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WO2024062259A1 - Vecteur rétroviral comprenant un rre inséré dans un intron - Google Patents

Vecteur rétroviral comprenant un rre inséré dans un intron Download PDF

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Publication number
WO2024062259A1
WO2024062259A1 PCT/GB2023/052462 GB2023052462W WO2024062259A1 WO 2024062259 A1 WO2024062259 A1 WO 2024062259A1 GB 2023052462 W GB2023052462 W GB 2023052462W WO 2024062259 A1 WO2024062259 A1 WO 2024062259A1
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Prior art keywords
retroviral
intron
rre
siv
vector
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Inventor
Jack Hickmott
Uta Griesenbach
Eric Alton
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Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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Priority to CA3268474A priority Critical patent/CA3268474A1/fr
Priority to JP2025517255A priority patent/JP2025533754A/ja
Priority to EP23783499.9A priority patent/EP4590696A1/fr
Priority to AU2023345979A priority patent/AU2023345979A1/en
Publication of WO2024062259A1 publication Critical patent/WO2024062259A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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
    • 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
    • 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
    • C12N2740/15045—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material
    • 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
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
    • C12N2840/445—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor for trans-splicing, e.g. polypyrimidine tract, branch point splicing

Definitions

  • the present invention relates to retroviral vectors modified to improve transgene expression.
  • the invention relates to retroviral vectors lacking an endogenous Rev response element
  • RRE and comprising an intron, particularly a chimeric intron into which an RRE has been inserted, as well as methods of production and uses thereof.
  • nucleic acids as medicine, or gene therapy, is a promising new treatment modality.
  • the present inventors have previously developed a lentiviral vector, which has been pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, comprising a promoter and a transgene.
  • the backbone of the vector is from a simian immunodeficiency virus (SIV), such as SIV1 or African green monkey SIV (SIV-AGM).
  • SIV-AGM African green monkey SIV
  • the backbone of a viral vector of the invention is from SIV-AGM.
  • the HN and F proteins function, respectively, to attach to sialic acids and mediate cell fusion for vector entry to target cells.
  • the present inventors discovered that this specifically F/HN-pseudotyped lentiviral vector can efficiently transduce airway epithelium, resulting in transgene expression sustained for periods beyond the proposed lifespan of airway epithelial cells. Importantly, the present inventors also found that re-administration does not result in a loss of efficacy. These features make the vectors of the present invention attractive candidates for treating diseases via their use in expressing therapeutic proteins: (i) within the cells of the respiratory tract; (ii) secreted into the lumen of the respiratory tract; and (iii) secreted into the circulatory system. However, even using this state-of-the-art platform technology, the levels of transgene expressed are at the lower predicted threshold required for clinical efficacy.
  • AAV vectors adeno-associated viral vectors
  • RNA genomes are subject to the same intron removal steps that mRNA is, such that introns are removed from RNA genomes during manufacturing, reducing the amount of protein an RNA viral vector can make.
  • the present inventors have for the first time demonstrated that it is possible to introduce an intron into a lentiviral genome, and that the introduction of such an intron can increase transgene expression.
  • the inventors have found that by removal of the endogenous Rev response element of a Simian Immunodeficiency Virus (SIV vector) pseudotyped with a VSV-G or F/HN envelope, and introduction of a ⁇ -globin/IgG intron with a precisely inserted SIV RRV into the SIV.VSV-G or SIV.F/HN genome increased AAT transgene expression by 686-fold or 501-fold, respectively, compared with corresponding vectors lacking an intron.
  • SIV vector Simian Immunodeficiency Virus
  • the inventors’ innovative approach has the potential to provide several clinically important advantages: (i) allowing gene therapies to more easily reach the required therapeutic window, making them more efficacious; (ii) lowering the dose of a gene therapy agent required for administration to a patient, making the gene therapy safer; and/or (iii) lowering the production costs (as less vector is needed per patient), solving a major challenge for clinical trials, & pharmaceutical companies, and health care providers.
  • the present invention provides a retroviral vector comprising an intron; wherein: (a) the endogenous Rev response element (RRE) of the retroviral genome is deleted; and (b) a retroviral RRE is inserted into the intron within 100 bp 5’ of the splice acceptor’s branch site.
  • the retroviral RRE may be inserted within 20 bp 5’ of the splice acceptor’s branch site.
  • the intron may be a chimeric intron, optionally selected from a ⁇ -globin/IgG chimeric intron or a chimeric intron from the CAGGS promoter.
  • the intron may be a viral intron, optionally selected from SV40 intron, CMV Intron A and adenovirus tripartite leader sequence intron.
  • the invention also preferably provides a retroviral vector comprising a chimeric intron; wherein: (a) the endogenous Rev response element (RRE) of the retroviral genome is deleted; and (b) a retroviral RRE is inserted into the chimeric intron.
  • the retroviral RRE inserted into the intron may be the endogenous RRE of the retroviral genome.
  • the RRE may be a Simian immunodeficiency virus (SIV) RRE.
  • the RRE may comprise or consist of a nucleic acid sequence having at least 90% identity to SEQ ID NO: 1.
  • the intron may be less than 1,000 bp in length, preferably less than 800 bp in length.
  • the chimeric intron may be a ⁇ -globin/IgG chimeric intron or a chimeric intron from the CAGGS promoter.
  • the chimeric intron may be a ⁇ - globin/IgG chimeric intron and the RRE inserted between (i) a splice donor site comprising or consisting of a nucleic acid sequence of TGAGTTTAAGGTAAGT (SEQ ID NO: 2); and (ii) a splice acceptor site comprising or consisting of a nucleic acid sequence of CTCTCCACAG (SEQ ID NO: 3).
  • the ⁇ - globin/IgG chimeric intron may comprise or consist of a nucleic acid sequence having at least 90% identity to SEQ ID NO: 4.
  • the intron may be a ⁇ -globin/IgG chimeric intron and the RRE an SIV RRE, and optionally wherein the chimeric intron comprising the RRE may comprise or consist of a nucleic acid sequence having at least 90% identity to SEQ ID NO: 5.
  • the intron may be between a promoter and a transgene operably linked to said promoter, wherein optionally the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, elongation factor 1a (EF1a) promoter, and a hybrid human CMV enhancer/EF1a (hCEF) promoter, preferably a hCEF promoter.
  • CMV cytomegalovirus
  • EF1a elongation factor 1a
  • hCEF hybrid human CMV enhancer/EF1a
  • the transgene may encode a therapeutic protein, wherein optionally said therapeutic protein is selected from: (a) a secreted therapeutic protein, optionally Alpha-1 Antitrypsin (AAT), Factor VIII, Surfactant Protein B (SFTPB), ADAMTS13, Factor VII, Factor IX, Factor X, Factor XI, von Willebrand Factor, Granulocyte- Macrophage Colony-Stimulating Factor (GM-CSF), Surfactant Protein C (SP-C), decorin, an anti- inflammatory protein and a monoclonal antibody against an infectious agent; or (b) CFTR, ABCA3, DNAH5, DNAH11, DNAI1, DNAI2, CSF2RA, CSF2RB and TRIM-72.
  • AAT Alpha-1 Antitrypsin
  • SFTPB Surfactant Protein B
  • ADAMTS13 Factor VII, Factor IX, Factor X, Factor XI, von Willebrand Factor, Granulocyte- Macrophage Colon
  • Any retroviral vector of the invention may be a lentiviral vector.
  • Said lentiviral vector may be selected from the group consisting of a Human immunodeficiency virus (HIV) vector, a Simian immunodeficiency virus (SIV) vector, a Feline immunodeficiency virus (FIV) vector, an Equine infectious anaemia virus (EIAV) vector, and a Visna/maedi virus vector.
  • Any retroviral vector of the invention may be pseudotyped with haemagglutinin- neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus or G glycoprotein from Vesicular Stomatitis Virus (G-VSV).
  • transgene expression may be increased by at least about 2-fold, preferably at least about 5-fold, more preferably at least about 10-fold compared with a corresponding vector which lacks a intron into which a retroviral RRE has been inserted.
  • the invention also provides a nucleic acid comprising or consisting of a intron into which a retroviral RRE has been inserted, wherein optionally (i) the intron; and/or (ii) the RRE are as defined herein.
  • the invention further provides a plasmid comprising a nucleic acid of the invention.
  • the invention also provides a retroviral vector, nucleic acid and/or plasmid of the invention, which is codon-optimised.
  • the invention further provides a composition comprising a retroviral vector, nucleic acid and/or plasmid of the invention, and a pharmaceutically-acceptable carrier.
  • the invention also provides a host cell comprising a retroviral vector, nucleic acid and/or plasmid of the invention.
  • the invention also provides a retroviral vector, nucleic acid plasmid or composition according as described herein for use in a method of treatment.
  • the invention also provides a method of producing a retroviral vector, said method comprising the following steps: (a) growing cells in suspension; (b) transfecting the cells with one or more plasmids; (c) adding a nuclease; (d) harvesting the lentivirus; (e) adding trypsin; and (f) purification; wherein the one or more plasmids comprises a vector genome plasmid which comprises a nucleic acid of the invention, and optionally (i) a promoter of the invention and/or (ii) a transgene of the invention.
  • the invention also provides a method of differentiating between a retroviral vector and a transgene expressed by said retroviral vector, said method comprising the steps of: (a1) transfecting cells with a retroviral vector of the invention; (b1) culturing the cells to allow transgene expression by the retrovirus; and (c1) quantifying RNA within the cells; or (a2) quantifying RNA within cells of a sample obtained from a patient who has undergone treatment with a retroviral vector, nucleic acid, plasmid or composition of the invention; wherein: (i) the amount of RNA comprising the chimeric intron into which a retroviral RRE has been inserted corresponds to the copy number of the retroviral vector; and (ii) the amount of RNA lacking the chimeric intron into which a retroviral RRE has been inserted corresponds to the amount of transgene mRNA; wherein optionally RNA is quantified by a PCR-based or in situ hybridisation-based assay.
  • FIG. 1 Rev response element (RRE) intron created by inserting rSIV RRE into a chimeric intron.
  • RRE Rev response element
  • A Schematic of the chimeric intron which is composed of the splice donor from an intron in Hemoglobin subunit B (black), and the splice acceptor from an intron in immunoglobulin gamma (grey).
  • B Schematic of the RRE intron, where the rSIV RRE (light grey) was inserted between the splice donor and splice acceptor.
  • Figure 2 A-H show schematic drawings of exemplary plasmids used for production of the vectors of the invention.
  • A Shows a schematic of an intron containing lentiviral vector genome plasmid (pDNA1) encoding an Alpha-1-Antitrypsin transgene. An intron has been inserted between the promoter (hCEF) and the RRE has been moved to within the intron.
  • B Shows a schematic of a plasmid encoding codon optimized SIV Gag and Pol (pDNA2a) for lentiviral production.
  • C Shows a schematic of a plasmid encoding SIV Gag and Pol (pDNA2a) for lentiviral production.
  • D Shows a schematic of a plasmid encoding SIV Rev for lentiviral production (pDNA2b).
  • (E) Shows a schematic of a plasmid encoding the fusion protein from Sendai virus (pDNA3a) for lentiviral production.
  • (F) Shows a schematic of a plasmid encoding the hemagglutinin-neuraminidase protein from Sendai virus (pDNA3b) for lentiviral production.
  • (G) Shows a schematic of a no intron lentiviral vector genome plasmid encoding an Alpha- 1-Antitrypsin transgene (pDNA1).
  • the RRE element is 5 ⁇ of the promoter (hCEF) between the Partial GAG and cPPT sequences.
  • FIG. 1 Shows a schematic of a plasmid encoding the VSV glycoprotein (pDNA3) for lentiviral production.
  • Figure 3 RRE Intron enhances AAT expression 10.6x.
  • Neg – negative control Each dot represents a different well of transduced HEK293T cells. A Mann-Whitney test was used for statistical analysis.
  • Figure 4 RRE Intron is correctly spliced in HEK293T cells.
  • DNA (A) and RNA (B) was extracted from HEK293Tcells transfected with plasmids encoding an Alpha-1-Antitrypsin (AAT) transgene without (pGM407 - No Intron) or with (pGM991 - Intron) the RRE intron.
  • AAT Alpha-1-Antitrypsin
  • B Reverse Transcriptase PCR of RNA extracted from transfected cells verified that the RRE intron was spliced during mRNA maturation.
  • Figure 5 RRE intron is packaged into rSIV.VSV-G lentivirus.
  • DNA was extracted from HEK293T cells transduced with rSIV.VSV-G lentivirus expressing AAT without (vGM290) and with (vGM291) an RRE intron.
  • NTC Non-transduced cells
  • PCR of the resulting DNA using primers that bind to either side of the intron reveal that the intron was packaged into vGM291, producing an 1127 bp product, whereas in the absence of an intron, vGM290 produces a smaller 371 bp product.
  • a no template control (n) as well as the lentiviral transfer plasmids for vGM290 (pGM407) and vGM291 (pGM991) were included.
  • FIG. 6 RRE intron is successfully spliced by transduced HEK293T cells.
  • RNA was extracted from HEK293T cells transduced with rSIV.VSV-G lentivirus expressing AAT without (vGM290) and with (vGM291) an RRE intron.
  • NTC Non-transduced cells
  • RT-PCR of the resulting RNA using primers that bind to either side of the intron reveal that the intron was spliced during expression of vGM291, producing the same 277 bp produced cells transduced with vGM290.
  • FIG. 7 A no template control (n) as well as the lentiviral transfer plasmids for vGM290 (pGM407) and vGM291 (pGM991) were included.
  • Figure 7 RRE Intron enhances AAT expression 686-fold in HEK293T cells.
  • HEK293T cells were transduced with rSIV.VSV-G lentivirus expressing AAT without (No Intron) and with (Intron) an RRE intron.
  • NTC Non- transduced cells
  • FIG. 8 An RRE Intron increases transgene transcription in HEK293T cells.
  • RT-ddPCR was performed on RNA extracted from transfected HEK293T cells. Inclusion of the intron increased the amount of mRNA (WPRE copies). WPRE transcript copies were standardized to the house keeping gene Beta-2-Microglobulin (B2M) (B). Inclusion of an RRE intron increases the amount of mRNA produced per plasmid copy (quantified by ddPCR from DNA extracted from transfected HEK293T cells). Each dot represents a different well of transduced HEK293T cells. Statistical analysis was performed with a Mann-Whitney test.
  • FIG. 9 RRE Intron enhances AAT expression 501-fold in HEK293T cells.
  • HEK293T cells were transduced with rSIV.F/HN lentivirus expressing AAT without (No Intron) and with (Intron) an RRE intron.
  • NTC Non-transduced cells
  • “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term “about” shall be understood herein as plus or minus ( ⁇ ) 5%, preferably ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.1%, of the numerical value of the number with which it is being used.
  • 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.
  • a "vector” or “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo.
  • a vector can be a linear or a circular molecule.
