WO2024201066A1 - Novel vector - Google Patents

Abstract

The invention provides inter alia optimised cDNA sequences encoding human IDUA, lentiviral vectors comprising said cDNA sequences and additional elements, which may improve therapeutic potential in regards to MPS-I treatment, of the lentiviral vectors.

Description

NOVEL VECTOR

Field of the invention

The invention relates to development of vectors for delivery and expression of therapeutic genes, including IDUA for the treatment of Hurler’s syndrome or the milder forms of IDUA deficiency, and to related aspects.

Background of the invention

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive genetic disease caused by dysfunctional variants in both alleles of the gene which encodes a-L-iduronidase (IDUA). IDUA is an essential enzyme for the degradation of glycosaminoglycans (GAGs), degrading iduronate sulfatase to heparan N-sulfatase. Mutation of the gene leads to deficiency of the IDUA protein’s enzymatic activity. Accumulation of GAGs, such as dermatan and heparan sulfates, in lysosomes leads to dysfunction of multiple tissues and organs.

The quantity of residual IDUA activity in cells determines the severity of the MPS-I disease. If the patient has between 1 and 2 % residual IDUA activity in cells they will suffer from a mild form of the disease, known as MPS-IS (Scheie syndrome). If the patient has between 0.5 and 1% residual IDUA activity in cells they will suffer from an intermediate form of the syndrome, leading to death before the age of 30. This is known as MPS-I H/S (Hurler-Scheie syndrome). If the patient only has 0 to 0.5% residual IDUA activity in cells, they will suffer from a severe disease phenotype, with death likely to occur in the first decade of life. This syndrome is categorised as MPS-IH, or Hurler Syndrome.

The current standard of care for MPS-I syndrome involves a haematopoietic stem cell transplant (HSCT) and/or enzyme replacement therapy. After HSCT, IDUA enzyme secreted by donor leukocytes circulating in the blood and in tissues rescues the recipients IDUA-deficient cells.

The aim of the invention is to provide vectors for the delivery and expression of therapeutic genes, including IDUA for the treatment of Hurler’s syndrome or the milder forms of IDUA deficiency.

Gene transfer can be limited to the liver by using a hepatocyte-specific transcription factor site linked to the transthyretin promoter (abbreviated as ET promoter). Alternatively, gene transfer can be limited to cells with a high proliferation rate, such as PBMCs, haematopoietic or stem cells, by using the eukaryotic elongation factor- 1 promoter (EF1a promoter). Summary of the invention

The present inventors have provided inter alia a lentiviral-based therapy for MPS-I. In particular embodiments, lentiviral vectors are provided which comprise elements further enhancing the vectors and rendering them particularly suitable for MPS-I treatment. Surprisingly, the inventors found that (a) the inclusion of these elements were not detrimental to viral vector titer and (b) the inclusion of these elements provided therapeutic function, while retaining the benefits of each of the elements (i.e. these elements did not have a detrimental effect on each other).

In one aspect the invention provides codon optimised cDNA-sequences encoding human and mouse IDIIA.

In a further aspect the invention provides a description of vectors for delivery and expression of therapeutic genes for use in the treatment of inherited genetic disorders.

In a further aspect the invention provides the use of a vector in the manufacture of a medicament for the treatment of MPS deficiencies.

In a further aspect the invention provides a method of treating genetic disorders in a subject which method comprises administering to the subject with vectors containing ubiquitous or tissue specific promoters.

In a further aspect the invention provides a pharmaceutical composition comprising vectors with the key design features present in the maps provided in this submission, as exemplified by the map present in Figure 1.

In a further aspect the invention provides a description of vectors and transfer vectors with additional features which may improve therapeutic potential by: immune system detection avoidance, suppression of transgene expression in antigen presenting cells, reduced oncogenic transformation potential, improved transgene expression, and/or improved biodistribution of the transgene product.

Further aspects of the invention will become clear from the text and figures which follows.

