIV-AGM originally isolated from African green monkeys, Cercopithecus aethiops
  • the invention relates to HIV vectors.
  • the HN and F proteins used to pseudotype the lentiviral vector of the invention are typically derived from a respiratory paramyxovirus.
  • the respiratory paramyxovirus is a Sendai virus (murine parainfluenza virus type 1).
  • Lentiviral (e.g. SIV) vectors such as those according to the invention, can integrate into the genome of transduced cells and lead to long-lasting expression, making them suitable for transduction of stem/progenitor cells in a target tissue of interest.
  • the lentiviral (e.g. SIV) vector of the present invention enables high levels of transgene expression, resulting in high levels of expression of a therapeutic protein.
  • the lentiviral (e.g. SIV) vector of the present invention typically provides high expression levels of a transgene when administered to a patient.
  • Expression may be measured by any appropriate method (qualitative or quantitative, preferably quantitative), and concentrations given in any appropriate unit of measurement, for example ng/ml or nM.
  • Expression of a transgene of interest may be given relative to the expression of the corresponding endogenous (defective) gene in a patient. Expression may be measured in terms of mRNA or protein expression.
  • the expression of the transgene of the invention such as a functional CFTR gene, may be quantified relative to the endogenous gene, such as the endogenous (dysfunctional) CFTR genes in terms of mRNA copies per cell or any other appropriate unit.
  • Expression levels of a transgene and/or the encoded therapeutic protein of the invention may be measured by conventional means.
  • expression levels of a transgene and/or an encoded therapeutic protein may be determined in a tissue sample, cell sample, and/or serum/plasma as appropriate.
  • a high and/or therapeutic expression level may therefore refer to the concentration in the tissue, cells and/or serum/plasma of the subject.
  • a high and/or therapeutic expression level may alternatively or additionally refer to the concentration in the lung, epithelial lining fluid.
  • Standard method for determining the expression levels of a transgene and/or an encoded therapeutic protein are well known in the art and including, RT-qPCR, ELISA and western blotting.
  • the transgene included in the vector of the invention may be modified to facilitate expression.
  • the transgene sequence may be in CpG-depleted (or CpG-fee) and/or codon-optimised form to facilitate gene expression.
  • Standard techniques for modifying the transgene sequence in this way are known in the art.
  • the lentiviral (e.g. SIV) vector of the present invention enables long-term transgene expression, resulting in long-term expression of a therapeutic protein.
  • the phrases “long-term expression”, “stable expression”, “sustained expression”, “long-lasting expression” and “persistent expression” are used interchangeably.
  • Long-term expression means expression of a therapeutic gene and/or protein, preferably at therapeutic levels, for at least 2 weeks at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks, at least 76 weeks, at least 104 weeks or more.
  • long-term expression means expression for at least 16 weeks or more, more preferably at least 24 weeks or more.
  • the lentiviral (e.g. SIV) vector of the present invention may be given as a single dose or as repeated doses.
  • Repeated doses may be administered twice-daily, daily, twice-weekly, weekly, at intervals of three to four weeks, monthly, every two months (every other month), every three months, every four months, every six months, yearly (annually), every two years, or more. Dosing may be continued for as long as required, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to for the lifetime of the patient to be treated. Preferably a single dose is administered. When repeat doses are used, dosing may be repeated at intervals of at six months or more.
  • the lentiviral e.g.
  • SIV vector typically comprises a promoter operably linked to a transgene, enabling expression of the transgene.
  • the promotor may be a constitutive promotor (i.e. an unregulated promoter that allows for continual transcription of its associated transgene in almost all tissues/cell types), a tissue- specific promotor (i.e. a promoter that only has activity in certain cell types), or an inducible or regulatable promotor (for example, the well-known tetracycline inducible promotors).
  • the promoter is a hybrid human CMV enhancer/EF1a (hCEF) promoter.
  • This hCEF promoter may lack the intron corresponding to nucleotides 570-709 and the exon corresponding to nucleotides 728-733 of the hCEF promoter.
  • a preferred example of an hCEF promoter sequence of the invention is provided by SEQ ID NO: 1.
  • the promoter may be a CMV promoter.
  • An example of a CMV promoter sequence is provided by SEQ ID NO: 2.
  • the promoter may be a human elongation factor 1a (EF1a) promoter.
  • An example of a EF1a promoter is provided by SEQ ID NO: 3.
  • Other promoters for transgene expression are known in the art and their suitability for the lentiviral (e.g.
  • SIV vector of the invention may be determined using routine techniques known in the art.
  • tissue-specific promoters is encompassed by the invention to enable tissue-specific expression of a transgene in a desired tissue or organ of interest.
  • Non-limiting examples of other promoters include UbC and UCOE.
  • the promoter may be modified to further regulate expression of the transgene of the invention.
  • the promoter included in the lentiviral (e.g. SIV) vector of the invention may be specifically selected and/or modified to further refine regulation of expression of the therapeutic gene. Again, suitable promoters and standard techniques for their modification are known in the art.
  • the lentiviral vector (particularly SIV F/HN vectors) of the invention comprise a hCEF promoter having low or no CpG dinucleotide content.
  • the hCEF promoter may have all CG dinucleotides replaced with any one of AG, TG or GT.
  • the hCEF promoter may be CpG-free.
  • a preferred example of a CpG-free hCEF promoter sequence of the invention is provided by SEQ ID NO: 1.
  • the absence of CpG dinucleotides further improves the performance of lentiviral (e.g. SIV) vector of the invention and in particular in situations where it is not desired to induce an immune response against an expressed antigen or an inflammatory response against the delivered expression construct.
  • the elimination of CpG dinucleotides reduces the occurrence of flu-like symptoms and inflammation which may result from administration of constructs.
  • the lentiviral (e.g. SIV) vector of the invention may be modified to allow shut down of gene expression. Standard techniques for modifying the vector in this way are known in the art. As a non- limiting example, Tet-responsive promoters are widely used.
  • the lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus of the invention may comprise any suitable transgene.
  • the lentiviral (e.g. SIV) vector may comprise a transgene that encodes a polypeptide or protein that is therapeutic for the treatment of disease.
  • F/HN pseudotyping was developed by the present inventors to enable efficient targeting of cells in the airway epithelium.
  • the present inventors have now shown that the F/HN pseudotyped lentiviral vector is surprisingly capable of transducing a range of other cell and tissue types thus expanding the potential use of this vector.