  • a vector of the invention may be viral or non-viral.
  • the terms "viral vector”, “retroviral vector” and “retroviral F/HN vector” are used interchangeably to mean a retroviral vector comprising a retroviral RNA sequence and pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise stated.
  • the terms “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.
  • intron refers to a nucleic acid sequence within a gene that is located between exons. Introns are transcribed along with the exons but are removed from the primary gene transcript by RNA splicing to leave mature mRNA.
  • Rev regulatory of virion
  • Rev is a trans-acting nuclear protein whose functional expression are required for retroviral replication. Specifically, the rev gene products are required for processing and translation of the gag and env mRNAs, and thus rev regulates the expression of the viral structural proteins.
  • the term “Rev-responsive element” (RRE) refers to a cis-acting anti-repression sequence in env, which is responsive to the rev gene product. mRNAs that contain an RRE can be exported from the nucleus to the cytoplasm for translation and virion packaging.
  • RRE and “RRE sequence” are used interchangeably herein.
  • plasmid refers to a common type of non-viral vector.
  • a plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.
  • a plasmid is circular and may be double-stranded.
  • nucleic acid cassette refers to mean a nucleic acid molecule that is capable of directing transcription.
  • a nucleic acid cassette includes, at the least, a promoter or a structure functionally equivalent to a promoter and a nucleic acid sequence to be transcribed.
  • a nucleic acid cassette includes, at the least, a promoter or a structure functionally equivalent to a promoter and a nucleic acid sequence encoding a protein of interest.
  • a nucleic acid cassette includes, at the least, a promoter or a structure functionally equivalent to a promoter, a nucleic acid sequence encoding a signal peptide and a nucleic acid encoding a therapeutic protein.
  • a nucleic acid cassette may include additional elements, such as an enhancer, and/or a transcription termination signal.
  • signal peptide As used herein the terms “signal peptide”, “signal sequence”, “targeting sequence”, “leader sequence” and “secretory signal” are used interchangeably to mean heterogenous peptide sequences that are found at the N-terminus of secreted proteins that are instrumental in initiating the secretion process.
  • signal peptides are found in proteins that are targeted to the endoplasmic reticulum and eventually destined to be either secreted or retained in the cell membrane of the cell, particularly as single-pass membrane proteins. Signal peptides are typically removed to produce the mature form of the protein.
  • Signal peptides are normally short peptides, typically about 5 to about 40 amino acids in length, such as about 5 to about 35, or about 10 to about 35 amino acids in length, preferably about 10 to about 30 or about 15 to about 30 amino acids in length.
  • a signal peptide may comprise a core of hydrophobic amino acids, said core typically being about 4 to about 20, such as about 5 to about 20, about 5 to about 16 or about 5 to about 15 amino acids in length).
  • a signal peptide is typically present at the N-terminus of a protein.
  • 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.
  • RRE Rev response element
  • a wildtype/unmodified vector will comprise an RRE within its genome, typically at a defined standard location.
  • Viral vectors of the present invention typically have genomes which lack their endogenous RRE.
  • the endogenous RRE may be inserted into an intron which is then itself introduced into the retroviral/lentiviral genome.
  • An exogenous RRE is from a different virus to the viral vector of the invention.
  • the viral vector may be an HIV vector, and the RRE may be a SIV RRE.
  • 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) is a biological readout of the number of host cells that get transduced under certain tissue culture/virus dilutions conditions, and is a measure of the number of “active” virus particles.
  • 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. Assumptions are then made that 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.
  • 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.
  • Protein and polypeptide are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • the terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing.
  • nucleic acid 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.
  • the terms “transgene” and “gene” are also used interchangeably and both terms encompass fragments or variants thereof encoding the target protein.
  • the 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.
  • 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.
  • the term 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.
  • variant amino acid sequences may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.
  • 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 “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.
  • Retroviral and Lentiviral Vectors The invention relates to a retroviral/lentiviral (e.g. SIV) vector.
  • Retroviral/lentiviral vectors of the invention can integrate into the genome of transduced cells and lead to long-lasting expression.
  • the term “retrovirus” refers to any member of the Retroviridae family of RNA viruses that encode the enzyme reverse transcriptase.
  • the term “lentivirus” refers to a family of retroviruses.
  • retroviruses suitable for use in the present invention include gamma retroviruses such as murine leukaemia virus (MLV) and feline leukaemia virus (FLV).
  • 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
  • HAV Human immunodeficiency virus
  • FV Feline immunodeficiency virus
  • EIAV Equine infectious anaemia virus
  • Visna/maedi virus lentiviral vectors and the production thereof.
  • a particularly preferred lentiviral vector is an SIV vector (including all strains and subtypes), such as a SIV-AGM (originally isolated from African green monkeys, Cercopithecus aethiops).
  • the invention relates to HIV vectors.
  • the retroviral/lentiviral e.g.
  • SIV vectors of the present invention are typically pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, or with G glycoprotein from Vesicular Stomatitis Virus (referred to as VSV-G or G-VSV).
  • HN hemagglutinin-neuraminidase
  • F fusion proteins from a respiratory paramyxovirus
  • VSV-G or G-VSV G glycoprotein from Vesicular Stomatitis Virus
  • the lentiviral (e.g. SIV) vectors of the present invention are pseudotyped with HN and F from a respiratory paramyxovirus.
  • the respiratory paramyxovirus is a Sendai virus (murine parainfluenza virus type 1).
  • the retroviral/lentiviral e.g.
  • SIV vectors of the present invention may be pseudotyped with proteins from another virus, provided that the pseudotyping proteins do not negatively impact the manufactured titre of the vector (or even result in an increased titre of the vector) and/or transgene expression (or even result in increased transgene expression).
  • Non-limiting examples of other proteins that may be used to pseudotype retroviral/lentiviral (e.g. SIV) vectors of the present invention include severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or modified forms thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • VSV-G and SARS-Cov2 spike protein used for pseudotyping are as those described in UK Patent Application Nos. 2118685.3 and International Application No. PCT/GB2022/050933, each of which is herein incorporated by reference in its entirety.
  • a retroviral/lentiviral (e.g. SIV) vector for use according to the invention may be integrase- competent (IC).
  • the lentiviral (e.g. SIV) vector may be integrase-deficient (ID).
  • Viral vectors of the invention, particularly retroviral/lentiviral (e.g. SIV) vectors as described herein may transduce one or more cell types as described herein to achieve long term transgene expression.
  • the retroviral/lentiviral (e.g. SIV) vectors of the present invention enable high levels of transgene expression.
  • retroviral/lentiviral (e.g. SIV) vectors of the present invention typically result in high levels (therapeutic levels) of expression of a therapeutic protein.
  • the nucleic acid sequence encoding a therapeutic protein to be included in a viral vector of the invention, particularly a retroviral/lentiviral (e.g. SIV) 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 genome of the retroviral/lentiviral (e.g. SIV) vector may be fully or partially CpG-depleted (or CpG-fee) and/or codon-optimised.
  • Retroviral/lentiviral e.g.
  • SIV vectors such as those of 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.
  • SIV stem/progenitor cells.
  • these cell types with regenerative capacity have been identified as responsible for maintaining specific cell lineages in the conducting airways and alveoli. These include basal cells and submucosal gland duct cells in the upper airways, club cells and neuroendocrine cells in the bronchiolar airways, bronchioalveolar stem cells in the terminal bronchioles and type II pneumocytes in the alveoli. Therefore, and without being bound by theory, it is believed that said retroviral/lentiviral (e.g.
  • SIV vectors bring about long term gene expression of the transgene of interest by introducing the transgene into one or more long-lived airway epithelial cells or cell types, such as basal cells and submucosal gland duct cells in the upper airways, club cells and neuroendocrine cells in the bronchiolar airways, bronchioalveolar stem cells in the terminal bronchioles type II pneumocytes in the alveoli, submucosal acinar cells, ionocytes, and type I pneumocytes.
  • retroviral/lentiviral e.g. SIV
  • modified retroviral/lentiviral e.g.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention may transduce one or more cells or cell lines with regenerative potential within the lung (including the airways and respiratory tract) to achieve long term gene expression.
  • the retroviral/lentiviral (e.g. SIV) vectors may transduce basal cells, such as those in the upper airways/respiratory tract. Basal cells have a central role in processes of epithelial maintenance and repair following injury.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention may be used to transduce isolated and expanded stem/progenitor cells ex vivo prior administration to a patient.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention are used to transduce cells within the lung (or airways/respiratory tract) in vivo.
  • the retroviral/lentiviral (e.g. SIV) are used to transduce cells within the lung (or airways/respiratory tract) in vivo.
  • SIV vectors of the invention demonstrate remarkable resistance to shear forces with only modest reduction in transduction ability when passaged through clinically- relevant delivery devices such as bronchoscopes, spray bottles and nebulisers.
  • the retroviral/lentiviral (e.g. SIV) vectors of the present invention enable high levels of transgene expression, resulting in high levels (therapeutic levels) of expression of a therapeutic protein.
  • the retroviral/lentiviral (e.g. SIV) vectors of the present invention typically provide high expression levels of a transgene when administered to a patient.
  • high expression and therapeutic expression are used interchangeably herein.
  • 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 in the lung tissue, epithelial lining fluid and/or serum/plasma as appropriate.
  • a high and/or therapeutic expression level may therefore refer to the concentration in the lung, epithelial lining fluid and/or serum/plasma.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention enables long-term transgene expression, resulting in long-term expression of a therapeutic protein.
  • the phrases “long-term expression”, “sustained expression”, “long-lasting expression” and “persistent expression” are used interchangeably.
  • the retroviral/lentiviral e.g.
  • SIV vectors of the present invention enable long-term transgene expression, resulting in long-term expression of a therapeutic protein, particularly by airway cells, as described herein.
  • Long-term expression means expression of a therapeutic gene and/or protein, preferably at therapeutic levels, for at least 45 days, at least 60 days, at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 730 days or more.
  • long-term expression means expression for at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days or more, more preferably at least 360 days, at least 450 days, at least 720 days or more.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may drive (increased) long- lasting expression of a therapeutic protein in an airway cell in vivo in a patient.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention drives expression of a therapeutic protein in an airway cell for at least 45 days, more preferably at least 90 days.
  • Repeated doses may be administered twice-daily, daily, twice-weekly, weekly, monthly, every two months, every three months, every four months, every six months, yearly, 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.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention exhibit enhanced expression of the therapeutic protein. Accordingly, the retroviral/lentiviral (e.g. SIV) vectors of the invention are capable of producing long-lasting, repeatable, high-level expression, particularly in airway cells, without inducing an undue immune response.
  • the invention relates to F/HN retroviral/lentiviral vectors comprising a promoter and a transgene, particularly SIV F/HN vectors.
  • the viral vectors of the invention may be made using any suitable process known in the art.
  • retroviral/lentiviral (e.g. SIV) vectors of the invention may be made using the methods disclosed in International Application No. PCT/GB2022/050524 which is herein incorporated by reference in its entirety.
  • the viral vectors of the invention, particularly 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).
  • cPPT central polypurine tract
  • WPRE Woodchuck hepatitis virus posttranscriptional regulatory elements
  • a retroviral/lentiviral (e.g. SIV) vector of the invention has been modified to (i) delete the endogenous RRE as described herein; and (ii) to introduce one or more intron into which a retroviral/lentiviral (e.g. SIV) RRE has been inserted.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention has a genome that has been modified to (i) delete the endogenous RRE as described herein; and (ii) to introduce one or more intron into which a retroviral/lentiviral (e.g. SIV) RRE has been inserted.
  • any reference herein to a retroviral/lentiviral (e.g. SIV) vector of the invention which comprises an intron with an RRE inserted into the intron applies equally and without reservation to a retroviral/lentiviral (e.g. SIV) vector of the invention genome which comprises an intron with an RRE inserted into the intron.
  • typically an intron comprising an RRE which is introduced into a retroviral/lentiviral (e.g. SIV) vector of the invention is appropriately spliced by target cells, aiding in the maturation of a stable mRNA molecule. This results in an increased expression of the coding regions of the retroviral/lentiviral (e.g. SIV) genome, including the transgene.
  • an intron comprising an RRE within retroviral/lentiviral (e.g. SIV) vector results in increased expression of the transgene, which may encode a therapeutic protein.
  • the introduction of an intron comprising an RRE into a retroviral/lentiviral (e.g. SIV) vector may increase expression of the therapeutic protein compared with expression of the therapeutic protein from a corresponding retroviral/lentiviral (e.g. SIV) vector without the RRE-comprising intron.
  • a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an RRE-comprising intron typically exhibits increased transgene expression compared with transgene expression from a corresponding retroviral/lentiviral (e.g. SIV) vector lacking said RRE-comprising intron.
  • a retroviral/lentiviral vector of the invention comprising an AAT transgene (SERPINA1) and ⁇ - globulin/IgG chimeric intron with an inserted RRE (e.g. the RRE-comprising ⁇ -globulin/IgG chimeric intron of SEQ ID NO: 5) may increase AAT expression compared with a corresponding retroviral/lentiviral (e.g.
  • SIV vector which comprises the AAT transgene but which lacks the ⁇ - globulin/IgG chimeric intron with an inserted RRE (e.g. the RRE-comprising ⁇ -globulin/IgG chimeric intron of SEQ ID NO: 5).
  • RRE e.g. the RRE-comprising ⁇ -globulin/IgG chimeric intron of SEQ ID NO: 5
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral (e.g. SIV) vector of the invention comprising an RRE-comprising intron may be as defined herein.
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an RRE-comprising intron may an increase of at least about 5-fold, an increase of at least about 10-fold, an increase of at least about 50-fold, an increase of at least about 100-fold, an increase of at least about 200-fold, an increase of at least about 500-fold, an increase of at least about 600-fold or more, typically compared with expression of the therapeutic protein from a corresponding retroviral/lentiviral (e.g. SIV) vector without the RRE-comprising intron.
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an RRE-comprising intron is at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about 500-fold, typically compared with expression of the therapeutic protein from a corresponding retroviral/lentiviral (e.g. SIV) vector without the RRE- comprising intron.
  • the RRE-comprising intron is a ⁇ -globulin/IgG chimeric intron comprising an RRE, such as the ⁇ -globulin/IgG chimeric RRE-comprising intron of SEQ ID NO: 5
  • said intron may increase transgene expression by a retroviral/lentiviral (e.g.
  • SIV vector of the invention comprising said ⁇ -globulin/IgG chimeric RRE-comprising intron by at least 600-fold, such as by about 686-fold compared with the expression of the transgene by a corresponding retroviral/lentiviral (e.g. SIV) vector without said ⁇ -globulin/IgG chimeric RRE-comprising intron.
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral (e.g. SIV) vector of the invention comprising an RRE-comprising intron may be as defined herein.