Summary of the sequences

SEQ ID NO: 1 - cDNA sequence encoding Human IDIIA

SEQ ID NO: 2 - Codon optimised cDNA sequence encoding human IDIIA

SEQ ID NO: 3 - Codon optimised cDNA sequence encoding human IDIIA with reduced GC content

SEQ ID NO: 4 - cDNA sequence encoding mouse IDIIA

SEQ ID NO: 5 - Codon optimised cDNA sequence encoding mouse IDIIA SEQ ID NO: 6 - cDNA Enhanced Green Fluorescent Protein NanoLuciferase EGFPNLuc

SEQ ID NOs: 7 to 18 - Full length vector sequences comprising microRNA-142 binding sites SEQ ID NOs: 19 to 30 - Full length vector sequences without microRNA-142 binding sites SEQ ID NO: 31 - Polypeptide sequence of human IDIIA

SEQ ID NO: 32 - Polypeptide sequence of mouse IDIIA

SEQ ID NO: 33 - Polynucleotide sequence of EF1a

SEQ ID NO: 34 - Polynucleotide sequence of ET

SEQ ID NO: 35 - Lentiviral 5’-LTR polynucleotide sequence

SEQ ID NO: 36 - Psi polynucleotide sequence

SEQ ID NO: 37 - gag (matrix-encoding) polynucleotide sequence

SEQ ID NO: 38 - CPPT Tract polynucleotide sequence

SEQ ID NO: 39 - Flap polynucleotide sequence

SEQ ID NO: 40 - WPRE polynucleotide sequence

SEQ ID NO: 41 - Lentiviral 3’-LTR polynucleotide sequence

SEQ ID NO: 42 - Lentiviral packaging (RRE) polynucleotide sequence

SEQ ID NO: 43 - HIV gag polypeptide sequence

SEQ ID NO: 44 - Polynucleotide sequence encoding a tEGFR

SEQ ID NO: 45 - Polynucleotide sequence of a genomic insulator (DNA)

SEQ ID NO: 46 - Polynucleotide sequence of a genomic insulator (RNA)

Summary of the figures

Figure 1 - Vector maps of designed vectors with microRNA 142 binding sites

Figure 2 - Vector maps of designed vectors

Figure 3 - Phase contrast and GFP microscopy of cells transformed with Vector 11 or Vector 12 Figure 4 - Phase contrast and GFP microscopy of 293T kidney cells transformed with Vector 17 Figure 5 - FACS analysis of 293T kidney cells transformed with Vector 17

Figure 6 - Phase and GFP microscopy of HepG2 HCC cells transformed with Vector 18

Figure 7 - FACS analysis of HepG2 HCC cells transformed with Vector 18

Figure 8 - IDUA protein expression in transformed cell lines. (A and B) Combination 1 -Vector 12; Combination 2-Vector 14; Combination 3-Vector 12; Combination 4-Vector 18; Combination 5- Vector 13.

Figure 9 - IDUA enzymatic activity in transformed cell lines. Combination 1-Vector 12;

Combination 2-Vector 14; Combination 3-Vector 12; Combination 4-Vector 11 ; Combination 5- Vector 13. Figure 10 - Quantification of intracellular GAGS in Hurler GM00798 cells after cross-correction with transformed cells. White bar- Untreated Hurler GM00798; Orange bar-Vector 17 control; Grey bar- Vector 11.

Figure 11 - Body weight of mice following treatment with vectors. Group 1 (PBS/Saline control); Group 2-Vector 13; Group 3-Vector 14; Group 4-Vector 17; Group 5- Vector 18.

Figure 12 - mIDUA gene expression in lung, liver, spleen and bone marrow of mice following treatment with vectors

Figure 13 - mIDUA activity in plasma of mice following treatment with vectors (Low dose LV treatment)

Figure 14 - IDUA enzymatic activity in mice (High dose LV treatment; PBS/Saline vs Vector 14 vs Vector 13)

Figure 15A - A liver-specific immunologically privileged vector that is restricted to the expression of A1AT in liver cells by the hTTR promoter and the miR-142 target site at 3’ UTR. This also includes truncated EGFR (tEGFR) as a safety measure to ensure the killing of transduced cells when needed with an anti-EGFR antibody (Wang et al. 2011).

Figure 15B - Illustration that miR-142 expression is liver cell specific and not expressed in monocytes. Murine RAW264.7 and human monocyte U937 cells were transduced with control pHIV-GFP and pHIV7-GFP-miR-142 and screened for GFP expression. Embedding miR-142 target site into the 3’ UTR inhibits GFP expression in RAW264.7 and U937 monocytic cell lines that express miR-142, which degrades the miR-142 containing GFP transcript.