  • SIV vector of the invention may comprise a transgene encoding a protein selected from: (i) a secreted therapeutic protein, optionally Alpha-1 Antitrypsin (AAT), Factor VIII, Surfactant Protein B (SFTPB), Surfactant Protein C (SFTPC), Factor V, Factor VII, Factor IX, Factor X, Factor XI, von Willebrand Factor, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), , decorin, an anti-inflammatory protein (e.g. IL-10 or TGF ⁇ ) or monoclonal antibody, a monoclonal antibody against an infectious agent (e.g.
  • AAT Alpha-1 Antitrypsin
  • SFTPB Surfactant Protein B
  • SFTPC Surfactant Protein C
  • GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor
  • decorin an anti-inflammatory protein (e.g. IL-10 or TGF ⁇ ) or monoclonal
  • transgenes that may be comprised in a /lentiviral (e.g. SIV) vector of the invention include genes related to or associated with other surfactant deficiencies and/or genes related to congenital metabolic disorders, such as lysosomal storage disorders and mucopolysaccharidoses. In some embodiments, the transgene is not FVIII.
  • the transgene is selected from CSF2, SERPINA1, DCN, TRIM72, ABACA3, BMPR2, FVIII and/or CFTR.
  • the transgene may encode an AAT.
  • An example of an SERPINA1 transgene is provided by SEQ ID NO: 10, or by the complementary sequence of SEQ ID NO: 11.
  • SEQ ID NO: 10 is a codon-optimized CpG depleted SERPINA1 transgene previously designed by the present inventors to enhance translation in human cells. Such optimisation has been shown to enhance gene expression by up to 15-fold.
  • Variants of same sequence which possess the same technical effect of enhancing translation compared with the unmodified (wild-type) SERPINA1 gene sequence are also encompassed by the present invention.
  • the polypeptide encoded by said SERPINA1 transgene may be exemplified by the polypeptide of SEQ ID NO: 12. Variants thereof (as described therein) are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to any one of SEQ ID NO:10, 11 or 12.
  • the transgene may encode a FVIII.
  • FVIII transgene examples include SEQ ID NOs: 13 and 14, or by the respective complementary sequences of SEQ ID NO: 15 and 16.
  • the polypeptide encoded by the FVIII transgene may be exemplified by the polypeptide of SEQ ID NO: 17 or 18. Variants thereof (as described therein) are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to any one of SEQ ID NOs: 13 to 18.
  • the transgene may encode a CFTR.
  • An example of a CFTR transgene is provided by SEQ ID NO: 4.
  • the polypeptide encoded by said CFTR transgene may be exemplified by the polypeptide of SEQ ID NO: 5. Variants thereof (as described therein) are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to SEQ ID NO: 4 or 5.
  • the transgene may encode GM-CSF.
  • An example of a human GM-CSF transgene (CSF2) is provided by SEQ ID NO: 6.
  • An example of a mouse GM-CSF transgene (CSF2) is provided by SEQ ID NO: 8.
  • a human GM-CSF transgene such as SEQ ID NO: 6, us used.
  • the polypeptide encoded by said CSF2 transgene may be exemplified by the polypeptide of SEQ ID NO: 7 (human) or 9 (mouse).
  • the transgene encodes a human GM-CSF polypeptide, such as SEQ ID NO: 7.
  • Variants thereof are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to any one of SEQ ID NOs: 6 to 9, preferably SEQ ID NOs: 6 and 7.
  • the transgene may encode decorin.
  • An example of a DCN transgene is provided by SEQ ID NO: 19.
  • the polypeptide encoded by said DCN transgene may be exemplified by the polypeptide of SEQ ID NO: 20. Variants thereof (as described therein) are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to SEQ ID NO: 19 or 20.
  • the transgene may encode TRIM72.
  • An example of a TRIM72 transgene is provided by SEQ ID NO: 21.
  • the polypeptide encoded by said TRIM72 transgene may be exemplified by the polypeptide of SEQ ID NO: 22.
  • Variants thereof are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to SEQ ID NO: 21 or 22.
  • the transgene may encode ABCA3.
  • An example of a ABACA3 transgene is provided by SEQ ID NO: 23.
  • the polypeptide encoded by said ABACA3 transgene may be exemplified by the polypeptide of SEQ ID NO: 24.
  • Variants thereof (as described therein) are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to SEQ ID NO: 23 or 24.
  • the transgene may encode BMPR2.
  • An example of a BMPR2 transgene is provided by SEQ ID NO: 25.
  • the polypeptide encoded by said BMPR2 transgene may be exemplified by the polypeptide of SEQ ID NO: 26.
  • Variants thereof are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100% to SEQ ID NO: 25 or 26.
  • the transgene of the invention may be any one or more of SFTPB, SFTPC, Factor V, Factor VII, Factor IX, Factor X and/or Factor XI, von Willebrand Factor, GM-CSF, ABCA3, BMPR2, TRIM72 or DCN , or other known related gene.
  • the transgene may encode a monoclonal antibody (mAb) against an infectious agent (bacterial, fungal or viral, e.g. the SARS-CoV-2 virus).
  • the transgene may encode a monoclonal antibody which targets an inflammatory mediator, for example, the transgene may encode an anti- TNF alpha antibody.
  • the transgene may encode a therapeutic protein implicated in an inflammatory, immune or metabolic condition.
  • a lentiviral (e.g. SIV) vector of the invention may be delivered to the cells of a target organ to allow production and processing of proteins to be secreted into circulatory system.
  • the transgene may encode for Factor VIII, Factor VII, Factor V, Factor IX, Factor X, Factor XI and/or von Willebrand’s factor.
  • Such a vector may be used in the treatment of diseases, particularly cardiovascular diseases and blood disorders, such as leukaemia or blood clotting disorders.
  • blood clotting deficiencies include diseases such as haemophilia (both haemophilia A, which is a deficiency of factor VIII, haemophilia B, which is a deficiency of factor IX, and/or haemophilia C, which is a deficiency of factor XI).
  • the transgene may encode an mAb against an infectious agent or a protein implicated in an inflammatory, immune or metabolic condition, such as, lysosomal storage disease.
  • a lentiviral vector may be delivered to the cells of a target organ to allow production and processing of protein for the treatment of congenital metabolic disorders, including but not limited to lysosomal storage disorders and mucopolysaccharidoses.
  • the lentiviral (e.g. SIV) vector of the invention may have no intron positioned between the promoter and the transgene. Similarly, there may be no intron between the promoter and the transgene in the vector genome (pDNA1) plasmid used to make said vector.
  • pDNA1 vector genome
  • the lentiviral (e.g. SIV) vector comprises a hCEF promoter and an SERPINA1 transgene, including those described herein.
  • said lentiviral e.g.
  • the lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene.
  • the lentiviral (e.g. SIV) vector comprises a hCEF or CMV promoter and a FVIII transgene, including those described herein.
  • said lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene.
  • the lentiviral (e.g. SIV) vector comprises a hCEF promoter and a CFTR transgene, including those described herein.
  • said lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene.
  • the lentiviral (e.g. SIV) vector comprises a hCEF or CMV promoter and an DCN transgene, including those described herein.
  • said lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene.
  • Such a lentiviral (e.g. SIV) vector may be produced by the method described herein, using a genome plasmid carrying the DCN transgene and a promoter.
  • the lentiviral (e.g. SIV) vector comprises a hCEF or CMV promoter and an TRIM72 transgene, including those described herein.
  • SIV vector may have no intron positioned between the promoter and the transgene.