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an RRE-comprising intron may an increase of at least about 100%, an increase of at least about 500%, an increase of at least about 1000%, an increase of at least about 5000%, an increase of at least about 10000%, an increase of at least about 20000%, an increase of at least about 50000%, an increase of at least about 60000% or more, typically compared with expression of the therapeutic protein from a corresponding retroviral/lentiviral (e.g. SIV) vector without the RRE- comprising intron.
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an RRE-comprising intron is at least about 1000%, more preferably at least about 10000%, even more preferably at least about 50000%, typically compared with expression of the therapeutic protein from a corresponding retroviral/lentiviral (e.g. SIV) vector without the RRE-comprising intron.
  • the RRE-comprising intron is a ⁇ -globulin/IgG chimeric intron comprising an RRE, such as the ⁇ -globulin/IgG chimeric RRE-comprising intron of SEQ ID NO: 5
  • said intron may increase transgene expression by a retroviral/lentiviral (e.g.
  • SIV vector of the invention comprising said ⁇ - globulin/IgG chimeric RRE-comprising intron by at least 60000%, such as by about 68600% compared with the expression of the transgene by a corresponding retroviral/lentiviral (e.g. SIV) vector without said ⁇ -globulin/IgG chimeric RRE-comprising intron.
  • typically an intron comprising an RRE which is introduced into a retroviral/lentiviral (e.g. SIV) vector of the invention is appropriately spliced by target cells, aiding in the maturation of a stable mRNA molecule. This results in an increased number of mRNA molecules (i.e.
  • retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid results in increased expression of the transgene, which may encode a therapeutic protein.
  • the introduction of an intron comprising an RRE into a retroviral/lentiviral (e.g. SIV) vector may increase the number of mRNA molecules produced by the retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid compared with the number of mRNA molecules produced from a corresponding retroviral/lentiviral (e.g.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention comprising an RRE- comprising intron typically results in an increase in the number of mRNA molecules produced by the retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid compared with the number of mRNA molecules produced from a corresponding retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid lacking said RRE-comprising intron.
  • a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an AAT transgene (SERPINA1) and ⁇ - globulin/IgG chimeric intron with an inserted RRE (e.g. the RRE-comprising ⁇ -globulin/IgG chimeric intron of SEQ ID NO: 5) may result in an increased number of mRNA molecules produced by the retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid compared with a corresponding retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid which comprises the AAT transgene but which lacks the ⁇ -globulin/IgG chimeric intron with an inserted RRE (e.g.
  • the increase in number of mRNA molecules produced by the retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid may be as defined herein.
  • the increase in expression of the therapeutic protein by a retroviral/lentiviral (e.g. SIV) vector of the invention comprising an RRE-comprising intron may an increase of at least about 2-fold, at least about 5-fold, at least about 7-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold or more, typically compared with the number of mRNA molecules produced by a corresponding retroviral/lentiviral (e.g.
  • the increase in number of mRNA molecules produced by the retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid is least about 10-fold, more preferably at least about 12-fold, even more preferably at least about 13-fold, typically compared with the number of mRNA molecules produced by a corresponding retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid without the RRE- comprising intron.
  • the RRE-comprising intron is a ⁇ - globulin/IgG chimeric intron comprising an RRE, such as the ⁇ -globulin/IgG chimeric RRE-comprising intron of SEQ ID NO: 5, said intron may increase the number of mRNA molecules produced by the retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid by at least 12-fold, such as by about 13.7-fold compared with the number of mRNA molecules produced by a corresponding retroviral/lentiviral (e.g.
  • retroviral/lentiviral e.g. SIV
  • an intron comprising an RRE which is introduced into a retroviral/lentiviral (e.g. SIV) vector of the invention is appropriately spliced by target cells, aiding in the maturation of a stable mRNA molecule. This results in an increased number of mRNA molecules (i.e. increased mRNA copy number) produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid.
  • retroviral/lentiviral e.g. SIV
  • SIV vector results in increased expression of the transgene, which may encode a therapeutic protein.
  • introduction of an intron comprising an RRE into a retroviral/lentiviral (e.g. SIV) vector may increase the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid compared with the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid from a corresponding retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid without the RRE-comprising intron.
  • a retroviral/lentiviral e.g.
  • SIV vector of the invention comprising an RRE-comprising intron typically results in an increase in the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid compared with the number of mRNA molecules produced from a corresponding retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid lacking said RRE-comprising intron.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention comprising an AAT transgene (SERPINA1) and ⁇ -globulin/IgG chimeric intron with an inserted RRE (e.g.
  • the RRE-comprising ⁇ - globulin/IgG chimeric intron of SEQ ID NO: 5 may result in an increased number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid compared with a corresponding retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid which comprises the AAT transgene but which lacks the ⁇ -globulin/IgG chimeric intron with an inserted RRE (e.g. the RRE-comprising ⁇ -globulin/IgG chimeric intron of SEQ ID NO: 5).
  • the increase in number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid may be as defined herein.
  • the increase in number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid of the invention comprising an RRE-comprising intron may an increase of at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 45- fold or more, typically compared with the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g.
  • the increase in number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid is least about 20-fold, more preferably at least about 30-fold, even more preferably at least about 40-fold, typically compared with the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid from a corresponding retroviral/lentiviral (e.g.
  • the RRE-comprising intron is a ⁇ -globulin/IgG chimeric intron comprising an RRE, such as the ⁇ -globulin/IgG chimeric RRE-comprising intron of SEQ ID NO: 5, said intron may increase the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g. SIV) vector and/or vector genome plasmid by at least 40-fold, such as by about 42.2-fold compared with the number of mRNA molecules produced per copy of retroviral/lentiviral (e.g.
  • the invention also provides host cells comprising a retroviral/lentiviral (e.g. SIV) vector of the invention.
  • a host cell is a mammalian cell, particularly a human cell or cell line.
  • Non-limiting examples of host cells include HEK293 cells (such as HEK293F or HEK293T cells) and 293T/17 cells.
  • Commercial cell lines suitable for the production of virus are also readily available (as described herein).
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention have been designed such that the (RNA) genome of said retroviral/lentiviral (e.g. SIV) vector comprises an intron that is not removed during manufacture of the vector, such that the final retroviral/lentiviral (e.g. SIV) vector comprises said intron, resulting in increased expression of the transgene upon transduction of target cells.
  • the endogenous RRE of the retroviral/lentiviral (e.g. SIV) vector is deleted from its position within the wildtype/unmodified retroviral/lentiviral (e.g.
  • SIV retroviral/lentiviral genome and an RRE inserted into the intron sequence.
  • Deletion of the endogenous RRE from its position within the wildtype/unmodified retroviral/lentiviral (e.g. SIV) genome may be complete or a partial deletion, provided that if the deletion is partial, the activity of the remaining RRE sequence is decreased or completely ablated.
  • partial deletion of the RRE sequence is sufficient provided that the activity of the remaining RRE sequence is insufficient for gene expression from the retroviral/lentiviral (e.g. SIV) genome, placing pressure on the retroviral/lentiviral (e.g. SIV) vector to rely on the activity of the RRE within the intron, and hence to retain the RRE inserted into the intron.
  • Reference herein to deletion of the endogenous RRE therefore encompasses both complete and partial deletion of the endogenous RRE.
  • Standard techniques are known in the art for the deletion of nucleic acid sequences from a nucleic acid (e.g. plasmid), and may be readily used by one of ordinary skill in the art to delete the endogenous RRE.
  • Any RRE may be inserted into the intron to be included in the genome of the retroviral/lentiviral (e.g. SIV) vector, provided said RRE is able to facilitate retroviral/lentiviral (e.g. SIV) gene expression in the absence of the endogenous RRE in its standard position within the wildtype/unmodified retroviral/lentiviral (e.g. SIV) genome.
  • the inserted RRE is a viral RRE, particularly a retroviral RRE, even more particularly a lentiviral RRE.
  • Standard techniques are known in the art for the insertion of nucleic acid sequences into a nucleic acid (e.g. plasmid), and may be readily used by one of ordinary skill in the art to insert an RRE into an intron according to the present invention.
  • the RRE to be inserted into an intron may be the endogenous RRE of the retroviral/lentiviral (e.g. SIV) vector.
  • the endogenous RRE may be deleted from within the wildtype/unmodified retroviral/lentiviral (e.g.
  • SIV retroviral/lentiviral genome and inserted into an intron which is itself introduced into a retroviral/lentiviral (e.g. SIV) vector of the invention.
  • the endogenous RRE is moved from its position within the wildtype/unmodified retroviral/lentiviral (e.g. SIV) genome, and inserted into an intron.
  • the endogenous SIV RRE has been deleted from within the wildtype/unmodified SIV genome, and an intron into which the endogenous SIV RRE has been inserted is itself introduced into the SIV vector.
  • the RRE to be inserted into an intron may be an exogenous RRE.
  • an exogenous RRE may be an RRE from a different retrovirus.
  • an exogenous RRE may be an RRE from a different lentivirus.
  • an HIV vector of the invention may have its endogenous HIV RRE deleted and an intron comprising a SIV RRE introduced into the HIV genome.
  • the RRE sequence inserted into an intron within a retroviral/lentiviral (e.g. SIV) vector of the invention is the same as the endogenous RRE sequence which is deleted from the retroviral/lentiviral (e.g. SIV) genome.
  • the RRE is from the same virus as the viral vector, but is inserted into the (chimeric) intron, rather than being present in its standard location within the viral genome.
  • the viral vector may be an SIV vector in which the SIV RRE has been deleted from the genome and a (chimeric) intron introduced into which an SIV RRE has been inserted.
  • the viral vector may be an HIV vector, and the RRE may be an HIV RRE, but the HIV RRE is inserted within a (chimeric) intron, rather than in the standard location of the HIV RRE within the HIV genome.
  • the RRE to be inserted into the intron may be less than 1,000 bp, such as less than 900 bp or less than 800 bp may be preferred. Without being bound by theory, it is believed that smaller RRE- comprising introns may be more suitable for general applicability, allowing for greater flexibility in terms of the additional elements to be included within the retroviral/lentiviral (e.g. SIV) genome. Particularly preferred are RRE of between about 750 bp to about 800 bp, such as about 760 bp, such as the exemplified SIV RRE of the invention.
  • the RRE to be inserted into the intron may be an SIV RRE.
  • the SIV RRE may comprise or consist of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 1.
  • the SIV RRE comprises or consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1. More preferably, the SIV RRE consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1.
  • the SIV RRE comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 1.
  • the RRE to be inserted into the intron may be an HIV RRE.
  • the HIV RRE may comprise or consist of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 50.
  • the HIV RRE comprises or consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 50. More preferably, the HIV RRE consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 50. Still more preferably, the HIV RRE comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 50. According to the invention, typically the RRE inserted into the intron does not form part of the mature mRNA expressed within the host/target cells, as the RRE will be spliced out as part of the intron. Introns A retroviral/lentiviral (e.g.
  • SIV vector of the invention has been modified to (i) delete the endogenous RRE as described herein; and (ii) to introduce one or more intron into which a retroviral/lentiviral (e.g. SIV) RRE has been inserted.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise one or more intron, such as one, two, three, four, five or more introns.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention comprises one or two introns, preferably one intron. Wildtype/unmodified retroviral/lentiviral (e.g. SIV) vector genomes do not comprise introns.
  • any and all references herein to retroviral/lentiviral (e.g. SIV) vectors/ vector genomes comprising one or more introns refers to retroviral/lentiviral (e.g. SIV) vectors/ vector genomes into which one or more introns have been introduced.
  • any intron comprised in a retroviral/lentiviral (e.g. SIV) vector/vector genome of the invention is an intron which has been introduced into said retroviral/lentiviral (e.g. SIV) vector/vector genome, as described herein.
  • the size of the one or more intron to be inserted is not particularly limited, provided that the retroviral/lentiviral (e.g.
  • the retroviral/lentiviral (e.g. SIV) vector/vector genome comprising the one or more intron is within the packing limit for the retroviral/lentiviral (e.g. SIV) vector/vector genome.
  • the retroviral/lentiviral (e.g. SIV) vector/vector genome will comprise other elements (including the genome backbone, transgene and transgene promoter) in addition to the intron
  • Retroviral/lentiviral (e.g. SIV) vectors typically have a packing limit of approximately 10 kb. Therefore, typically the size of the one or more intron to be inserted is less than about 5,000 bp, accounting for the other elements that must be present within the retroviral/lentiviral (e.g. SIV) genome. Introns of less than 1,000 bp, such as less than 900 bp or less than 800 bp may be preferred. Without being bound by theory, it is believed that smaller introns of this type may be more suitable for general applicability, allowing for greater flexibility in terms of the additional elements to be included within the retroviral/lentiviral (e.g. SIV) genome.
  • introns of between about 750 bp to about 800 bp, such as about 770 bp, such as the exemplified ⁇ -globulin/IgG chimeric intron of the invention are particularly preferred.
  • the one or more intron may be introduced at any position within the retroviral/lentiviral (e.g. SIV) genome.
  • the one or more intron is introduced at a position within the retroviral/lentiviral (e.g. SIV) genome that does not disrupt the function of the retroviral/lentiviral (e.g. SIV) genome or any part thereof.
  • the one or more intron may be inserted at any position within the retroviral/lentiviral (e.g.
  • the intron is introduced between a transgene and the promoter operably linked to said transgene.
  • a retroviral/lentiviral (e.g. SIV) vector/genome of the invention comprises an intron between the transgene and the promoter operably linked to said transgene.
  • the RRE-comprising intron does not comprise the transgene to be expressed.
  • the sequence of the intron to be introduced is not particularly limited. Indeed, as exemplified herein, the present inventors have shown that the retroviral RRE remains functional in different contexts, and thus that RRE function does not depend on the specific intron sequence.
  • any appropriate intron may be used according to the present invention.
  • any intron may have an RRE sequence inserted, and said intron/RRE introduced into a retroviral/lentiviral (e.g. SIV) vector/genome of the invention.
  • the intron may be a naturally occurring, recombinant, or artificial, such as a chimeric intron.
  • the intron may be a viral intron.
  • Non-limiting examples of introns include: SV40 intron, Ef1- ⁇ intron 1, CMB intron A and adenovirus tripartite leader sequence intron.
  • the intron may be a chimeric intron or a non-chimeric intron.
  • the intron is a chimeric intron, with the chimeric ⁇ -globulin/IgG intron exemplified herein being particularly preferred.
  • the intron is a chimeric intron, such that retroviral/lentiviral (e.g. SIV) vectors of the invention comprise a chimeric intron.
  • a chimeric intron is an artificial intron which comprises or consists of sequences from two or more different introns.
  • Non-limiting examples of chimeric introns include a ⁇ -globulin/IgG chimeric intron and the chimeric intron from the CAGGS promoter.
  • the latter comprises the splice donor from chicken ⁇ -actin and the splice acceptor from rabbit ⁇ -globulin.