Figure 15C - TTR promoter and embedded miR142 target site in the 3’ UTR restricts transgene expression in phagocytic cells. HEK293 and RAW264.7 cells were transfected with GFP-Fluc vector containing the miR-142 site in the 3’ UTR, and luciferase expression was assessed 48 hours post-transfection.

Figure 15D - To assess liver-specific expression, HEK293 cells were transduced with a vector containing GFP-Fluc expressed from an mTTR promoter. The percentage of positive cells was assessed at days, 0, 5, and 11 post-transduction.

Figure 16A - Surface expression of CD47. A dual-CD47/CD55 vector was generated and assessed for over-expression of CD47/CD55 from producer HEK293 cells.

Figure 16B - Surface expression of CD55.

Figure 16C - High expression of both CD47 and CD55 was observed on the same transfected cells. Detailed description of the invention

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

Polypeptides are organic polymers consisting of a number of amino acid residues bonded together in a chain. As used herein, ‘polypeptide’ is used interchangeably with ‘protein’ and ‘peptide’.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein.

As used herein, "expression" refers to the two-step process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

"Expression is controlled by" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element that contributes to the initiation of, or promotes, transcription. "Operatively linked" intends that the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, this invention provides promoters operatively linked to the downstream sequences.

The term "encode" as it is applied to polynucleotides refers to a polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom (which can also be referred as the ‘sense’ strand.

The term "promoter" as used herein means a control sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or a transgene, are controlled. Promoters may be constitutive, inducible, repressible, or tissue-specific. Promoters may contain genetic elements at which regulatory proteins and molecules such as RNA polymerase and transcription factors may bind. It is known in the art that the nucleotide sequences of such promoters may be modified in order to increase or decrease the efficiency of mRNA transcription. See, e.g., Gao et al. (2018) (modifying TATA box of 7SK, U6 and HI promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase Il-dependent mRNA transcription). Synthetically-derived promoters may be used for ubiquitous or tissue specific expression. Further, virus-derived promoters, some of which are noted above, may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters. In embodiments, the promoter is used together with an enhancer to increase the transcription efficiency.

An enhancer is a regulatory element that increases the expression of a target sequence. The enhancer or promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer or promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e. , molecular biological techniques) such that transcription of that gene is directed by the linked enhancer or promoter. It is understood in the art that enhancers can operate from a distance and irrespective of their orientation relative to the location of an endogenous or heterologous promoter. It is thus further understood that an enhancer operating at a distance from a promoter is thus “operably linked” to that promoter irrespective of its location in the vector or its orientation relative to the location of the promoter.

A “transgene” is a gene that has, or is intended for, transfer from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code.

A “genetic insulator” or “insulator” is a cis-regulatory element that functions in two ways which may or may not be mutually exclusive. “Barrier” insulators prevent the spread of “heterochromatin” and subsequent silencing of enhancers, promoters, or gene bodies within euchromatin. “Enhancer-blocking” insulators inhibit distal enhancers from influencing neighbouring genes. Depending on locations, enhancer-blocking insulators may also inhibit local enhancers from acting on distal or neighbouring genes. It has previously been found that certain CTFC sequences in the human genome can serve as effective insulators (WO2015138852A1). Insulators can be beneficial in gene therapy. Barrier Insulators - Lowering the chance of transgene silencing over time. Enhancer-blocking Insulators - Attenuating transgene enhancer activity on neighbouring genes which lowers the insertional mutagenesis potential (activation of nearby proto-oncogenes) of transgene integration. Additionally, insulators may act by attenuation of transcriptional elongation beyond a defined transcriptional stop site. Attenuation of transcriptional elongation can increase the level of transgene expression as erroneously elongated transcripts may be quickly targeted for degradation by the cell. However, genetic insulator sequences, when present in lentiviral vectors, are known to negatively impact infectious titre. Lower infectious titres in turn requires a higher concentration of viral vector to be administered to a patient resulting in a more costly (production) and dangerous (concentrationdependent immune response) therapy.