  • a lentiviral (e.g. SIV) vector may be produced by the method described herein, using a genome plasmid carrying the TRIM72 transgene and a promoter.
  • the lentiviral (e.g. SIV) vector comprises a hCEF or CMV promoter and an ABACA3 transgene, including those described herein.
  • said lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene.
  • the lentiviral (e.g. SIV) vector may be produced by the method described herein, using a genome plasmid carrying the ABACA3 transgene and a promoter.
  • the lentiviral (e.g. SIV) vector comprises a hCEF or CMV promoter and a BMPR2 transgene, including those described herein.
  • said lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene.
  • Such a lentiviral (e.g. SIV) vector may be produced by the method described herein, using a genome plasmid carrying the BMPR2 transgene and a promoter.
  • SIV) vector as described herein comprises a transgene.
  • the transgene comprises a nucleic acid sequence encoding a gene product, e.g., a protein, particularly a therapeutic protein.
  • the nucleic acid sequence encoding SERPINA1, GM-CSF, FVIII,CFTR, decorin, TRIM72, ABAC3 or BMPR2 comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the SERPINA1, GM-CSF, FVIII,CFTR, decorin, TRIM72, ABAC3 or BMPR2 nucleic acid sequence respectively, examples of which are described herein.
  • the nucleic acid sequence encoding SERPINA1, GM-CSF, FVIII,CFTR, decorin, TRIM72, ABAC3 or BMPR2 comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the SERPINA1, GM-CSF, FVIII, CFTR, decorin, TRIM72, ABAC3 or BMPR2 nucleic acid sequence respectively, examples of which are described herein.
  • the nucleic acid sequence encoding SERPINA1 is provided by SEQ ID NO: 10, or by the complementary sequence of SEQ ID NO: 11, the nucleic acid sequence encoding FVIII is provided by SEQ ID NO: 13 or 14, or by the respective complementary sequence of SEQ ID NO: 15 or 16, and/or the nucleic acid sequence encoding CFTR is provided by SEQ ID NO: 4, the nucleic acid sequence encoding GM-CSF is provided by SEQ ID NO: 6 or 8, preferably SEQ ID NO: 6, the nucleic acid sequence encoding decorin is provided by SEQ ID NO: 19, the nucleic acid sequence encoding TRIM72 is provided by SEQ ID NO: 21, the nucleic acid sequence encoding ABCA3 is provided by SEQ ID NO: 24, and/or the nucleic acid sequence encoding BMPR2 is provided by SEQ ID NO: 25,or variants thereof.
  • the amino acid sequence of the AAT, FVIII,CFTR, GM-CSF, decorin, TRIM72 and/or ABCA3 polypeptide encoded by the respective SERPINA1, FVIII, CFTR, CSF2, DCN, TRIM72, and/or ABACA3 transgene may comprise (or consist of) an amino acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the functional AAT, FVIII,CFTR, GM-CSF, decorin, TRIM72 and/or ABCA3 polypeptide sequence respectively.
  • the transgene may include a nucleic acid sequence encoding for a signal peptide (such as the endogenous signal peptide of a secreted protein), or may exclude a nucleic acid sequence encoding for a signal peptide.
  • the therapeutic protein may include a signal peptide (such as the endogenous signal peptide of a secreted protein), or may exclude a signal peptide. Where appropriate, endogenous signal peptides have been identified in the sequence information section herein. All disclosure herein relates to both transgenes and therapeutic proteins including and excluding signal peptides unless explicitly stated.
  • sequence identity of variants, and/or lengths of fragments may be based on the sequence with or without a signal peptide.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention may comprise a central polypurine tract (cPPT) and/or the Woodchuck hepatitis virus posttranscriptional regulatory elements (WPRE).
  • An exemplary WPRE sequence is provided by SEQ ID NO: 26.
  • the invention also provides a method of expressing a transgene in a target cell, comprising delivering a pharmaceutical composition as defined herein into the target cell.
  • Said delivering may comprise integrating the lentiviral (e.g. SIV) vector of the pharmaceutical composition into the genome of the target cell.
  • the present inventors have unexpectedly shown that sustained, high- level transgene expression can be achieved in a variety of tissue types through intravenous administration of a F/HN pseudotyped lentiviral vector.
  • the present invention allows for greater flexibility in the choice of transgene for delivery, and hence in the choice of disease or disorder to be treated according to the invention, particularly when compared with transgenes that can be delivered/diseases treated by direct respiratory administration as taught in the art.
  • the pharmaceutical composition of the present invention particularly when delivered intravenously, can be used in gene therapy.
  • the present invention a pharmaceutical composition
  • a pharmaceutical composition comprising: (i) a lentiviral vector pseudotyped with hemagglutinin- neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, and which comprises a transgene; and (ii) a pharmaceutically-acceptable carrier; wherein said composition is for intravenous administration.
  • Said composition may be formulated for intravenous administration as described herein.
  • the invention provides a pharmaceutical composition of the present invention for use in a method of treating or preventing a disease, wherein said pharmaceutical composition is administered intravenously.
  • the present invention provides a pharmaceutical composition as described herein for use in a method of gene therapy, wherein the composition is administered intravenously.
  • Intravenous administration allows for the lentiviral (e.g. SIV) vectors to be dispersed systemically throughout the body by means of the circulatory system.
  • the lentiviral (e.g. SIV) vectors may be targeted to cells, tissues and organs throughout the body.
  • target cells for the lentiviral (e.g. SIV) vectors of the invention include liver cells, spleen cells, kidney cells, lung cells, bone marrow cells, gonad cells, cells of the central nervous system (CNS), including brain cells and/or cardiovascular cells.
  • the target cells are the cells of the liver, spleen and/or bone marrow.
  • target cells of the invention include liver cells, spleen cells and/or endothelial cells.
  • liver cells may be selected from hepatocytes, stellate fat- storing cells, Kupffer cells and/or liver endothelial cells, preferably hepatocytes.
  • spleen cells may be selected from lymphocytes (T cells, particularly helper T cells and B cells), macrophages, and/or splenic endothelial cells.
  • kidney cells may be selected from glomerulus parietal cells, glomerulus podocytes, proximal tubule brush border cells , Loop of Henle thin segment cells, thick ascending limb cells, kidney distal tubule cells, collecting duct principal cells, collecting duct intercalated cells and/or interstitial kidney cells.
  • lung cells may be selected from pulmonary endothelial cells, epithelial cells, basal cells, submucosal gland duct cells, club cells, neuroendocrine cells, bronchioalveolar stem cells, submucosal acinar cells, ionocytes, type I pneumocytes and/or type II pneumocytes.
  • bone marrow cells may be selected from both haematopoietic cells and/or stromal cells.
  • Bone marrow haematopoietic cells may be selected from myelopoietic cells (e.g. myeloblasts, promyelocytes, neutrophilic myelocytes, eosinophilic myelocytes, neutrophilic metamyelocytes, eosinophilic metamyelocytes, neutrophilic band cells, eosinophilic band cells, segmented neutrophils, segmented eosinophils, segmented basophils and/or mast cells), erythropoietic cells (e.g.