  • a chimeric intron according to the present invention is a ⁇ -globulin/IgG chimeric intron, such as that exemplified herein.
  • a ⁇ -globulin/IgG chimeric intron which comprises or consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 4.
  • the ⁇ -globulin/IgG chimeric intron comprises or consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4.
  • the ⁇ -globulin/IgG chimeric intron consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4. Still more preferably, the ⁇ -globulin/IgG chimeric intron comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 4.
  • a chimeric intron from the CAGGS promoter may comprise or consist of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 48.
  • the chimeric intron from the CAGGS promoter comprises or consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 48.
  • the chimeric intron from the CAGGS promoter consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 48. Still more preferably, the chimeric intron from the CAGGS promoter comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 48.
  • the RRE is introduced at a position within the intron which does not disrupt the splice donor and/or acceptor sites of the intron, to allow for the intron to be spliced correctly within a target cell.
  • the splice donor and/or acceptor sites of a particular intron may be readily determined using routine methods and techniques, for example as described in Desmet et al.
  • an RRE may preferably be inserted within 20 bp 5' of the splice acceptor’s branch site (and polypyrimidine tract), such 20 bp 5' of the splice acceptor’s branch site, 19 bp 5’ of the splice acceptor’s branch site, 18 bp 5' of the splice acceptor’s branch site or less, with 18 bp 5' of the splice acceptor’s branch site being particularly preferred.
  • an RRE may be inserted at a location between about 5-20 bp 5' of the splice acceptor’s branch site (and polypyrimidine tract), such as between about 10-20 bp 5' of the splice acceptor’s branch site or between about 15-20 bp 5' of the splice acceptor’s branch site, with RRE insertion 18 bp 5' of the splice acceptor’s branch site being preferred.
  • the RRE insertion site devised by the present inventors differs from insertion sites attempted in the art, which are typically closer to the splice donor site than the splice acceptor site.
  • the RRE insertion site of the invention is also typically designed such that any other regulatory sequences within the intron are not disrupted by RRE insertion.
  • the intron is a chimeric intron, the RRE may be inserted at a junction between the sequences from the two or more different introns.
  • the RRE may be inserted at the junction between the ⁇ -globulin intron sequence (the 5’ portion of the chimeric ⁇ -globulin/IgG chimeric intron) and the IgG intron sequence (the 3’ portion of the ⁇ -globulin/IgG chimeric intron).
  • the RRE may be inserted between a splice donor site and a splice acceptor site, wherein (a) the splice donor site comprises or consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 2 (from ⁇ -globulin); and/or (b) the splice acceptor site comprises or consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
  • the splice donor site comprises or consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2; and/or (b) the splice acceptor site comprises or consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3.
  • the splice donor site consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2; and/or (b) the splice acceptor site consists of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3.
  • the splice donor site comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 2; and/or (b) the splice acceptor site comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 3.
  • the intron may be introduced into the retroviral/lentiviral (e.g.
  • the intron is introduced into the retroviral/lentiviral (e.g. SIV) vector/genome in the forward orientation.
  • the intron is a ⁇ -globin/IgG chimeric intron and the RRE is an SIV RRE.
  • the ⁇ -globin/IgG chimeric intron comprising a SIV RRE may comprise or consist of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 5.
  • the ⁇ - globin/IgG chimeric intron comprising a SIV RRE comprises or consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5.
  • the ⁇ - globin/IgG chimeric intron comprising a SIV RRE consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5. Still more preferably, the ⁇ -globin/IgG chimeric intron comprising a SIV RRE comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 5.
  • An intron into which an RRE has been inserted according to the present invention may be referred to interchangeably herein as an “RRE-comprising intron”, an “intron comprising an RRE”, “an intron with an inserted RRE”, and an “intron with an introduced RRE”.
  • the intron comprising an RRE within a retroviral/lentiviral (e.g. SIV) vector/genome is appropriately spliced by target cells, aiding in the maturation of a stable mRNA molecule. This results in an increased expression of the coding regions of the retroviral/lentiviral (e.g. SIV) genome, including the transgene.
  • the introduction of an intron comprising an RRE within retroviral/lentiviral (e.g. SIV) vector/genome results in increased expression of the transgene, which may encode a therapeutic protein.
  • Said method may comprise or consist of the steps of (a) identifying the splice donor and splice acceptor sequences within the intron; and (b) inserting the RRE into the intron such that the splice donor and split acceptor sequences remain intact.
  • the method may further comprise one or more steps to delete the endogenous RRE from the retroviral/lentiviral (e.g. SIV) genome.
  • Standard techniques insert and/or delete nucleic acid sequences from a nucleic acid (e.g. plasmid) are known in the art, and may be used by one of ordinary skill to insert the RRE-comprising intron and/or to delete the endogenous RRE according to the invention.
  • a retroviral/lentiviral (e.g. SIV) vector the invention typically comprises a transgene encoding for a therapeutic protein.
  • a therapeutic protein is one which has potential utility in the treatment or prevention of a disease or condition, such as those describe herein.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention comprises a transgene encoding a protein which has a therapeutic effect on a disease or condition to be treated.
  • SIV vector of the invention may comprise a transgene encoding a therapeutic protein which is a functional or wild-type form of a protein which is present in a patient to be treated in a dysfunctional form (whether the dysfunction is inherent or acquired).
  • inherent dysfunction refers to a protein which is innately dysfunctional due to genetic factors
  • incquired dysfunction refers to a protein which is dysfunctional due to environmental or other factors after birth.
  • CFTR is an example of a protein which is inherently dysfunctional in patients with cystic fibrosis.
  • a retroviral/lentiviral e.g.
  • SIV vector of the invention may comprise a transgene encoding a therapeutic protein which is a functional or wild-type form of a protein which is present in a patient, but which that has become dysfunctional due to a genetic disease, such as a genetic respiratory disease.
  • the retroviral/lentiviral (e.g. SIV) vectors of the present invention are useful in the treatment of diseases via their use in expressing therapeutic proteins in target cells, wherein the therapeutic protein exerts its therapeutic effects: (i) within the target cells; (ii) by secretion from said cells into the surrounding tissue; or (iii) for secretion from said cells into the circulatory system.
  • a retroviral/lentiviral e.g.
  • SIV vector of the present invention may be pseudotyped to target airway cells of the respiratory tract (e.g. by pseudotyping with F and HN proteins from a respiratory paramyxovirus such as a Sendai virus), as described herein.
  • retroviral/lentiviral (e.g. SIV) vectors of the invention are useful in the treatment of diseases via their use in expressing therapeutic proteins in airway cells: (i) within the respiratory tract; (ii) for secretion from said cells into the lumen of the respiratory tract; and (iii) for secretion from said cells into the circulatory system.
  • the therapeutic protein may be selected from: (a) a secreted therapeutic protein, optionally alpha-1-antitrypsin (AAT), Factor VIII, Surfactant Protein B (SFTPB), Factor VII, Factor IX, Factor X, Factor XI, von Willebrand Factor, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Surfactant Protein C (SP-C), an anti-inflammatory protein (e.g. IL-10 or TGG ⁇ ) or monoclonal antibody, an anti-inflammatory decoy and a monoclonal antibody against an infectious agent; or (b) CFTR, CSF2RA, CSF2RB and ATP-binding cassette sub-family member A (ABCA3).
  • AAT alpha-1-antitrypsin
  • SFTPB Surfactant Protein B
  • SP-C Surfactant Protein C
  • an anti-inflammatory protein e.g. IL-10 or TGG ⁇
  • monoclonal antibody an anti-inflammatory decoy
  • therapeutic proteins include AAT, GM-CSF, FVIII, CFTR, decorin, TRIM72 and ABCA3.
  • the transgene may encode: (i) a therapeutic protein that is secreted into epithelial lining fluid and/or blood); (ii) a therapeutic protein that is secreted into blood); or (iii) a therapeutic membrane protein).
  • Preferred examples of these classes of transgenes include (i) AAT; (ii) FVIII; and (iii) CFTR.
  • the therapeutic protein is not an antibody, particularly not a monoclonal antibody and/or not a ⁇ -globulin gene.
  • the therapeutic protein may be selected from: (a) a secreted therapeutic protein, optionally alpha-1-Antitrypsin (AAT), Factor VIII, Surfactant Protein B (SFTPB), Factor VII, Factor IX, Factor X, Factor XI, von Willebrand Factor, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Surfactant Protein C (SP-C), an anti- inflammatory protein (e.g. IL-10, TGG ⁇ , or TNF-alpha) and an anti-inflammatory decoy; or (b) CFTR, CSF2RA, CSF2RB and ATP-binding cassette sub-family member A (ABCA3).
  • AAT alpha-1-Antitrypsin
  • SFTPB Surfactant Protein B
  • SP-C Surfactant Protein C
  • an anti- inflammatory protein e.g. IL-10, TGG ⁇ , or TNF-alpha
  • CFTR CSF2RA, CSF2RB and
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention are particularly efficient at driving the expression, secretion and/or membrane insertion of proteins (e.g. therapeutic proteins as described herein) by airway cells.
  • proteins e.g. therapeutic proteins as described herein
  • retroviral/lentiviral (e.g. SIV) vectors are F/HN pseudotyped viral vectors of the invention (as described herein), which are efficient at targeting cells in the airway epithelium.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention are typically delivered to cells of the respiratory tract, including the cells of the airway epithelium.
  • retroviral/lentiviral vectors of the invention are typically delivered to airway cells as described herein. Accordingly, the retroviral/lentiviral (e.g. SIV) vectors of the invention are particularly suited for treatment of diseases or disorders of the airways, respiratory tract, or lung. Typically, the retroviral/lentiviral (e.g. SIV) vectors of the invention may be used for the treatment of a genetic respiratory disease.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a transgene encoding a polypeptide or protein that is therapeutic for the treatment of such diseases, particularly a disease or disorder of the airways, respiratory tract, or lung.
  • transgene and therapeutic protein of the invention are not limited, one of ordinary skill in the art will be able to identify therapeutic proteins which may be usefully delivered according to the invention, particularly in the context of genetic diseases, particularly genetic respiratory diseases and diseases or disorders of the airways, respiratory tract, or lung such as those described herein. Accordingly, a retroviral/lentiviral (e.g.
  • SIV vectors of the invention may comprise a nucleic acid sequence encoding a therapeutic protein selected from: (a) a secreted therapeutic protein, optionally alpha-1-antitrypsin (AAT), Factor VIII, Surfactant Protein B (SFTPB), ADAMTS13, Factor VII, Factor IX, Factor X, Factor XI, von Willebrand Factor, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Surfactant Protein C (SP-C), an anti-inflammatory protein (e.g.
  • IL-10 TGG ⁇ , or TNF- alpha
  • monoclonal antibody an anti-inflammatory decoy and a monoclonal antibody against an infectious agent
  • CFTR CSF2RA, CSF2RB, ATP-binding cassette sub-family member A (ABCA3), DNAH5, DNAH11, DNAI1, and DNAI2.
  • ABCA3 ATP-binding cassette sub-family member A
  • therapeutic proteins include AAT, GM-CSF, FVIII, CFTR, ADAMTS13, SFTPB, decorin, TRIM72 and ABCA3.
  • the therapeutic protein encoded by a retroviral/lentiviral (e.g. SIV) vector of the invention may be an AAT.
  • An example of an AAT therapeutic transgene (SERPINA1) is provided by SEQ ID NO: 6, or by the complementary sequence of SEQ ID NO: 7.
  • SEQ ID NO: 6 is a codon-optimized CpG depleted AAT transgene (SERPINA1) 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 (as defined herein) which possess the same technical effect of enhancing translation compared with the unmodified (wild-type) AAT gene sequence are also encompassed by the present invention.
  • the therapeutic protein encoded by said AAT transgene may be exemplified by the polypeptide of SEQ ID NO: 8. 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: 6, 7 or 8.
  • the therapeutic protein encoded by a retroviral/lentiviral (e.g. SIV) vector of the invention may be an FVIII. Examples of a FVIII therapeutic transgene are provided by SEQ ID NOs: 9 and 10, or by the respective complementary sequences of SEQ ID NO: 11 and 12.
  • the polypeptide encoded by the FVIII transgene may be exemplified by the polypeptide of SEQ ID NO: 13 or 14. 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: 9 to 14.
  • the therapeutic protein encoded by a retroviral/lentiviral (e.g. SIV) vector of the invention is a CFTR.
  • An example of a CFTR transgene is provided by SEQ ID NO: 15.
  • the polypeptide encoded by said CFTR transgene may be exemplified by the polypeptide of SEQ ID NO: 16.
  • 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: 15 or 16.
  • the therapeutic protein encoded by a retroviral/lentiviral (e.g. SIV) vector of the invention may be GM-CSF.
  • a GM-CSF transgene may comprise or consist of SEQ ID NO: 17 (human).
  • the polypeptide encoded by the GM-CSF transgene may be exemplified by the polypeptide of SQE ID NO: 18 (human).
  • 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: 17 and 18.
  • the transgene may encode decorin.
  • An example of a DCN transgene is provided by SEQ ID NO: 21.
  • the polypeptide encoded by said DCN transgene may be exemplified by the polypeptide of SEQ ID NO: 22.
  • 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: 21 or 22.
  • the transgene may encode TRIM72.
  • An example of a TRIM72 transgene is provided by SEQ ID NO: 23.
  • the polypeptide encoded by said TRIM72 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 ABCA3.
  • An example of a ABCA3 transgene is provided by SEQ ID NO: 25.
  • the polypeptide encoded by said ABCA3 transgene may be exemplified by the polypeptide of SEQ ID NO: 26. 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: 25 or 26.
  • the transgene may encode SFTPB.
  • An example of a SFTPB transgene is provided by SEQ ID NO: 40.
  • the polypeptide encoded by said SFTPB transgene may be exemplified by the polypeptide of SEQ ID NO: 41.
  • 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: 40 or 41.
  • the transgene may encode ADAMTS13.
  • An example of a ADAMTS13 transgene is provided by SEQ ID NO: 42.
  • the polypeptide encoded by said ADAMTS13 transgene may be exemplified by the polypeptide of SEQ ID NO: 43.
  • 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: 42 or 43.
  • the therapeutic protein encoded by a retroviral/lentiviral (e.g. SIV) vector of the invention may be encoded by any one of SFTPB, SFTPC, ADAMTS13, Factor V, Factor VII, Factor IX, Factor X and/or Factor XI, von Willebrand Factor, GM-CSF, ABCA3, TRIM72 or DCN, or other known related gene.
  • the therapeutic protein may be AAT, SFTPB, or GM-CSF.
  • the therapeutic protein may be a monoclonal antibody (mAb) against an infectious agent (bacterial, fungal or viral, e.g.
  • the therapeutic protein may be anti-TNF alpha.
  • the therapeutic protein may be one implicated in an inflammatory, immune or metabolic condition.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may be delivered to the cells of the respiratory tract to allow production of proteins to be secreted into circulatory system.