A “micro-RNA target site” refers to a sequence within an RNA, often the 3’ UTR, that a miRNA, as part of the RNA-induced silencing complex (RISC), recognizes and binds to leading to post transcriptional silencing of the target RNA. It has previously been found that incorporating mir-142 target sites within an expressed transgene or exogenous RNA leads to suppression of expression of the gene or RNA product in antigen producing cells (APCs) (Brown, et al. 2006). Thus, mir-142 target sites in transgene transcripts are thought to improve gene therapies by attenuating the immune system’s detection and clearance of transgene expressing cells. “microRNA-142-3p”, “miR-142”, “miR-142BS”, “miR-142 target site” are used interchangeably herein.

A “therapeutic transgene” is a transgene encoding a correctly functioning protein which may be used in transduction of cells in a subject suffering from a genetic disorder in which a defective version of the protein is produced.

The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Expression vectors include viral vectors (e.g., replication defective retroviruses, lentiviral vectors, adenoviruses, Sendai viruses and adeno-associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. Another type of vector includes RNA molecules, e.g., mRNA and stabilised RNA, to carry coding genetic information to the cells.

A ’’fusion protein” or fusion polypeptide is the result of one discrete polypeptide being functionally linked to another polypeptide through a peptide bond during protein translation. Fusion proteins are made by functionally linking the underlying DNA sequences encoding both polypeptides in such a way that the translation of the resulting transcribed mRNA into polypeptide creates the first polypeptide chain followed by what was the original N-terminus of the second polypeptide chain to what was the original C-terminus of the first polypeptide. The C-terminus of the first and N-terminus of the second polypeptide chains can be truncated by multiple amino acids. Additionally, a linker amino acid sequence may be included to link the two original polypeptide chains together as an alternative to direct linkage between original N- and C- termini.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell, for example, when said progeny are employed to make a cell line or cell bank which is then optionally stored, provided, sold, transferred, or employed to manufacture a polypeptide, antibody or fragment thereof as described herein.

With respect to general recombinant techniques, vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif) and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of cloned transgenes to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.

Alternatively, consensus ribosome binding sites (Kozak Sequence) can be inserted immediately 5' of the start codon to enhance expression. A "viral vector" is defined as a recombinantly produced virus or viral particle that contains a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors and the like. A viral vector is capable of infecting and transducing a cell.

A “lentiviral vector” is a viral vector based on a lentivirus, comprising one or more genetic elements from a lentivirus. The vector may comprise a transgene for transduction of a cell. The genetic elements may have undergone modification, such as substitutions, deletions or insertions, relative to their native lentiviral sequence. Nonetheless, the genetic elements are ‘from’ a lentivirus and the vector remains a lentiviral vector. Suitably the genetic elements are substantially similar to their native counterparts. Suitably, the genetic elements share at least 50%, such as at least 70%, such as at least 90%, such as at least 99% identity with their native sequences. The genetic elements are operably combined. A lentiviral vector is capable of infection of a host cell, integration into the genome of a host cell and transduction of the cell, allowing for stable transgene expression. A lentiviral vector comprises lentiviral structural elements necessary for infection of a host cell, e.g. the five major lentiviral structural proteins and 3-4 non-structural proteins: gp120 surface envelope protein Sil, gp41 transmembrane envelope protein TM, P24 capsid protein CA, P17 matrix protein MA, P7/P9 capsid protein NC and will also typically comprise reverse transcriptase, integrase, protease and dllTPase enzymes; Tat and Rev gene regulatory proteins, and Nef, Vpr, Vif, Vpu/Vpx and p6 accessory proteins.

A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses, such as baculoviruses, adenoviruses and retroviruses; bacteriophage, cosmid, plasmid, and fungal vectors; and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Liposomes that also comprise, consist essentially of, or consist of a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins disclosed herein are other non-limiting techniques. A “lentiviral transfer vector” is a vector comprising one or more genetic elements from a lentivirus and optionally a transgene, which can be used alongside packaging genes (e.g. in the form of one or more packaging plasmids) to transfect a host cell to produce lentiviral vectors. Generally, the transfer plasmid contains a transgene along with the essential cis-acting elements, while essential trans-acting genes provided separately (in trans) by the packaging plasmids.