  • myelopoietic cells e.g. myeloblasts, promyelocytes, neutrophilic myelocytes, eosinophilic myelocytes, neutrophilic metamyelocytes, eosinophilic metamyelocytes, neutrophilic band
  • pronormoblasts basophilic normoblasts, polychromatic normoblasts and/or orthochromatic normoblasts
  • megakaryocytes plasma cells, reticular cells, lymphocytes and/or monocytes.
  • bone marrow haematopoietic cells include monocytes.
  • Bone marrow stromal cells may be selected from mesenchymal stem cells (MSCs), macrophages, fibroblasts, adipocytes, osteoblasts, osteoclasts, endothelial stem cells and/or endothelial cells.
  • MSCs mesenchymal stem cells
  • macrophages macrophages
  • fibroblasts fibroblasts
  • adipocytes adipocytes
  • osteoblasts osteoclasts
  • endothelial stem cells and/or endothelial cells.
  • bone marrow stromal cells include MSCs and/or macrophages.
  • gonad cells may be male or female gonad cells, and may be selected from gem cells, Sertoli cells, peritubular myoid cells, Leydig cells, interstitial macrophages, interstitial epithelial cells, germinal epithelial cells, follicular cells and/or granulosa cell.
  • cells of the CNS may be brain cells, which may optionally be selected from neurons, glial cells, astrocytes, oligodendrocytes and/or microglia.
  • cells of the cardiovascular system include red blood cells, white blood cells (e.g.
  • Target tissues or organs for the lentiviral (e.g. SIV) vectors of the invention include the liver, spleen, kidney, lung and respiratory tract, bone marrow, gonad (ovaries or testes), brain or central nervous system and/or cardiovascular system (including the heart and/or circulatory system).
  • target tissues or organs are the liver, spleen and/or vascular endothelium.
  • SIV vectors allows for any of the above mentioned cells, tissues and/or organs to be used as factories for the expression of a transgene, and hence the production of a therapeutic protein.
  • the therapeutic protein may have a therapeutic effect within the cells, tissues and/or organs to which the lentiviral (e.g. SIV) vectors are targeted.
  • a lentiviral (e.g. SIV) vector of the invention is targeted to the liver, where the transgene is expressed and the therapeutic protein produced, the therapeutic protein may have a therapeutic effect on one or more cell type of the liver.
  • the therapeutic protein having been produced by the cells of the liver, may be secreted or released into the circulatory system, and may have a therapeutic effect in the circulatory system and/or travel in the circulatory system to target cells, tissue and/or organ distinct from the liver.
  • Intravenous administration of lentiviral (e.g. SIV) vectors according to the invention potentially allows for the use of transgenes which encode proteins which may be more efficiently processed/post-translationally modified by organs/tissues other than the lungs or respiratory tract.
  • the invention allows for the use of transgene which encode proteins which may be more efficiently processed/post-translationally modified by the liver, kidneys, spleen, and/or vascular endothelium.
  • von Willebrand Factor is an example of a protein which undergoes processing in endothelial cells and requires storage vesicles specific to endothelial cells.
  • the half-life of FVIII in the circulation is severely reduced without association with soluble von Willebrand Factor, and so systemic expression of FVIII is therapeutically desirable.
  • intravenous administration of lentiviral (e.g. SIV) vectors according to the invention has the potential to allow for increased processing and/or post-translational modification compared with processing and/or post-translational modification of the same transgene by cells of the lung.
  • any increase in processing and/or post-translational modification following intravenous administration of a lentiviral (e.g. SIV) vector compared with processing and/or post-translational modification of the same transgene by cells of the lung may be an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or more compared with the dose of the same F/HN pseudotyped lentiviral (e.g. SIV) vector when administered by a respiratory (e.g. nasal) route.
  • any increase in processing and/or post-translational modification following intravenous administration of a lentiviral e.g.
  • SIV vector compared with processing and/or post-translational modification of the same transgene by cells of the lung may be an increase of at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times or more compared with processing and/or post-translational modification of the same transgene by cells of the lung.
  • any increase in processing and/or post-translational modification following intravenous administration of a lentiviral (e.g. SIV) vector is compared with processing and/or post-translational modification of the same transgene administered in the same lentiviral (e.g. SIV) vector to the lung.
  • Intravenous administration of F/HN pseudotyped lentiviral compared with processing and/or post-translational modification of the same transgene by cells of the lung.
  • SIV vectors according to the invention may allow for increased efficiency in the delivery and expression of transgenes compared with administration by a respiratory route.
  • the invention has the potential to allow for lower doses of lentiviral (e.g. SIV) vector to be administered.
  • the use of lower doses may provide advantages such as improved safety and/or reduced cost of gene therapy (through reduced manufacturing costs).
  • the decrease may be as defined above.
  • the required dose of F/HN pseudotyped lentiviral (e.g. SIV) vectors for intravenous administration according to the invention may be decreased compared with the dose of the same vector when administered by a respiratory (e.g. nasal) route.
  • the dose of an F/HN pseudotyped lentiviral (e.g. SIV) vectors for intravenous administration according to the invention may be decreased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or more compared with the dose of the same F/HN pseudotyped lentiviral (e.g. SIV) vector when administered by a respiratory (e.g. nasal) route.
  • the required dose of an F/HN pseudotyped lentiviral e.g.
  • SIV vectors for intravenous administration according to the invention may be decreased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more compared with the dose of the same F/HN pseudotyped lentiviral (e.g. SIV) vector when administered by a respiratory (e.g. nasal) route. Accordingly, as intravenous administration of F/HN pseudotyped lentiviral (e.g. SIV) vectors according to the invention may allow for a lower dose of the vector to be used compared with respiratory administration, a lentiviral (e.g.
  • SIV vector for intravenous administration may be administered at a dose of about 1.0 x 10 8 to about 1.0 x 10 9 TU/ml, typically at a dose of about 1.0 x 10 8 to about 6.0 x 10 8 TU/ml.
  • the concentration of lentiviral vector within the pharmaceutical composition is between about 2.0 x 10 8 to about 6 x 10 8 TU/ml, such as about 2.0 x 10 8 to about 4.5 x 10 8 TU/ml.
  • the dose may be given in TU/kg.
  • the disease to be treated may be chronic or acute.
  • the pharmaceutical composition of the present invention when delivered by intravenous administration to a subject may be used to deliver any transgene useful in gene therapy.
  • the pharmaceutical composition of the present invention when delivered by intravenous administration to a subject can also be used in methods of gene therapy to promote secretion of therapeutic proteins.
  • the lentiviral vector of the invention enables the use of a subject’s tissues and/or organs as “factories” to produce therapeutic protein that is then secreted and may enter the systemic circulation, where it can travel to cells/tissues of interest to elicit a therapeutic effect.