  • the therapeutic protein may be any one of Factor VII, Factor VIII, Factor IX, Factor X, Factor XI and/or von Willebrand’s factor.
  • Such a retroviral/lentiviral (e.g. SIV) vector of the invention may be used in the treatment of diseases, particularly cardiovascular diseases and blood disorders, preferably blood clotting deficiencies such as haemophilia.
  • the retroviral/lentiviral (e.g. SIV) vector comprises a promoter operably linked to a transgene, enabling expression of the transgene.
  • 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: 27.
  • the promoter may be a CMV promoter.
  • An example of a CMV promoter sequence is provided by SEQ ID NO: 28.
  • the promoter may be a human elongation factor 1a (EF1a) promoter.
  • An example of a EF1a promoter is provided by SEQ ID NO: 29.
  • Other promoters for transgene expression are known in the art and their suitability for the retroviral/lentiviral (e.g. SIV) vectors of the invention determined using routine techniques known in the art. Non-limiting examples of other promoters include UBC and UCOE. As described herein, the promoter may be modified to further regulate expression of the transgene of the invention.
  • the promoter included in the retroviral/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.
  • suitable promoters and standard techniques for their modification are known in the art.
  • CpG-free promoters suitable for use in the present invention are described in Pringle et al. (J. Mol. Med. Berl. 2012, 90(12): 1487-96), which is herein incorporated by reference in its entirety.
  • the retroviral/lentiviral vectors (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: 27.
  • the absence of CpG dinucleotides typically further improves the performance of retroviral/lentiviral (e.g. SIV) vectors 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, particularly when administered to the airways.
  • the retroviral/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.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a hCEF promoter and a CFTR transgene, including those described herein.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a hCEF promoter and an AAT transgene (SERPINA1), including those described herein.
  • SIV vector of the invention may comprise a hCEF or CMV promoter and an FVIII transgene, including those described herein.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a hCEF or CMV promoter and an DCN transgene, including those described herein.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a hCEF or CMV promoter and an TRIM72 transgene, including those described herein.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a hCEF or CMV promoter and an ABCA3 transgene, including those described herein.
  • SIV vector of the invention may comprise a hCEF or CMV promoter and an SFTPB transgene, including those described herein.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise a hCEF or CMV promoter and an ADAMTS13 transgene, including those described herein.
  • the retroviral/lentiviral (e.g. SIV) vector of the invention comprises a nucleic acid encoding a therapeutic protein (said nucleic acid is referred to interchangeably herein as a transgene).
  • the nucleic acid sequence encodes a gene product, e.g., a protein, particularly a therapeutic protein.
  • SIV vector may comprise a transgene encoding an AAT, GM-CSF, FVIII, SFTPB, ADAMTS13, CFTR, decorin, TRIM72 or ABCA3 and said transgene 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 AAT, GM-CSF, FVIII, SFTPB, ADAMTS13, CFTR, decorin, TRIM72 or ABCA3 transgene respectively, examples of which are described herein.
  • the transgene encoding AAT, GM-CSF, FVIII, SFTPB, ADAMTS13, CFTR, decorin, TRIM72 or ABCA3 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 AAT, GM-CSF, FVIII, SFTPB, ADAMTS13, CFTR, decorin, TRIM72 or ABCA3 nucleic acid sequence respectively, examples of which are described herein.
  • the nucleic acid sequence encoding CFTR may be provided by SEQ ID NO: 15, the nucleic acid sequence encoding AAT may be provided by SEQ ID NO: 6, or by the complementary sequence of SEQ ID NO: 7 and/or the nucleic acid sequence encoding FVIII may be provided by SEQ ID NO: 11 or 12, or by the respective complementary sequences of SEQ ID NO: 13 or 14, and/or the nucleic acid sequence encoding SFTPB may be provided by SEQ ID NO: 40, and/or the nucleic acid sequence encoding ADAMTS13 may be provided by SEQ ID NO: 42, and/or the nucleic acid sequence encoding GM-CSF may be provided by SEQ ID NO: 17, the nucleic acid sequence encoding decorin may be provided by SEQ ID NO: 21, the nucleic acid sequence encoding TRIM72 may be provided by SEQ ID NO: 23, and/or the nucleic acid sequence encoding ABCA3 may be provided by SEQ ID NO: 25, or variants thereof.
  • the amino acid sequence of the therapeutic protein may be a functional variant having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the functional protein.
  • an AAT, FVIII, SFTPB, ADAMTS13, CFTR, GM-CSF, decorin, TRIM72 and/or ABCA3 polypeptide encoded by the respective AAT, FVIII, SFTPB, ADAMTS13, CFTR, CSF2, DCN, TRIM72, and/or ABCA3 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, SFTPB, ADAMTS13, CFTR, GM-CSF, decorin, TRIM72 and/or ABCA3 polypeptide sequence respectively.
  • the transgene encoding for a therapeutic protein may include a nucleic acid sequence encoding for the endogenous signal peptide of the therapeutic protein, or may exclude a nucleic acid sequence encoding for this signal peptide. All disclosure herein relates to both transgenes and therapeutic proteins including and excluding endogenous signal peptides unless explicitly stated. By way of non-limiting example, sequence identity of variants, and/or lengths of fragments may be based on the sequence with or without a signal peptide.
  • a retroviral/lentiviral (e.g. SIV) vector of the invention typically further comprises a Rev protein.
  • This Rev protein is typically provided by (encoded by) one of the plasmids used in the manufacture of the retroviral/lentiviral (e.g. SIV) vector, as described herein.
  • the Rev protein may be provided by the Rev plasmid (pDNA2b), wherein separate plasmids are used to provide the Gag-Pol and Rev proteins, or the Rev protein may be provided by the Rev-Gag- Pol plasmid, when a single plasmid is used to provide the Gag-Pol and Rev proteins.
  • An exemplary pDNA2b plasmid, as described herein is pGM299, as shown in Figure 2D and with a sequence represented by SEQ ID NO: 33.
  • Rev protein is the rSIV Rev protein which comprises or consists of the amino acid sequence of SEQ ID NO: 44.
  • This Rev protein is encoded by the pGM299 plasmid.
  • Nucleic Acids The present invention also provides a nucleic acid comprising or consisting of an intron (e.g. a chimeric intron) into which an RRE has been introduced, as described herein. Any intron as described herein may be comprised in a nucleic acid of the invention. Similarly, any RRE as described herein may be comprised in a nucleic acid of the invention. The intron and RRE may each be selected independently, e.g. from those described herein.
  • nucleic acid of the invention may comprise any intron as described herein, for example a ⁇ -globin/IgG chimeric intron comprising or consisting of SEQ ID NO: 4, or a variant thereof, as described herein.
  • a nucleic acid of the invention may comprise any RRE as described herein, for example a SIV RRE comprising or consisting of SEQ ID NO: 1, or a variant thereof, as described herein.
  • a nucleic acid of the invention comprises or consists of a ⁇ -globin/IgG chimeric intron and an SIV RRE.
  • a nucleic acid of the invention may comprise or consist of a ⁇ -globin/IgG chimeric intron comprising a SIV RRE which comprise or consist of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 5.
  • a nucleic acid of the invention may comprise or consist of a ⁇ -globin/IgG chimeric intron comprising a SIV RRE which comprises or consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5. More preferably, a nucleic acid of the invention may comprise or consist of a ⁇ -globin/IgG chimeric intron comprising a SIV RRE which consists of nucleic acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5.
  • a nucleic acid of the invention may comprise or consist of a ⁇ -globin/IgG chimeric intron comprising a SIV RRE which comprises or consists, particularly consists of, the nucleic acid sequence of SEQ ID NO: 5.
  • a nucleic acid of the invention may further comprise a transgene, typically which encodes a therapeutic protein as described herein. Any and all disclosure herein in relation to transgenes in the context of retroviral/lentiviral (e.g. SIV) vectors of the invention applies equally and without reservation to transgenes in the context of nucleic acids of the invention.
  • the therapeutic protein encoded by the transgene is AAT or CFTR.
  • a nucleic acid of the invention comprising an RRE-comprising intron may exhibit increased expression of the transgene compared with a corresponding nucleic acid lacking said intron.
  • the disclosure herein relating to the increase in transgene expression by a retroviral/lentiviral (e.g. SIV) vector of the invention comprising an RRE-comprising intron applies equally and without reservation to the increase in transgene expression exhibited by a nucleic acid of the invention.
  • the increase in expression of the therapeutic protein by a nucleic acid of the invention comprising an RRE-comprising intron may an increase of at least about 5- fold, an increase of at least about 10-fold, an increase of at least about 50-fold, an increase of at least about 100-fold, an increase of at least about 200-fold, an increase of at least about 500-fold, an increase of at least about 600-fold or more, typically compared with expression of the therapeutic protein from a corresponding nucleic acid without the RRE-comprising intron.
  • a nucleic acid of the invention enables long-term transgene expression, resulting in long-term expression of a therapeutic protein.
  • Long-term expression means expression of a therapeutic protein, preferably at therapeutic levels, for at least 45 days, at least 60 days, at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 730 days or more.
  • long-term expression means expression for at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days or more, more preferably at least 360 days, at least 450 days, at least 720 days or more.
  • a nucleic acid of the invention may drive (increased) long-lasting expression of a therapeutic protein in an airway cell in vivo in a patient.
  • a nucleic acid of the invention drives expression of a therapeutic protein in an airway cell for at least 45 days, more preferably at least 90 days.
  • the nucleic acid of the nucleic acid may be as defined herein.
  • the nucleic acid may comprise DNA and/or RNA.
  • the nucleic acid is DNA.
  • a nucleic acid of the invention may optionally be codon optimised for expression in a particular cell type, for example, eukaryotic cells (e.g. mammalian cells, yeast cells, insect cells or plants cells) or prokaryotic cells (e.g.
  • codon optimised refers to the replacement of at least one codon within a base polynucleotide sequence with a codon that is preferentially used by the host organism in which the polynucleotide is to be expressed. Typically, the most frequently used codons in the host organism are used in the codon-optimised polynucleotide sequence. Methods of codon optimisation are well known in the art. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • nucleic acid that encodes a therapeutic protein of the invention includes all polynucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • a nucleic acid cassette of the invention typically comprises a promoter operably linked to the nucleic acid sequence encoding the therapeutic protein.
  • the promoter is configured to express the nucleic acid sequence encoding the signal peptide and/or the nucleic acid sequence encoding the therapeutic protein.
  • the disclosure herein in relation to promoters in the context of retroviral/lentiviral (e.g. SIV) vectors of the invention applies equally to nucleic acids of the invention.
  • the promoter is a hCEF promoter as described herein.
  • the nucleic acids of the invention may include at least one part of a vector, in particular, regulatory elements.
  • the promoter e.g.
  • the hCEFI promoter within a nucleic acid cassette of the invention may be used to express more than one polypeptide, including one or more therapeutic proteins.
  • the nucleic acid may comprise a nucleic acid sequence which, when transcribed, gives rise to multiple polypeptides, for instance a transcript may contain multiple open reading frames (ORFs) and also one or more Internal Ribosome Entry Sites (IRES) to allow translation of ORFs after the first ORF.
  • a transcript may be polycistronic, i.e. it may be translated to give a polypeptide which is subsequently cleaved to give a plurality of polypeptides.
  • a nucleic acid of the invention may comprise multiple promoters and hence give rise to a plurality of transcripts and hence a plurality of polypeptides, including a plurality of therapeutic proteins.
  • Nucleic acids may, for instance, express one, two, three, four or more polypeptides via a promoter (e.g. hCEFI) or promoters.
  • a nucleic acid may comprise one or more translation initiation sequences (TIS). Translation initiation plays an important role in mRNA translation, canonically a methionyl tRNA unique for initiation (Met-tRNAi) identifies the AUG start codon and triggers the downstream translation process.
  • Non-canonical start codons e.g.
  • the nucleic acids of the present invention may comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, a termination signal that ends the production of an RNA transcript is contemplated according to the present invention.
  • a terminator may be necessary in vivo to achieve desirable message levels. In eukaryotic systems, a terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site.
  • RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
  • a terminator typically comprises a signal for the cleavage of the RNA, and it is preferred that the terminator signal promotes polyadenylation of the message.
  • the terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • the nucleic acids of the invention are capable of expressing the therapeutic protein in a given host cell. Any appropriate host cell may be used, such as mammalian, bacterial, insect, yeast, and/or plant host cells. In addition, cell-free expression systems may be used. Such expression systems and host cells are standard in the art.
  • nucleic acid cassettes and vectors of the invention are capable of expressing the therapeutic protein in airway cells, as described herein in relation to retroviral/lentiviral (e.g. SIV) vectors of the invention.
  • the nucleic acids of the invention may be made using any suitable process known in the art. Thus, the nucleic acids may be made using chemical synthesis techniques. Alternatively, the nucleic acids of the invention may be made using molecular biology techniques. A nucleic acid of the invention may be used in the production of a retroviral/lentiviral (e.g. SIV) vector, as described herein.
  • a nucleic acid of the invention may be a plasmid which is used in the manufacture of a retroviral/lentiviral (e.g. SIV) vector.
  • a nucleic acid of the invention may be comprised in a retroviral/lentiviral (e.g. SIV) vector.
  • a nucleic acid of the invention may be in the form of a DNA vector, such as a DNA plasmid.
  • the vector(s) may be an RNA vector, such as a mRNA vector or a self-amplifying RNA vector.
  • the DNA and/or RNA vector(s) of the invention may be capable of expression in eukaryotic and/or prokaryotic cells.
  • the DNA and/or RNA vector(s) are capable of expression in a cell of a subject, for example, a cell of a mammalian or avian subject to be immunised.
  • the nucleic acids of the invention are capable of expressing the therapeutic protein in airway cells (as described herein).
  • a non-viral vector of the present invention may be a phage vector, such as an AAV/phage hybrid vector as described in Hajitou et al., Cell 2006; 125(2) pp. 385-398; herein incorporated by reference.
  • Nucleic acids of the present invention e.g. DNA or RNA vectors
  • Non-viral plasmids cannot replicate in the subject to be treated, as they lack the viral genetic material which hijacks the body's normal production machinery. However they are capable of replicating in appropriate host cells, such as yeasts or bacteria including E. coli, and particularly airway cells as defined herein.
  • the term "plasmid” as used herein refers to a construction comprised of genetic material designed to direct transformation of a targeted cell.
  • the plasmid contains a plasmid backbone.
  • a "plasmid backbone” as used herein contains multiple genetic elements positionally and sequentially oriented with other necessary genetic elements such that the nucleic acid in the nucleic acid can be transcribed and when necessary translated in the transfected cells.
  • the plasmid backbone can contain one or more unique restriction sites within the backbone.
  • the plasmid may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced.
  • the plasmid can confer some well-defined phenotype on the host organism which is either selectable or readily detected.
  • the plasmid or plasmid backbone may have a linear or circular configuration.