  • the production of such secreted proteins does not rely on specific disease target cells being transduced, which is a significant advantage and achieves high levels of protein expression.
  • the lentiviral vector of the invention may transduce the liver of a subject, expressing a therapeutic protein which has its therapeutic effects within the circulation.
  • the pharmaceutical compositions of the invention when delivered by intravenous administration may be used to treat or prevent a broad range of diseases including: a genetic respiratory disease, a haematological disease, an ophthalmological disease, a neurological disease, an autoimmune disease, an infectious disease or a neoplastic disease.
  • the lentiviral (e.g. SIV) vector enters the circulatory system and is able to quickly reach target cells, tissues or organs, preferably at high concentration.
  • the pharmaceutical compositions of the invention may be used to treat diseases where the therapeutic protein expressed by the transgene has its effect within the transduced cell, tissue or organ, or, at the very least, within the local area of the transduced cell, tissue or organ.
  • a lentiviral vector of the invention harbouring a functional copy of the CFTR gene may be used to ameliorate or prevent lung disease in CF patients, independent of the underlying mutation.
  • the lentiviral (e.g. SIV) vector may transduce a lung endothelial cell and elicit expression of the functional CFTR gene therein.
  • Intravenous administration of a lentiviral (e.g. SIV) vector of the invention harbouring a CFTR transgene also allows the vector to transduce the pancreatic duct epithelium and elicit functional expression of the CFTR gene therein, and hence ameliorate or prevent pancreatic disease in CF patients.
  • the pharmaceutical composition of the invention may be used to treat cystic fibrosis (CF), typically by gene therapy with a CFTR transgene as described herein, wherein the composition is administered intravenously.
  • a lentiviral (e.g. SIV) vector may comprise a transgene encoding for GM-CSF.
  • the vector may travel to and target cells in the bone marrow of a patient, where the vector may transduce bone marrow cells and drive GM-CSF expression.
  • the GM- CSF may act within the bone marrow to stimulate the production of white blood cells, particularly macrophages and eosinophils.
  • such vectors may be useful in the treatment of cancer, or following chemotherapy.
  • a lentiviral (e.g. SIV) vector may comprise a transgene encoding for BMPR2.
  • the vector may travel to and target cells in the pulmonary endothelium of a patient, where the vector may transduce endothelial cells and drive BMPR2 expression.
  • Expression of BMPR2 within the pulmonary endothelium may be useful in the treatment of pulmonary hypertension.
  • a pharmaceutical composition of the invention when administered intravenously may be used to treat Alpha-1 Antitrypsin (AAT) deficiency, typically by gene therapy with a SERPINA1 transgene as described herein.
  • AAT Alpha-1 Antitrypsin
  • AAT is a secreted anti-protease that is produced mainly in the liver and then trafficked to the lung, with smaller amounts also being produced in the lung itself.
  • the main function of AAT is to bind and neutralise/inhibit neutrophil elastase.
  • Gene therapy with AAT according to the present invention is relevant to AAT deficient patients, as well as in other lung diseases such as CF or chronic obstructive pulmonary disease (COPD), and offers the opportunity to overcome some of the problems encountered by conventional enzyme replacement therapy, providing stable, long-lasting expression in the target tissue (lung/nasal epithelium), ease of administration and unlimited availability.
  • Intravenous administration of the pharmaceutical composition of the invention facilitates the transduction of a wide range of cell types by the lentiviral vector of the invention.
  • the transduced cell, tissue or organ can then act as a factory to produce the therapeutic protein which is then efficiently processed and secreted into the systemic circulation.
  • AAT gene therapy may therefore also be beneficial in other disease indications, non-limiting examples of which include type 1 and type 2 diabetes, acute myocardial infarction, ischemic heart disease, rheumatoid arthritis, inflammatory bowel disease, transplant rejection, graft versus host (GvH) disease, multiple sclerosis, liver disease, cirrhosis, vasculitides and infections, such as bacterial and/or viral infections.
  • AAT has numerous other anti-inflammatory and tissue-protective effects, for example in pre- clinical models of diabetes, graft versus host disease and inflammatory bowel disease.
  • the production of AAT in a variety of cells, tissue or organs following transduction according to the present invention may, therefore, be more widely applicable, including to these indications.
  • Other examples of diseases that may be treated with gene therapy of a secreted protein according to the present invention include cardiovascular diseases and blood disorders, particularly blood clotting deficiencies such as haemophilia (A, B or C), von Willebrand disease and Factor VII deficiency.
  • Other blood disorders that may be treated according to the invention include blood cancers, such as leukaemia.
  • the disease to be treated may be a haematological disease, such as haemophilia and the transgene may be selected from Factor VIII, Factor V, Factor IX, Factor X and/or Factor XI.
  • diseases or disorders to be treated include, acute lung injury, Surfactant Protein B (SFTB) deficiency, Pulmonary Alveolar Proteinosis (PAP, hereditary and/or acquired), pulmonary hypertension, Chronic Obstructive Pulmonary Disease (COPD), pulmonary surfactant metabolism dysfunction 2 (SMDP3), pulmonary surfactant metabolism dysfunction 3 (SMDP3) or another surfactant deficiency, acute respiratory distress syndrome (ARDS), COVID-19, a pulmonary fibrotic disease (including idiopathic pulmonary fibrosis), a pulmonary allergic condition, asthma, lung cancer or a dysplastic change in the lungs, haemophilia and/or inflammatory, infectious, immune or metabolic conditions, such as lysosomal storage diseases or a pulmonary bacterial, viral or fungal infection, or any other lung disease or disorder.
  • SFTB Surfactant Protein B
  • PAP Pulmonary Alveolar Proteinosis
  • COPD Chronic Obstructive Pulmonary Disease
  • SMDP3 pulmonary surfact
  • compositions of the invention typically provide high expression levels of a therapeutic protein when administered intravenously to a patient.
  • Expression may be measured by any appropriate method (qualitative or quantitative, preferably quantitative), and concentrations given in any appropriate unit of measurement, for example ng/ml or nM.
  • Expression or secretion of a therapeutic protein of interest may be given in absolute terms.
  • expression or secretion of a therapeutic protein may be given in relative terms, for example relative to the expression or secretion of the corresponding endogenous (defective) gene or relative to expression of the therapeutic protein compared with expression of the therapeutic protein as achieved using the same vector administered by conventional respiratory means.
  • Expression may be measured in terms of mRNA or protein expression.
  • the expression of the therapeutic protein of the invention may be quantified relative to the endogenous protein or gene, such as the endogenous (dysfunctional) CFTR genes in terms of protein concentration, mRNA copies per cell or any other appropriate unit.
  • Expression levels of a nucleic acid encoding a therapeutic protein and/or the expression or secretion of the encoded therapeutic protein of the invention may be measured ex vivo (e.g. in the conditioned media used to culture the cells or within the cells themselves) or in vivo (e.g. in tissue of the transduced organ and/or serum/plasma) as appropriate.
  • a high and/or therapeutic expression level may therefore refer to the concentration in the tissue of the transduced organ and/or serum/plasma.
  • the invention provides pharmaceutical composition as described herein for use in a method of gene therapy, wherein said pharmaceutical composition is administered intravenously.
  • the invention provides pharmaceutical composition as described herein for use in a method of gene therapy, wherein said pharmaceutical composition is administered intravenously and wherein the gene therapy treat or prevent a genetic respiratory disease, a haematological disease, an ophthalmological disease, a neurological disease, an autoimmune disease, an infectious disease or a neoplastic disease.
  • the gene therapy may treat or prevent Alpha 1-antitrypsin Deficiency (AATD),pulmonary hypertension, acute lung injury, Surfactant Protein B (SFTB) deficiency, Pulmonary Alveolar Proteinosis (PAP, hereditary and/or acquired), Chronic Obstructive Pulmonary Disease (COPD), pulmonary surfactant metabolism dysfunction 2 (SMDP2), pulmonary surfactant metabolism dysfunction 3 (SMDP3) or another surfactant deficiency, acute respiratory distress syndrome (ARDS), COVID-19, a pulmonary fibrotic disease (including idiopathic pulmonary fibrosis), a pulmonary allergic condition, asthma, lung cancer or a dysplastic change in the lungs, haemophilia and/or inflammatory, infectious, immune or metabolic conditions, particularly congenital or inherited metabolic disorders such as lysosomal storage diseases, mucopolysaccharidoses or mitochondrial disorders, or a pulmonary bacterial infection, or any other lung disease or disorder.
  • AATD Alpha 1-antitrypsin Def
  • the invention also provides a pharmaceutical composition of the invention as described herein for use in a method of gene therapy, wherein said composition is administered intravenously and wherein the lentiviral vector integrates into the genome of a target cell (as defined herein).
  • the lentiviral (e.g. SIV) vectors of the invention are capable of driving stable transgene expression within a target cell, tissue or organ following intravenous administration.
  • stable expression is as defined herein, and typically means expression of a therapeutic gene and/or protein, preferably at high levels, for at least 2 weeks, preferably for at least 16 weeks or more, more preferably at least 24 weeks or more. Such stable expression may be achieved using a single (intravenous) administration of the lentiviral (e.g.
  • a transgene exhibits stable expression in a target cell, tissue or organ following a single (intravenous) administration of a lentiviral (e.g. SIV) vector as described herein.
  • a therapeutic indication of the invention may comprise the intravenous administration of a lentiviral (e.g. SIV) vector as a single dose or as repeated doses (as defined herein). Preferably a single dose is administered.
  • Intravenous administration of a lentiviral (e.g. SIV) vector of the invention may be by any type of intravenous administration.
  • intravenous administration may be by bolus administration, infusion and/or as a secondary i.v. administration.
  • An intravenous bolus dose (also referred to as an i.v. push) is a single dose that may be administered rapidly or over the course of a few minutes (e.g. from about 0 to 5 minutes, such as about 1 to 5 minutes, about 1 to 3 minutes).
  • a bolus of i.v. fluids (also referred to as an i.v. flush) may be administered immediately after the bolus dose of the vector to further force the vector dose into the blood stream.
  • An intravenous infusion may be used when it is desirable to maintain a steady concentration of the vector within the blood. Infusions may be either continuous (where one infusion begins immediately after completion of the preceding infusion), or intermittent (where a gap is left between completion of one infusion and beginning the next).
  • a secondary intravenous administration may be used when a lentiviral (e.g. SIV) vector as described herein is to be administered in addition to (and often concomitantly with) an additional (primary) therapeutic agent.
  • the lentiviral (e.g. SIV) vector flows through the primary therapeutic agent (e.g. through the i.v. bag holding the dose of the primary therapeutic agent to be administered). This allows for a single i.v. line to be used to administer both agents.
  • Venous access for intravenous administration of a lentiviral (e.g. SIV) vector according to the invention may be by any appropriate means, examples of which are standard in the art.
  • venous access for intravenous administration of a lentiviral (e.g. SIV) vector according to the invention may be direct venous access, peripheral cannula, a peripheral or central line, a peripherally inserted central catheter, a tunnelled line or an implantable port.
  • the invention provides a method of gene therapy, said method comprising administering a pharmaceutical composition of the invention to a patient in need thereof, wherein the composition is administered intravenously.
  • the invention provides a method of treating a disease, said method comprising administering a pharmaceutical composition of the invention to a patient in need thereof, wherein the composition is administered intravenously. Any disease described herein may be treated according to the invention.
  • the invention provides a method of treating or preventing a genetic respiratory disease, a haematological disease, an ophthalmological disease, a neurological disease, an autoimmune disease, an infectious disease or a neoplastic disease using a pharmaceutical composition of the invention, wherein the composition is administered intravenously.
  • the disease to be treated may be alpha 1-antitrypsin deficiency, haemophilia or cystic fibrosis.
  • the invention also provides pharmaceutical compositions according to the present invention for use in a method of treating a disease, wherein said composition is administered intravenously. Any disease described herein may be treated according to the invention.
  • the invention provides a pharmaceutical composition according to the present invention for use in a method of treating or preventing a genetic respiratory disease, a haematological disease, an ophthalmological disease, a neurological disease, an autoimmune disease, an infectious disease or a neoplastic disease, wherein said composition is administered intravenously.
  • the disease to be treated may be alpha 1- antitrypsin deficiency, haemophilia or cystic fibrosis.
  • the invention also provides the use of a lentiviral (e.g. SIV) vector as described herein in the manufacture of a medicament for use in gene therapy, wherein the medicament is for intravenous administration.
  • the invention also provides the use of a lentiviral (e.g.
  • the pharmaceutical composition described herein is for intravenous administration.
  • intravenous administration is intended to encompass any means of administering a pharmaceutical composition of the invention to the venous system of a subject.
  • the pharmaceutical composition may be administered by bolus administration (i.e. a single injection intravenously) or infusion administration (i.e.
  • the pharmaceutical composition according to the present invention may be formulated (e.g. formulated specifically) for intravenous administration.