  • the components of a plasmid can contain, but is not limited to, a DNA molecule incorporating: (1) the plasmid backbone; (2) a sequence encoding a signal peptide; (3) a sequence encoding a therapeutic protein; and (4) regulatory elements for transcription, translation, RNA stability and replication
  • the purpose of the plasmid in human gene therapy for the efficient delivery of nucleic acid sequences to, and expression of therapeutic proteins in, a cell or tissue.
  • the purpose of the plasmid is to achieve high copy number, avoid potential causes of plasmid instability and provide a means for plasmid selection.
  • a nucleic acid of the invention contains the necessary elements for expression of the transgene comprised in the nucleic acid.
  • Expression includes the efficient transcription of an inserted gene, nucleic acid sequence, or nucleic acid within the plasmid.
  • a DNA plasmid may be CpG-free, or be optimised to reduce CpG dinucleotides as described herein.
  • a DNA plasmid of the invention may be codon-optimised as described herein. Methods of preparing plasmid DNA are well known in the art. Typically, they are capable of autonomous replication in an appropriate host or producer cell. Host cells containing (e.g. transformed, transfected, or electroporated with) the plasmid may be prokaryotic or eukaryotic in nature, either stably or transiently transformed, transfected, or electroporated with the plasmid.
  • Suitable host cells include bacterial, yeast, fungal, invertebrate, and mammalian cells.
  • the host cell is bacterial; more preferably E. coli.
  • Host cells can then be used in methods for the large scale production of the plasmid.
  • the cells are grown in a suitable culture medium under favourable conditions, and the desired plasmid isolated from the cells, or from the medium in which the cells are grown, by any purification technique well known to those skilled in the art; e.g. see Sambrook et al, supra. Any appropriate delivery means can be used to deliver a non-viral vector (e.g. plasmid) of the invention to a target cell or patient.
  • a non-viral vector e.g. plasmid
  • Suitable delivery means are known in the art and within the routine skill of one of ordinary skill in the art.
  • Non-limiting examples include the use of cationic lipids, polymers (e.g. polyethyleneimine and poly-L-lysine) and electroporation.
  • Preferably cationic lipids may be used to deliver non-viral (e.g. plasmid) vectors of the invention to target cells or to a patient.
  • Non-limiting examples of cationic lipids suitable for use according to the invention are GL67A and lipofectamine.
  • the cationic lipid mixture GL67A is a mixture of three components - GL67 (Cholest-5-en-3-ol (3 ⁇ )-,3-[(3-aminopropyl)[4-[(3- aminopropyl)amino]butyl]carbamate], (CAS Number: 179075-30-0)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and DMPE-PEG5000 (1,2-Dimyristoyl-sn- Glycero-3-Phosphoethanolamine-N-[methoxy (Polyethylene glycol)5000]). These components are formulated at a 1:2:0.05 molar ratio to form GL67A.
  • Lipofectamine consists of a 3:1 mixture of DOSPA (2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) and DOPE.
  • DOSPA 2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate
  • DOPE 2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate
  • DOPE 2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate
  • the invention also provides host cells comprising a nucleic acid (e.g.
  • Non-limiting examples of host cells include HEK293 cells (such as HEK293F or HEK293T cells) and 293T/17 cells. Commercial cell lines suitable for the production of virus are also readily available (as described herein). Methods of Production Methods for the production of retroviral/lentiviral (e.g. SIV) vectors of the invention as also described herein.
  • SIV retroviral/lentiviral
  • retroviral vectors comprising a retroviral/lentiviral RNA sequence comprising (i) codon substitutions and (ii) a reduced number of modified retroviral/lentiviral open reading frames (ORFs) do not negatively impact the manufactured vector titre, transgene expression and/or integration of the retroviral/lentiviral RNA sequence into the host/target cell genome, and can even result in an increase in vector titre, transgene expression and/or integration of the retroviral/lentiviral RNA sequence.
  • ORFs modified retroviral/lentiviral open reading frames
  • retroviral/lentiviral vectors can be produced with the endogenous RRE of the retroviral/lentiviral (e.g. SIV) genome deleted, and an intron with a retroviral/lentiviral (e.g. SIV) inserted therein introduced into the retroviral/lentiviral (e.g. SIV) genome, and that this can increase transgene expression.
  • the present invention provides a method of producing a retroviral/lentiviral (e.g. SIV) vector from which (i) the endogenous RRE of the retroviral/lentiviral (e.g.
  • retroviral/lentiviral genome has been deleted, and (ii) an intron with a retroviral/lentiviral (e.g. SIV) inserted therein has been introduced.
  • a retroviral/lentiviral e.g. SIV
  • HN hemagglutinin-neuraminidase
  • F fusion proteins from a respiratory paramyxovirus or with VSV-G, and which comprises a promoter and a transgene.
  • retroviral/lentiviral vector is a lentiviral vector, with Simian immunodeficiency virus (SIV) vectors being particularly preferred.
  • the method of the invention may be a scalable GMP-compatible method.
  • the method of the invention allows the generation of retroviral/lentiviral (e.g. SIV) vectors as described herein, which exhibit high levels of transgene expression.
  • a method of the invention produces retroviral/lentiviral (e.g. SIV) vectors as described herein that exhibit increased transgene expression compared with a corresponding retroviral/lentiviral (e.g. SIV) vector which lacks an RRE-comprising intron according to the invention.
  • the increase in transgene expression by a retroviral/lentiviral e.g.
  • SIV vector of the invention may be an increase of at least about 5-fold, an increase of at least about 10-fold, an increase of at least about 50-fold, an increase of at least about 100-fold, an increase of at least about 200-fold, an increase of at least about 500-fold, an increase of at least about 600-fold or more, typically compared with expression of the transgene by a corresponding retroviral/lentiviral (e.g. SIV) vector which lacks an RRE-comprising intron according to the invention produced by the same method. More preferably, the increase in transgene expression by a retroviral/lentiviral (e.g.
  • SIV vector of the invention may be at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about 500-fold, typically compared with expression of the transgene by a corresponding retroviral/lentiviral (e.g. SIV) vector which lacks an RRE-comprising intron according to the invention produced by the same method.
  • the RRE-comprising intron is a ⁇ -globulin/IgG chimeric intron comprising an RRE, such as the ⁇ -globulin/IgG chimeric RRE-comprising intron of SEQ ID NO: 5
  • said intron may increase transgene expression by a retroviral/lentiviral (e.g.
  • SIV vector of the invention comprising said ⁇ -globulin/IgG chimeric RRE-comprising intron by at least 600-fold, such as by about 686-fold compared with the expression of the transgene by a corresponding retroviral/lentiviral (e.g. SIV) vector without said ⁇ -globulin/IgG chimeric RRE-comprising intron.
  • a method of the invention typically allows the generation of retroviral/lentiviral (e.g. SIV) vectors comprising a modified retroviral/lentiviral (e.g. SIV) RNA sequence with high levels of vector integration into the host/target cell genome.
  • a method of the invention may allow the generation of high titre purified retroviral/lentiviral (e.g. SIV) vectors comprising a modified retroviral/lentiviral (e.g. SIV) RNA sequence.
  • retroviral/lentiviral e.g. SIV
  • the production of retroviral/lentiviral (e.g. SIV) vectors typically employs one or more plasmids which provide the elements needed for the production of the vector: the genome for the retroviral/lentiviral vector, the Gag-Pol, Rev, F and HN. Multiple elements can be provided on a single plasmid.
  • each element is provided on a separate plasmid, such that there five plasmids, one for each of the vector genome, the Gag-Pol, Rev, F and HN, respectively.
  • a single plasmid may provide the Gag-Pol and Rev elements, and may be referred to as a packaging plasmid (pDNA2).
  • the remaining elements may be provided by separate plasmids (pDNA1, pDNA3a, pDNA3b respectively), such that four plasmids are used for the production of a retroviral/lentiviral (e.g. SIV) vector according to the invention.
  • pDNA1, pDNA3a and pDNA3b may be as described herein in the context of the five-plasmid method.
  • retroviral/lentiviral e.g. SIV
  • VSV-G another envelope protein
  • a method of the invention typically employs one or more plasmids which provide the elements needed for the production of the vector: the genome for the retroviral/lentiviral vector, the Gag-Pol (pDNA2a), Rev (pDNA2b), and envelope (e.g. VSV-G) (pDNA3).
  • Multiple elements can be provided on a single plasmid.
  • each element is provided on a separate plasmid, such that there four plasmids, one for each of the vector genome, the Gag-Pol, Rev and envelope (e.g. VSV-G), respectively.
  • pDNA1, pDNA2a and pDNA2b may be as described herein in the context of the five-plasmid method for retroviral/lentiviral vectors pseudotyped with F and HN proteins.
  • a single plasmid may provide the Gag-Pol and Rev elements, and may be referred to as a packaging plasmid (pDNA2).
  • the remaining elements may be provided by separate plasmids (pDNA1 and pDNA3 respectively), such that three plasmids are used for the production of a retroviral/lentiviral (e.g. SIV) vector according to the invention.
  • pDNA1 may be as described herein in the context of the five/four-plasmid methods.
  • the vector genome plasmid encodes all the genetic material that is packaged into the final retroviral/lentiviral vector, including the transgene.
  • the vector genome plasmid may be designated herein as “pDNA1”, and typically comprises the transgene and the transgene promoter.
  • the RRE-comprising intron is typically comprised within the vector genome plasmid.
  • the RRE-comprising intron is typically incorporated from the vector genome plasmid into the retroviral/lentiviral (e.g. SIV) RNA sequence.
  • the other four plasmids are manufacturing plasmids encoding the Gag-Pol, Rev, F and HN proteins. These plasmids may be designated “pDNA2a”, “pDNA2b”, “pDNA3a” and “pDNA3b” respectively.
  • the lentivirus is SIV, such as SIV1, preferably SIV-AGM.
  • the F and HN proteins are derived from a respiratory paramyxovirus, preferably a Sendai virus.
  • the five plasmids are characterised by Figures 2A- 2F, thus pDNA1 is the pGM991 plasmid of Figure 2A, pDNA2a is the pGM691 plasmid of Figure 2B or the pGM297 plasmid of Figure 2C, pDNA2b is the pGM299 plasmid of Figure 2D, pDNA3a is the pGM301 plasmid of Figure 2E and pDNA3b is the pGM303 plasmid of Figure 2F, or variants thereof any of these plasmids (as described herein).
  • pGM407 (as shown in Figure 2G) is an unmodified version of the vector genome plasmid from which pGM991 is derived.
  • the plasmid as defined in Figure 2A is represented by SEQ ID NO: 30; the plasmid as defined in Figure 2B is represented by SEQ ID NO: 31; the plasmid as defined in Figure 2C is represented by SEQ ID NO: 32; the plasmid as defined in Figure 2D is represented by SEQ ID NO: 33; the plasmid as defined in Figure 2E is represented by SEQ ID NO: 34; the plasmid as defined in Figure 2F is represented by SEQ ID NO: 35; and the plasmid as defined in Figure 2G is represented by SEQ ID NO: 36.
  • variants of these plasmids are also encompassed by the present invention.
  • variants having at least 90% such as at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5 or 100% sequence identity to any one of SEQ ID NOs: 30 to 36 are encompassed.
  • all of the plasmids contribute to the formation of the final retroviral/lentiviral (e.g. SIV) vector, although only the vector genome plasmid provides nucleic acid sequence comprised in the retroviral/lentiviral (e.g. SIV) RNA sequence.
  • manufacture of the retroviral/lentiviral e.g.
  • the vector genome plasmid provides the enhancer/promoter, Psi, RRE-comprising intron, cPPT, mWPRE, SIN LTR, SV40 polyA (see Figure 1A), which are important for virus manufacture.
  • pGM991 as a non-limiting example of a pDNA1
  • the CMV enhancer/promoter, SV40 polyA, colE1 Ori and KanR are involved in manufacture of the retroviral/lentiviral (e.g. SIV) vector of the invention, but are not found in the final retroviral/lentiviral (e.g. SIV) vector.
  • the cPPT central polypurine tract
  • RRE-comprising intron inserted between hCEF and the AAT transgene
  • hCEF hCEF
  • AAT transgene
  • mWPRE from pGM991
  • SIV retroviral/lentiviral vector
  • SIN LTR long terminal repeats, SIN/IN self- inactivating
  • Psi packetaging signal
  • pGM407 (from which pGM991 is derived) lacks the RRE- comprising intron, but comprises the endogenous SIV RRE, which is positioned 5’ of the hCEF promoter and between the partial Gag and cPPT sequences.
  • pDNA1 corresponding elements from the other vector genome plasmids
  • the four plasmids are characterised by Figures 2A-2F, thus pDNA1 is the pGM991 plasmid of Figure 2A, pDNA2a is the pGM691 plasmid of Figure 2B or the pGM297 plasmid of Figure 2C, pDNA2b is the pGM299 plasmid of Figure 2D, pDNA3 is the pMD2.G plasmid of Figure 2H, or variants thereof any of these plasmids (as described herein).
  • the plasmid as defined in Figure 2A is represented by SEQ ID NO: 30; the plasmid as defined in Figure 2B is represented by SEQ ID NO: 31; the plasmid as defined in Figure 2C is represented by SEQ ID NO: 32; the plasmid as defined in Figure 2D is represented by SEQ ID NO: 33; the plasmid as defined in Figure 2H is represented by SEQ ID NO: 49.
  • Variants (as defined herein) of these plasmids are also encompassed by the present invention.
  • variants having at least 90% having at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5 or 100% sequence identity to any one of SEQ ID NOs: 30 to 33 and 49 are encompassed.
  • the F and HN proteins from pDNA3a and pDNA3b preferably Sendai F and HN proteins
  • the VSV-G from pDNA3 are important for infection of target cells with the final retroviral/lentiviral (e.g. SIV) vector, i.e. for entry of a patient’s epithelial cells (typically lung or nasal cells as described herein).
  • a method of the invention may comprise or consist of the following steps: (a) growing cells in suspension; (b) transfecting the cells with one or more plasmids; (c) adding a nuclease; (d) harvesting the retrovirus/lentivirus (e.g.
  • the one or more plasmids may comprise or consist of: a vector genome plasmid pDNA1; a Gag-Pol plasmid (e.g.
  • the pDNA1 may be pGM991.
  • the pDNA2a may be pGM297 or pGM691, preferably pGM691.
  • the pDNA2b may be pGM299.
  • the pDNA3a may be pGM301.
  • the pDNA3b may be pGM303.
  • the pDNA1 is pGM991; the pDNA2a is pGM691; the pDNA2b is pGM299; the pDNA3a is pGM301; and the pDNA3b is pGM303.
  • the one or more plasmids may comprise or consist of: a vector genome plasmid pDNA1; a Gag-Pol plasmid (e.g.
  • the pDNA1 may be pGM991.
  • the pDNA2a may be pGM297 or pGM691, preferably pGM691.
  • the pDNA2b may be pGM299.
  • the pDNA3 may be pMD2.G. Any combination of pDNA1, pDNA2a, pDNA2b, and pDNA3 may be used.
  • the pDNA1 is pGM991; the pDNA2a is pGM691; the pDNA2b is pGM299; the pDNA3a is pGM301; and the pDNA3 is pMD2.G.
  • Any appropriate ratio of vector genome plasmid: Gag-Pol plasmid: Rev plasmid: F plasmid: HN plasmid may be used to further optimise (increase) the retroviral/lentiviral (e.g. SIV) titre produced.
  • the ratio of vector genome plasmid: Gag-Pol plasmid: Rev plasmid: F plasmid: HN plasmid may by in the range of 10-40:-4-20:3-12:3-12:3-12, typically 15-20:7-11:4-8:4- 8:4-8, such as about 18-22:7-11:4-8:4-8:4-8, 19-21:8-10:5-7:5-7:5-7.
  • the ratio of vector genome plasmid: Gag-Pol plasmid: Rev plasmid: F plasmid: HN plasmid is about 20:9:6:6:6.
  • the ratio of vector genome plasmid: Gag-Pol plasmid: Rev plasmid: VSV-G plasmid is about 20:9:6:12.
  • Steps (a)-(f) of the method are typically carried out sequentially, starting at step (a) and continuing through to step (f).
  • the method may include one or more additional step, such as additional purification steps, buffer exchange, concentration of the retroviral/lentiviral (e.g. SIV) vector after purification, and/or formulation of the retroviral/lentiviral (e.g. SIV) vector after purification (or concentration).
  • Each of the steps may comprise one or more sub-steps.
  • harvesting may involve one or more steps or sub-steps, and/or purification may involve one or more steps or sub-steps.
  • Any appropriate cell type may be transfected with the one or more plasmids (e.g. the five-, four- or three- plasmids described herein) to produce a retroviral/lentiviral (e.g. SIV) vector of the invention.
  • plasmids e.g. the five-, four- or three- plasmids described herein
  • retroviral/lentiviral vector of the invention e.g. SIV
  • mammalian cells particularly human cell lines are used.
  • Non-limiting examples of cells suitable for use in the methods of the invention are HEK293 cells (such as HEK293F or HEK293T cells) and 293T/17 cells.
  • Commercial cell lines suitable for the production of virus are also readily available (e.g.
  • the cells may be grown in animal-component free media, including serum-free media.
  • the cells may be grown in a media which contains human components.
  • the cells may be grown in a defined media comprising or consisting of synthetically produced components.
  • Any appropriate transfection means may be used according to the invention. Selection of appropriate transfection means is within the routine practice of one of ordinary skill in the art. By way of non-limiting example, transfection may be carried out by the use of PEIPro TM , Lipofectamine2000 TM or Lipofectamine3000 TM . Any appropriate nuclease may be used according to the invention.
  • nuclease is an endonuclease.
  • the nuclease may be Benzonase® or Denarase®.
  • the addition of the nuclease may be at the pre-harvest stage or at the post-harvest stage, or between harvesting steps.
  • the gag-pol genes used in the production of a retroviral/lentiviral (e.g. SIV) vectors of the invention may be codon-optimised.
  • the gag-pol genes within the pDNA2a plasmid may be codon-optimised.
  • codon-optimised gag-pol genes may comprise or consist of the nucleic acid sequence of SEQ ID NO: 37, or a variant thereof (as defined herein).
  • the codon-optimised gag-pol genes of the invention may comprise or consist of a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 37, preferably at least 95%, identity to SEQ ID NO: 37.
  • the codon-optimised gag-pol genes may consist of the nucleic acid sequence of SEQ ID NO: 37.
  • the preferred pDNA2a, pGM691 comprises the codon-optimised gag-pol genes of SEQ ID NO: 37.
  • the gag-pol genes e.g. SIV gag-pol genes
  • codon-optimised gag-pol genes are typically operably linked to a promoter to facilitate expression of the gag-pol proteins.
  • Any suitable promoter may be used, including those described herein in the context of promoters for the transgene.
  • the promoter is a CAG promoter, as used on the exemplified pGM691 plasmid.
  • An exemplary CAG promoter is set out in SEQ ID NO: 38.
  • the codon-optimised gag-pol genes of SEQ ID NO: 37 comprise a translational slip, and so do not form a single conventional open reading frame. Codon-optimised gag-pol genes (or nucleic acids comprising or consisting thereof) and plasmids comprising said genes or nucleic acids are advantageous in the production of retroviral/lentiviral (e.g. SIV) vectors using methods of the invention, as they allow for the production of high titre retroviral/lentiviral (e.g. SIV) vectors.
  • retroviral/lentiviral e.g. SIV
  • said codon-optimised gag-pol genes or nucleic acids comprising or consisting thereof and plasmids comprising said genes or nucleic acids can be used to produces a titre of retroviral/lentiviral (e.g. SIV) vector that is at least equivalent to the titre of retroviral/lentiviral (e.g. SIV) vector produced by a corresponding method which does not use codon-optimised gag-pol genes, as described herein.
  • Codon-optimised gag-pol genes are further disclosed in PCT/GB2022/050524, which is herein incorporated by reference in its entirety.
  • the invention also provides a retroviral/lentiviral (e.g. SIV) vector obtainable by a method of the invention.
  • the retroviral/lentiviral (e.g. SIV) vector obtainable by a method of the invention is produced at a high-titre, as described herein. Titre may be measured in terms of transducing units, as defined here. Accordingly, the retroviral/lentiviral (e.g. SIV) vector obtainable by a method of the invention is produced at a high-titre, as described herein. Titre may be measured in terms of transducing units, as defined here. Accordingly, the retroviral/lentiviral (e.g.
  • SIV vectors of the invention may optionally be at a titre of at least about 2.5x10 6 TU/mL, at least about 3.0x10 6 TU/mL, at least about 3.1x10 6 TU/mL, at least about 3.2x10 6 TU/mL, at least about 3.3x10 6 TU/mL ⁇ at least about 3.4x10 6 TU/mL, at least about 3.5x10 6 TU/mL, at least about 3.6x10 6 TU/mL, at least about 3.7x10 6 TU/mL, at least about 3.8x10 6 TU/mL, at least about 3.9x10 6 TU/mL, at least about 4.0x10 6 TU/mL or more.
  • the retroviral/lentiviral (e.g. SIV) vector is produced at a titre of at least about 3.0x10 6 TU/mL, or at least about 3.5x10 6 TU/mL.
  • the production of high-titre retroviral/lentiviral (e.g. SIV) vectors may impart other desirable properties on the resulting vector products. For example, without being bound by theory, it is believed that production at high titres without the need for intense concentration by methods such as TFF results in a higher quality vector product than retroviral/lentiviral (e.g.
  • the gag-pol genes e.g. codon-optimised gag-pol genes
  • the codon-optimised gag-pol genes used are matched to the retroviral/lentiviral vector being produced.
  • the codon-optimised gag-pol genes used are HIV gag-pol genes.
  • the codon-optimised gag-pol genes used are SIV gag-pol genes.
  • the codon-optimised gag-pol genes used are SIV gag-pol genes.
  • the retroviral/lentiviral (e.g. SIV) vectors of the invention (i) lack the endogenous RRE; and (ii) comprise an RRE-comprising intron.
  • the vector genome plasmid used in the production of a retroviral/lentiviral (e.g. SIV) vector of the invention may be modified to (i) delete the endogenous RRE and (ii) introduce an RRE-comprising intron. Any disclosure herein in relation to retroviral/lentiviral (e.g.
  • SIV vectors (i) lacking the endogenous RRE and (ii) comprising an RRE-comprising intron, applies equally and without reservation to the vector genome plasmids (pDNA1) described herein, which may be used in the production of retroviral/lentiviral (e.g. SIV) vectors of the invention.
  • trypsin refers to both trypsin and equivalents thereof.
  • An equivalent enzyme is one with the same or essentially the same cleavage specificity as trypsin. Trypsin cleavage activity may be defined as cleavage C-terminal to arginine or lysine residues, typically exclusively C-terminal to arginine or lysine residues.
  • the trypsin activity may preferably be provided by an animal origin free, recombinant enzyme such as TrypLE SelectTM.
  • the addition of trypsin may be at the pre-harvest stage or at the post-harvest stage, or between harvesting steps.
  • Any appropriate purification means may be used to purify the retroviral/lentiviral (e.g. SIV) vector.
  • suitable purification steps include depth/end filtration, tangential flow filtration (TFF) and chromatography.
  • the purification step typically comprises at least on chromatography step.
  • Non-limiting examples of chromatography steps that may be used in accordance with the invention include mixed-mode size exclusion chromatography (SEC) and/or anion exchange chromatography.
  • Elution may be carried out with or without the use of a salt gradient, preferably without.
  • This method may be used to produce the retroviral/lentiviral (e.g. SIV) vectors of the invention, such as those comprising a CFTR, AAT and/or FVIII gene as described herein.
  • the retroviral/lentiviral (e.g. SIV) vector of the invention comprises any of the above-mentioned genes, or the genes encoding the above-mentioned proteins.
  • the method may use any combination of one or more of the specific plasmid constructs provided by Figures 2A-2F or 2H to provide a retroviral/lentiviral (e.g. SIV) vector of the invention.
  • retroviral/lentiviral vectors and nucleic acids e.g. plasmids
  • the retroviral/lentiviral vectors and nucleic acids e.g. plasmids
  • the retroviral/lentiviral vectors and nucleic acids e.g. plasmids
  • the F/HN-pseudotyped retroviral/lentiviral e.g.
  • SIV vectors of the invention are capable of: (i) airway transduction without disruption of epithelial integrity; (ii) persistent gene expression; (iii) lack of chronic toxicity; and (iv) efficient repeat administration.
  • Long term/persistent stable gene expression preferably at a therapeutically-effective level, may be achieved using repeat doses of a vector of the present invention. Alternatively, a single dose may be used to achieve the desired long-term expression.
  • the retroviral/lentiviral (e.g. SIV) vectors and nucleic acids (e.g. plasmids) of the present invention can be used in gene therapy.
  • the efficient airway cell uptake properties of the retroviral/lentiviral e.g.
  • SIV vectors and nucleic acids (e.g. plasmids) of the invention make them highly suitable for treating respiratory tract diseases.
  • the retroviral/lentiviral (e.g. SIV) vectors and nucleic acids (e.g. plasmids) of the invention can also be used in methods of gene therapy to promote secretion of therapeutic proteins.
  • the invention provides secretion of therapeutic proteins into the lumen of the respiratory tract or the circulatory system.
  • administration of a retroviral/lentiviral (e.g. SIV) vector or nucleic acid e.g.
  • plasmid of the invention and its uptake by airway cells may enable the use of the lungs (or nose or airways) as a “factory” to produce a therapeutic protein that is then secreted and enters the general circulation at therapeutic levels, 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.
  • other diseases which are not respiratory tract diseases such as cardiovascular diseases and blood disorders, particularly blood clotting deficiencies, can also be treated by the retroviral/lentiviral (e.g. SIV) vectors and nucleic acids (e.g.
  • Retroviral/lentiviral (e.g. SIV) vectors and nucleic acids (e.g. plasmids) of the invention can effectively treat a disease by providing a transgene for the correction of the disease. For example, inserting a functional copy of the CFTR gene to ameliorate or prevent lung disease in CF patients, independent of the underlying mutation. Accordingly, retroviral/lentiviral (e.g. SIV) vectors and nucleic acids (e.g. plasmids) of the invention may be used to treat cystic fibrosis (CF), typically by gene therapy with a CFTR transgene as described herein. As another example, retroviral/lentiviral (e.g.
  • SIV vectors and nucleic acids (e.g. plasmids) of the invention may be used to treat Alpha-1 Antitrypsin (AAT) deficiency, typically by gene therapy with a AAT 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 is relevant to AAT deficient patient, 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 (in which AAT isolated from human blood and administered intravenously every week), providing stable, long-lasting expression in the target tissue (lung/nasal epithelium), ease of administration and unlimited availability.
  • Transduction with a retroviral/lentiviral (e.g. SIV) vector of the invention or transfection with a nucleic acid (e.g. plasmid) of the invention may lead to secretion of the recombinant protein into the lumen of the lung as well as into the 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.
  • AAT in the lung and/or nose following transduction according to the present invention may, therefore, be more widely applicable, including to these indications.
  • 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.
  • diseases or disorders to be treated include Primary Ciliary Dyskinesia (PCD), acute lung injury, Surfactant Protein B (SFTB) deficiency, Pulmonary Alveolar Proteinosis (PAP), Chronic Obstructive Pulmonary Disease (COPD) and/or inflammatory, infectious, immune or metabolic conditions, such as lysosomal storage diseases.
  • PCD Primary Ciliary Dyskinesia
  • SFTB Surfactant Protein B
  • PAP Pulmonary Alveolar Proteinosis
  • COPD Chronic Obstructive Pulmonary Disease
  • inflammatory, infectious, immune or metabolic conditions such as lysosomal
  • the invention provides a method of treating a disease, the method comprising administering a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) of the invention to a subject.
  • a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) is produced using a method of the present invention.
  • Any disease described herein may be treated according to the invention.
  • the invention provides a method of treating a lung disease using a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) of the invention.
  • the disease to be treated may be a chronic disease.
  • a method of treating CF is provided.
  • the invention also provides a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) as described herein for use in a method of treating a disease.
  • retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) is produced using a method of the present disclosure.
  • Any disease described herein may be treated according to the invention.
  • the invention provides a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) of the invention for use in a method of treating a lung disease.
  • the disease to be treated may be a chronic disease.
  • a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) for use in treating CF is provided.
  • the invention also provides the use of a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) as described herein in the manufacture of a medicament for use in a method of treating a disease.
  • the retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) is produced using a method of the present disclosure. Any disease described herein may be treated according to the invention.
  • the invention provides the use of a retroviral/lentiviral (e.g.
  • SIV vector or nucleic acid (e.g. plasmid) of the invention for the manufacture of a medicament for use in a method of treating a lung disease.
  • the disease to be treated may be a chronic disease.
  • a retroviral/lentiviral (e.g. SIV) vector or nucleic acid (e.g. plasmid) in the manufacture of a medicament for use in a method of treating CF is provided.
  • Formulation and administration The retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) of the invention may be administered in any dosage appropriate for achieving the desired therapeutic effect.
  • a retroviral/lentiviral (e.g. SIV) vector include 1x10 8 transduction units (TU), 1x10 9 TU, 1x10 10 TU, 1x10 11 TU or more.
  • the invention also provides compositions comprising the retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) described above, and a pharmaceutically-acceptable carrier.
  • pharmaceutically acceptable carriers include water, saline, and phosphate- buffered saline.
  • the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) of the invention may be administered by any appropriate route. It may be desired to direct the compositions of the present invention (as described above) to the respiratory system of a subject.