  • the pharmaceutical composition may be formulated for general use (e.g. the formulation is not specific for intravenous administration and may be substantially the same as a formulation suitable for, say, intra-nasal administration).
  • the pharmaceutical composition of the invention comprises a a lentiviral vector as described herein and a pharmaceutically-acceptable carrier.
  • Non-limiting examples of pharmaceutically acceptable carriers include TSSM formulation buffer, water, saline, and phosphate- buffered saline.
  • TSSM formulation buffer is well known to the skilled person (the precise composition being available in Matet et al. 2017, Translational Research, 188: 40-57) and comprises tromethamine, sodium chloride, sucrose and mannitol.
  • the pharmaceutical composition may also be in a lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • compositions may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long-term storage.
  • a preservative such as thiomersal or sodium azide
  • the pharmaceutical composition of the invention may be administered in any dosage appropriate for achieving the desired therapeutic effect.
  • the dose to be administered may depend on factors such as the transgene to be expressed, the specific lentiviral (e.g. SIV) vector to be used, the target cells/tissue/organ and/or the disease to be treated. Appropriate dosages may be determined by a clinician or other medical practitioner using standard techniques and within the normal course of their work.
  • the concentration of lentiviral vector within the pharmaceutical composition may be about 1.0 x 10 8 to 6.0 x 10 8 TU/ml, such as between 2.0 x 10 8 to 4.5 x 10 8 TU/ml.
  • the dose of a pharmaceutical composition may be given in TU/kg.
  • Intravenous administration of the pharmaceutical composition of the invention achieves high levels of expression of the transgene. Accordingly, the pharmaceutical composition of the invention may be administered intravenously as a single dose.
  • repeated doses of the pharmaceutical composition of the invention may be administered to the subject.
  • a pharmaceutical composition of the invention may be administered intravenously at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, every other month, every 3 months, every 6 months, annually, or at longer intervals. Dosing may be continued for as long as required, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to for the lifetime of the patient to be treated
  • the frequency of the dosing can readily be determined by the skilled person.
  • the further dose of the pharmaceutical composition of the invention may be warranted when the expression levels of the transgene and/or therapeutic protein fall below a pre-determined level and/or the patients disease relapses (i.e. the patient exhibits symptoms of the disease that were previously being managed by the gene therapy).
  • Individuals receiving repeated doses of the pharmaceutical composition of the invention may receive them by different administration routes.
  • a patient undergoing gene therapy may receive a first dose of the pharmaceutical composition of the invention by intravenous administration according to the invention and a second dose by intra-nasal administration. Any two or more pharmaceutical compositions of the invention may be administered separately, sequentially or simultaneously.
  • two pharmaceutical compositions of the invention or more pharmaceutical compositions of the invention may be administered separately, simultaneously or sequentially.
  • the two pharmaceutical compositions of the invention may be administered in the same or different compositions.
  • two or more retroviral/lentiviral (e.g. SIV) vectors may be delivered in a single pharmaceutical composition of the invention.
  • the invention also provides a method of producing a pharmaceutical composition as defined herein. Said method typically comprises formulating a lentiviral (e.g. SIV) vector as defined herein with a pharmaceutically-acceptable carrier to produce the pharmaceutical composition in an intravenous formulation.
  • SEQUENCE HOMOLOGY Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D.
  • Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match- Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E.
  • % sequence identity between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences.
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues.
  • the polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4- azaphenyl-alanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.
  • an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • the non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.
  • Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci.2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989).
  • Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett.309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
  • phage display e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204
  • region-directed mutagenesis e.g., region-directed mutagenesis
  • SEQ ID NO: 15 Complementary strand to the exemplified FVIII transgene (N6)
  • Exemplified Human TRIM72 polypeptide SEQ ID NO: 23 Exemplified Human ABACA3 (ABCA3) transgene
  • SEQ ID NO: 24 Exemplified Human ABCA3 polypeptide SEQ ID NO: 25 Exemplified Human BMPR2 transgene
  • Example 2 Comparison of secreted protein levels in serum following intra-nasal and i.v. administration of SIV-F/HN vector. A long-term study (12 month duration) was carried out to investigate the expression of a secreted reporter gene, gaussia luciferase, following a single intravenous administration of SIV-F/HN vector harbouring the gaussia luciferase transgene.
  • transgene expression in serum 52 weeks post-dosing (gaussia luciferase; expressed as relative light units (RLU)) following intravenous delivery was increased compared to intra-nasal delivery at the two higher doses.
  • Example 3 Tissue distribution following i.v. delivery of SIV-F/HN vector.
  • a tissue distribution study was carried out to investigate the expression of reporter gene, firefly luciferase, in different tissues following administration of the SIV-F/HN vector harbouring the firefly luciferase transgene by intra-nasal vs. intravenous administration.
  • luciferase expressed as logarithm relative light units per mg of protein (log(RLU/mg protein)) was widely distributed when intravenously administered compared to highly localised expression following intra-nasal administration.
  • significantly higher levels of luciferase protein were expressed in the liver and spleen of intravenously treated animals versus intra-nasally treated animals.
  • Example 4 Expression of AAT following i.v. delivery
  • Wild type C57BL/6J mice received a single dose of SIV-F/HN vector harbouring a human SERPINA1 transgene (vGM109) by intravenous injection.
  • the vector was administered at a concentration of 2x10 8 TU/mouse and control mice delivered TSSM.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une composition pharmaceutique comprenant un vecteur lentiviral et un support pharmaceutiquement acceptable, pour une administration intraveineuse, et des utilisations médicales et des méthodes de traitement par administration intraveineuse de ladite composition pharmaceutique.
PCT/GB2022/050928 2021-04-13 2022-04-13 Administration de vecteurs de thérapie génique WO2022219332A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP22719987.4A EP4323532A1 (fr) 2021-04-13 2022-04-13 Administration de vecteurs de thérapie génique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB2105276.6A GB202105276D0 (en) 2021-04-13 2021-04-13 Delivery of gene therapy vectors
GB2105276.6 2021-04-13