  • Efficient transmission of a therapeutic/prophylactic composition or medicament to the site of infection in the respiratory tract may be achieved by oral or intra-nasal administration, for example, as aerosols (e.g. nasal sprays), or by catheters.
  • the retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) of the invention are stable in clinically relevant nebulisers, inhalers (including metered dose inhalers), catheters and aerosols, etc.
  • the retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids e.g.
  • plasmids of the invention are formulated for administration to the lungs by any appropriate means, e.g. they may be formulated for intratracheal administration, intranasal administration, aerosol delivery, or direct injection or delivery to the lungs (e.g. delivered by catheter). Other modes of delivery, e.g. intravenous delivery, are also encompassed by the invention.
  • the nose is a preferred production site for a therapeutic protein using a retroviral/lentiviral (e.g. SIV) vector and/or nucleic acid (e.g.
  • plasmid of the invention for at least one of the following reasons: (i) extracellular barriers such as inflammatory cells and sputum are less pronounced in the nose; (ii) ease of vector administration; (iii) smaller quantities of vector/nucleic acid required; and (iv) ethical considerations.
  • transduction of nasal epithelial cells with a retroviral/lentiviral (e.g. SIV) vector or transfection with a nucleic acid (e.g. plasmid) of the invention may result in efficient (high-level) and long-lasting expression of the therapeutic transgene of interest.
  • nasal administration of a retroviral/lentiviral (e.g. SIV) vector or a nucleic acid e.g.
  • Formulations for intra-nasal administration may be in the form of nasal droplets or a nasal spray.
  • An intra-nasal formulation may comprise droplets having approximate diameters in the range of 100-5000 ⁇ m, such as 500-4000 ⁇ m, 1000-3000 ⁇ m or 100-1000 ⁇ m.
  • the droplets may be in the range of about 0.001-100 ⁇ l, such as 0.1-50 ⁇ l or 1.0-25 ⁇ l, or such as 0.001-1 ⁇ l.
  • the aerosol formulation may take the form of a powder, suspension or solution. The size of aerosol particles is relevant to the delivery capability of an aerosol.
  • the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli.
  • the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli.
  • the particles may have diameters in the approximate range of 0.1-50 ⁇ m, preferably 1-25 ⁇ m, more preferably 1-5 ⁇ m.
  • Aerosol particles may be for delivery using a nebulizer (e.g. via the mouth) or nasal spray.
  • An aerosol formulation may optionally contain a propellant and/or surfactant.
  • compositions comprising a vector of the invention, in particular where intranasal delivery is to be used, may comprise a humectant.
  • Suitable humectants include, for instance, sorbitol, mineral oil, vegetable oil and glycerol; soothing agents; membrane conditioners; sweeteners; and combinations thereof.
  • the compositions may comprise a surfactant.
  • Suitable surfactants include non-ionic, anionic and cationic surfactants. Examples of surfactants that may be used include, for example, polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides, such as for example, Tween 80, Polyoxyl 40 Stearate, Polyoxy ethylene 50 Stearate, fusieates, bile salts and Octoxynol.
  • a subsequent administration of a retroviral/lentiviral (e.g. SIV) vector and/or a nucleic acid (e.g. plasmid) may be performed.
  • the administration may, for instance, be at least a week, two weeks, a month, two months, six months, a year or more after the initial administration.
  • a retroviral/lentiviral (e.g. SIV) vector and/or a nucleic acid (e.g. plasmid) of the invention may be administered at least once a week, once a fortnight, once a month, every two months, every six months, annually or at longer intervals.
  • administration is every six months, more preferably annually.
  • the retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) may, for instance, be administered at intervals dictated by when the effects of the previous administration are decreasing. Any two or more retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) of the invention may be administered separately, sequentially or simultaneously.
  • two or more retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) wherein at least one retroviral/lentiviral (e.g. SIV) vector and/or nucleic acid (e.g.
  • plasmid is a retroviral/lentiviral (e.g. SIV) vector and/or nucleic acid (e.g. plasmid) of the invention, may be administered separately, simultaneously or sequentially.
  • two or more retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) of the invention may be administered in such a manner.
  • the two may be administered in the same or different compositions.
  • the two retroviral/lentiviral (e.g. SIV) vectors and/or nucleic acids (e.g. plasmids) may be delivered in the same composition.
  • Retroviral/lentiviral vectors which when transcribed, produce an mRNA which is identical in sequence to the retroviral/lentiviral (e.g. SIV) genome.
  • retroviral/lentiviral vector genome the prodrug
  • the transcribed mRNA which will then be translated to produce the therapeutic protein.
  • the present invention relates to retroviral/lentiviral (e.g.
  • the retroviral/lentiviral (e.g. SIV) genome (the prodrug) contains the sequence of an RRE-comprising intron.
  • This RRE-comprising intron is spliced out during transcription of the retroviral/lentiviral (e.g. SIV) genome, resulting in mRNA which lacks the RRE-comprising intron, and hence has a different nucleic acid sequence compared with the retroviral/lentiviral (e.g. SIV) genome from which it was derived.
  • the invention provides a means of discriminating between the retroviral/lentiviral (e.g. SIV) vector genome (the prodrug) and the mRNA (first step towards the active therapeutic). This may be useful, for example, during the production of the retroviral/lentiviral (e.g. SIV) vector, during its use in vitro and/or for evaluating clinical efficacy of the retroviral/lentiviral (e.g. SIV) vector.
  • the present invention provides a method for differentiating between a retroviral/lentiviral (e.g. SIV) vector and a transgene expressed by said retroviral vector, said method comprising or consisting of the steps of (a) transfecting cells with a retroviral/lentiviral (e.g. SIV) vector of the invention; (b) culturing the cells to allow transgene expression by the retroviral/lentiviral (e.g. SIV) vector; and (c) quantifying RNA within the cells; wherein (i) the amount of RNA comprising the intron into which a retroviral/lentiviral (e.g.
  • the present invention provides a method for differentiating between a retroviral/lentiviral (e.g. SIV) vector and mRNA transcribed from said retroviral/lentiviral (e.g. SIV) vector, said method comprising or consisting of the steps of (a) transfecting cells with a retroviral/lentiviral (e.g.
  • SIV retroviral/lentiviral vector of the invention
  • culturing the cells to allow transcription of the genome of the retroviral/lentiviral (e.g. SIV) vector e.g. SIV
  • quantifying RNA within the cells wherein (i) the amount of RNA comprising the intron into which a retroviral/lentiviral (e.g. SIV) RRE has been inserted corresponds to the copy number of the retroviral/lentiviral (e.g. SIV) vector; and (ii) the amount of RNA lacking the intron into which a retroviral/lentiviral (e.g. SIV) RRE has been inserted corresponds to the amount of mRNA transcribed from the retroviral/lentiviral (e.g.
  • SIV vector genome, and wherein optionally the amount of mRNA transcribed corresponds to the level of transgene expression.
  • Said methods may involve the quantification of RNA by any appropriate technique, examples of which are known in the art and may be selected by a skilled person without undue burden.
  • said method may involve the quantification of RNA by a PCR-based and/or in situ hybridisation-based assay.
  • Such PCR-based methods may comprise the use of two sets of primer pairs.
  • the first primer pair includes one primer which binds to a sequence outside the intron and another primer which binds to a sequence inside the intron. This first primer pair is capable of detecting and quantifying non-spliced retroviral/lentiviral (e.g. SIV) vectors.
  • the second primer pair comprises two primers which bind outside of the intron on either side and, therefore only quantifies spliced retroviral/lentiviral (e.g. SIV) vectors.
  • retroviral/lentiviral e.g. SIV
  • the second primer pair is specific for the mRNA transcribed from the retroviral/lentiviral (e.g. SIV) vector while the first primer pair will detect retroviral/lentiviral (e.g. SIV) vector genomes and integrated retroviral/lentiviral (e.g. SIV) DNA.
  • 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 labelling, 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
  • Exemplified FVIII transgene (N6) SEQ ID NO: 10
  • SEQ ID NO: 12
  • Example 1 Design and production of a ⁇ -globin/IgG chimeric intron and insertion into an SIV vector genome plasmid
  • a functional ⁇ -globin/IgG chimeric intron comprising an SIV RRE was designed using SnapGene® Software (version 5.3.2, Insightful Science, San Diego, CA, USA; available at www.snapgene.com).
  • the chimeric intron sequence of the pCI Mammalian Expression Vector (Cat. E1731, Promega, Madison, WI, USA) was analysed using the Basic Local Alignment Search Tool (BLAST, National Centre for Biotechnology Information, Bethesda, MD, USA; https://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • BLAST Basic Local Alignment Search Tool
  • a region homologous to the Hemoglobin Subunit B gene, as well as the splice donor, branch site, polypyrimidine tract, and splice acceptor were mapped onto the chimeric intron.
  • the Rev Response Element (RRE) from r.SIV (Griesenbach et al.
  • the synthesized Ultramer was double digested with KpnI-HF and NheI restriction enzymes (Cat.# R3142 and R3131, New England BioLabs, Ipswich, MA, USA) following manufacture’s protocols, size separated on a 1% agarose gel, purified with a Monarch® DNA gel extraction kit (Cat.# T1020, New England BioLabs), and ligated with T4 DNA ligase (Cat.# M0202, New England BioLabs) following manufacture’s protocols. Ligation products were heat shock transformed into 5 ⁇ competent Escherichia coli (Cat. #2987, New England BioLabs), plated on LB plates with 50 ⁇ g/mL of kanamycin, and incubated overnight at 37°C.
  • Figure 2G shows a plasmid map of the pGM407 SIV vector genome plasmid, which comprises an AAT transgene under the control of a hCEF promoter.
  • the pGM407 plasmid lacks an intron and comprises a SIV RRE.
  • Figure 2A shows a plasmid map of a plasmid derived from pGM407 which has had the SIV RRE deleted and the ⁇ -globin/IgG chimeric intron comprising an SIV RRE of Figure 1 inserted between with hCEF promoter and the AAT transgene, as described above.
  • Example 2 Transfection of HEK293T cells with a plasmid comprising a ⁇ -globin/IgG chimeric intron with an inserted SIV RRE increases AAT transgene expression HEK293T cells were transfected with a plasmid lacking the endogenous SIV RRE and comprising (i) an AAT transgene under the control of an hCEF promoter and (ii) a ⁇ -globin/IgG chimeric intron with an inserted SIV RRE as produced in Example 1 (pGM991).
  • transfection of HEK293T cells with pGM991 significantly increased AAT expression compared with transfection of HEK293T cells with a corresponding plasmid comprising the endogenous SIV RRE and lacking the ⁇ -globin/IgG chimeric intron with an inserted SIV RRE (pGM407).
  • an increase in AAT expression of 10.6-fold was observed on transfection with the plasmid lacking the endogenous SIV RRE and comprising the ⁇ -globin/IgG chimeric intron with an inserted SIV RRE compared with transfection with a corresponding plasmid comprising the endogenous SIV RRE and lacking said intron/RRE.
  • PCR of DNA extracted from the transfected HEK293T cells was carried out using primers that bind to either side of the intron: As shown in Figure 4A, the intron in pGM991 was present and a large 1127 bp product was detected. In contrast, pGM407 produced a smaller product of 371 bp. RNA was extracted from the HEK293T cells transfected with either pGM991 or pGM407.
  • RT- PCR was carried out on the extracted RNA using primers which bind to either side of the intron: As shown in Figure 4B, the intron was spliced successfully by the transfected HEK293T cells, such that both pGM991 and pGM407 produced the same 277 bp RNA transcript.
  • Example 3 SIV vector comprising a ⁇ -globin/IgG chimeric intron with an inserted SIV RRE is correctly packaged and spliced HEK293T cells were transduced with (a) a VSV-G pseudotyped SIV vector (SIV.VSV-G) lacking the endogenous SIV RRE and comprising (i) an AAT transgene under the control of an hCEF promoter and (ii) a ⁇ -globin/IgG chimeric intron with an inserted SIV RRE as produced in Example 1 (vGM291); or (b) a SIV.VSV-G vector comprising the endogenous SIV RRE and lacking this chimeric intron-RRE (vGM290).
  • VSV-G pseudotyped SIV vector SIV.VSV-G
  • Non-transduced cells were used as controls. PCR of DNA extracted from the transduced HEK293T was carried out using primers which bind to either side of the intron: As shown in Figure 5, vGM291 was properly packaged to produce SIV.VSV-G particles, with an 1127 bp product being detected. In contrast, vGM290 produced a smaller product of 371 bp. RNA was extracted from the transduced HEK293T cells transduced with either vGM291; or vGM290.
  • RT-PCR was carried out on the extracted RNA using primers which bind to either side of the intron: As shown in Figure 6, the intron was spliced successfully by the transduced HEK293T cells, such that both vGM290 and vGM291 produced the same 277 bp RNA transcript. The effect of inclusion of the ⁇ -globin/IgG chimeric intron with an inserted SIV RRE on AAT transgene expression was also investigated.
  • transduction of HEK293T cells with this SIV.VSV-G vector significantly increased AAT expression compared with transduction of HEK293T cells with a corresponding SIV.VSV-G vector comprising the endogenous SIV RRE and lacking the ⁇ - globin/IgG chimeric intron with an inserted SIV RRE (vGM290).
  • vGM290 ⁇ -globin/IgG chimeric intron with an inserted SIV RRE
  • RT-ddPCR Reverse transcription droplet digital PCR
  • Example 5 SIV.FN/H vector comprising a ⁇ -globin/IgG chimeric intron with an inserted SIV RRE increases AAT transgene transcription
  • the experiment in Example 3 was repeated with an F/HN pseudotyped SIV vector, rather than a VSV-G pseudotyped SIV vector.
  • transduction of HEK293T cells with a SIV.F/HN vector containing the ⁇ -globin/IgG chimeric intron significantly increased AAT expression compared to HEK293T cells transduced with a corresponding SIV.F/HN vector comprising the endogenous SIV RRE and lacking the ⁇ -globin/IgG chimeric intron (vGM294).
  • vGM295 a SIV.F/HN vector containing the ⁇ -globin/IgG chimeric intron
  • ⁇ -globin/IgG chimeric intron with an inserted SIV RRE facilitates the maturation of a stable mRNA molecule, resulting in increased mRNA transcript levels, and consequently increased transgene expression, and this effect is observed in multiple different pseudotyped SIV vectors. Whilst this is exemplified with AAT, it is expected that similar advantages will be achieved using other transgenes.

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Abstract

La présente invention concerne des vecteurs rétroviraux modifiés permettant d'améliorer l'expression transgénique. En particulier, l'invention concerne des vecteurs rétroviraux dépourvus d'un élément de réponse Rev (RRE) endogène et comprenant un intron, en particulier un intron chimérique, dans lequel un RRE a été inséré, ainsi que leurs méthodes de production et leurs utilisations.
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