Publications (1)

Publication Number Publication Date
WO2022219332A1 true WO2022219332A1 (fr) 2022-10-20

Family

ID=75949506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2022/050928 WO2022219332A1 (fr) 2021-04-13 2022-04-13 Administration de vecteurs de thérapie génique

Country Status (3)

Country Link
EP (1) EP4323532A1 (fr)
GB (1) GB202105276D0 (fr)
WO (1) WO2022219332A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006204A1 (fr) 1990-09-28 1992-04-16 Ixsys, Inc. Banques de recepteurs heteromeres a expression en surface
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
EP1291419A1 (fr) * 2000-06-01 2003-03-12 Dnavec Research Inc. Vecteur de retrovirus de pseudo-type contenant une proteine de membrane possedant une activite d'hemagglutinine
WO2015177501A1 (fr) * 2014-05-21 2015-11-26 Imperial Innovations Limited Vecteurs lentiviraux

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
WO1992006204A1 (fr) 1990-09-28 1992-04-16 Ixsys, Inc. Banques de recepteurs heteromeres a expression en surface
EP1291419A1 (fr) * 2000-06-01 2003-03-12 Dnavec Research Inc. Vecteur de retrovirus de pseudo-type contenant une proteine de membrane possedant une activite d'hemagglutinine
WO2015177501A1 (fr) * 2014-05-21 2015-11-26 Imperial Innovations Limited Vecteurs lentiviraux

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
"Wiley Series in Probability and Statistics", July 2002, JOHN WILEY & SONS, article "Bayesian Methods for Nonlinear Classification and Regression"
ALTSCHUL ET AL., BULL. MATH. BIO., vol. 48, 1986, pages 603 - 16
BOWIESAUER, PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 2152 - 6
C. E. LAWRENCE ET AL.: "Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment", SCIENCE, vol. 262, no. 5131, 1993, pages 208 - 214, XP001152872, DOI: 10.1126/science.8211139
CHUNG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 10145 - 9
CHUNG ET AL., SCIENCE, vol. 259, 1993, pages 806 - 9
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 5
DERBYSHIRE ET AL., GENE, vol. 46, 1986, pages 145
ELLMAN ET AL., METHODS ENZYMOL., vol. 202, 1991, pages 301
ERIC DEPIEREUXERNEST FEYTMANS: "Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences", CABIOS, vol. 8, no. 5, 1992
GHOSE, ARUP K. ET AL.: "Software, Tools, and Applications in Drug Discovery", article "Combinatorial Library Design and Evaluation Principles"
HENIKOFFHENIKOFF, PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 10915 - 19
IVO VAN WALLE ET AL.: "Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences", BIOINFORMATICS, vol. 20, no. 9, 2004, pages 1428 - 1435
KANDEL, ABRAHAM ET AL.: "Computer-Assisted Reasoning in Cluster Analysis", 11 May 1995, PRENTICE HALL PTR
KOIDE ET AL., BIOCHEM., vol. 33, 1994, pages 7470 - 6
KRZANOWSKI, WOJTEK: "Oxford Statistical Science Series, No 22 (Paper", December 2000, OXFORD UNIVERSITY PRESS, article "Principles of Multivariate Analysis: A User's Perspective"
LOWMAN ET AL., BIOCHEM., vol. 30, 1991, pages 10832 - 7
MATET ET AL., TRANSLATIONAL RESEARCH, vol. 188, 2017, pages 40 - 57
NER ET AL., DNA, vol. 7, 1988, pages 127
NORMAN ET AL.: "Applied Regression Analysis", April 1998, WILEY-INTERSCIENCE
OSAMU GOTOH: "Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments", J. MOL. BIOL., vol. 264, no. 4, 1996, pages 823 - 838
PRINGLE, J. MOL. MED. BERL., vol. 90, no. 12, 2012, pages 1487 - 96
REIDHAAR-OLSONSAUER, SCIENCE, vol. 241, 1988, pages 53 - 7
ROBERTSON ET AL., J. AM. CHEM. SOC., vol. 113, 1991, pages 2722
SMITH ET AL., J. MOL. BIOL., vol. 224, 1992, pages 899 - 904
TURCATTI ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 19991 - 8
VOS ET AL., SCIENCE, vol. 255, 1992, pages 306 - 12
WITTEN, IAN H. ET AL.: "Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations", 11 October 1999, MORGAN KAUFMANN
WLODAVER ET AL., FEBS LETT, vol. 309, 1992, pages 59 - 64
WOLD ET AL.: "Chemometrics-Mathematics and Statistics in Chemistry", 1984, D. REIDEL PUBLISHING COMPANY, article "Multivariate data analysis in chemistry"
WYNNRICHARDS, PROTEIN SCI, vol. 2, 1993, pages 395 - 403

Also Published As

Publication number Publication date
EP4323532A1 (fr) 2024-02-21
GB202105276D0 (en) 2021-05-26

Similar Documents

Publication Publication Date Title
JP2000069982A (ja) ヒト血管内皮増殖因子２
CA2220912A1 (fr) Facteur de croissance humain du type ccn
JP2008542418A (ja) 自己免疫疾患及び移植片拒絶反応を治療するためのＩｄｅＳプロテイナーゼ（化膿連鎖球菌由来）の使用
JP2003009889A (ja) インターロイキン−１β転換酵素様アポトーシスプロテアーゼ−３および４
JP2011172588A (ja) Ｄｐ７５をコードするｄｎａおよびその使用のためのプロセス
JP2021511030A (ja) Ｃ３ｂ結合ポリペプチド
WO2022219336A1 (fr) Thérapie cellulaire
US20030045489A1 (en) Methods for modulating angiogenesis
JP2000508894A (ja) 細胞外／上皮増殖因子ｈｃａｂａ５８ｘ
CN112888777A (zh) 用于造血细胞的基因修饰的方法
EP4323516A1 (fr) Peptides signal
US6165794A (en) Suppression of proteolytic activity by dysfunctional protease formation
WO2022219332A1 (fr) Administration de vecteurs de thérapie génique
JP2019530705A (ja) 腎がんの治療のための方法及び医薬組成物
US7074399B2 (en) Treatment of inflammation with p20
CZ180895A3 (en) Polynucleotide sequence, dna vector, host, expressive system, polypeptide, system, use of the polypeptide, medicament, antibody, purification procedure and hybridoma
CA3174156A1 (fr) Acides nucleiques d'insuline et de glucokinase modifies pour le traitement du diabete
CA3208936A1 (fr) Vecteurs retroviraux
US20240024515A1 (en) Combination treatment
JPH11506908A (ja) トランスフォーミング増殖因子αＨＩＩ
JP2000508531A (ja) 細胞外／上皮増殖因子様タンパク質
WO2023118871A1 (fr) Vecteurs lentiviraux pseudotypés
JP2001517064A (ja) トランスフォーミング増殖因子アルファｈｉ
JPH11507502A (ja) ヒトクリプチン増殖因子
WO2003014340A2 (fr) Gene et proteine associes aux histones desacetylases

Legal Events

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

Ref document number: 22719987

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2022719987

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022719987

Country of ref document: EP

Effective date: 20231113