A polynucleotide disclosed herein can be delivered to a cell or tissue using a gene delivery vehicle. "Gene delivery," "gene transfer," "transducing," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector- mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

A "composition" is intended to mean a combination of active polypeptide, polynucleotide or antibody, and another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

A "pharmaceutical composition" is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives.

References to “subject” or “patient” refer to a subject, in particular a mammalian subject, to be treated. Mammalian subjects include humans, non-human primates, farm animals (such as cows), sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats or mice. In some embodiments, the subject is a human or a mouse. Most suitably the subject is a human. The term "tissue" is used herein to refer to tissue of a living or deceased organism or any tissue derived from or designed to mimic a living or deceased organism. The tissue may be healthy, diseased, and/or have genetic mutations. The biological tissue may include any single tissue (e.g., a collection of cells that may be interconnected), or a group of tissues making up an organ or part or region of the body of an organism. The tissue may comprise, consist essentially of, or consist of a homogeneous cellular material or it may be a composite structure such as that found in regions of the body including the thorax which for instance can include lung tissue, skeletal tissue, and/or muscle tissue. Exemplary tissues include, but are not limited to those derived from liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart and intestines, including any combination thereof.

The term “truncated EGFR” (tEGFR) refers to a polypeptide coding for human epidermal growth factor receptor where the sequences that encode the extracellular N-terminal ligand binding domains and the intracellular receptor tyrosine kinase activity domains have either been removed or made defective by partial removal (i.e. removed or defective due to sequence truncation). tEGFR is useful for sorting or targeting cells which have been transduced with a lentiviral vector due to tEGFR being expressed on the cell’s surface (X. Wang 2011 , which is incorporated by reference for the purpose of the tEGFR sequences disclosed therein). In one embodiment, tEGFR is encoded by the polynucleotide sequence of SEQ ID NO: 44 or a variant thereof. Additionally, antibody I antibody drug conjugates have been made to specifically target these cells to remove them from a population should they become problematic (e.g. tumorigenic).

A “internal ribosomal entry site” (IRES) refers to a nucleic acid sequence that allows an mRNA to be translated from a site that is no proximal to its 5’ cap. IRES elements are often added in such a way that a transgene of interest and another transgene of interest may be expressed from a single mRNA transcript that is transcribed from a transgene cassette.

As used herein, “treating a disease or disorder” means reducing the frequency and/or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

As used herein, the term "administer" or "administration" intends to mean delivery of a substance to a subject such as an animal or human. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and other animals, treating veterinarian.

As used herein, the term “about” includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between”, includes the values of the specified boundaries.

“Potency” is a measure of the activity of a therapeutic agent expressed in terms of the amount required to produce an effect of given intensity. A highly potent agent evokes a greater response at low concentrations compared to an agent of lower potency that evokes a smaller response at low concentrations. Potency is a function of affinity and efficacy. Efficacy refers to the ability of therapeutic agent to produce a biological response upon binding to a target and the quantitative magnitude of this response. The term half maximal effective concentration (EC50) refers to the concentration of a therapeutic agent which causes a response halfway between the baseline and maximum after a specified exposure time. The therapeutic agent may cause inhibition or stimulation. It is commonly used, and is used herein, as a measure of potency.

Lentiviral vectors

Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have emerged as promising tools for both gene therapy and immunotherapy purposes, because they exhibit several advantages over other viral systems. In particular, lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are capable of transducing non-dividing cells, in particular dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6): 913-24), allowing stable transduction and antigen presentation through the endogenous pathway.

Lentiviruses are linked by similarities in genetic composition, molecular mechanisms of replication and biological interactions with their hosts. They are best known as agents of slow disease syndromes that begin insidiously after prolonged periods of subclinical infection and progress slowly; thus, they are referred to as the "slow" viruses (Narayan et al. 1989). They have the same basic organization as all retroviruses but are more complex due to the presence of accessory genes (e.g., vif, vpr, vpu, nef, tat, and rev), which play key roles in lentiviral replication in vivo.

Lentiviruses represent a genus of slow viruses of the Retroviridae family, which includes the human immunodeficiency viruses (HIV), the simian immunodeficiency virus (SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection. Persistent replication of the viruses in their hosts depends on their ability to circumvent host defences.

To produce lentiviral vectors, the transfer plasmid, which comprises a gene of interest and lentiviral genetic elements, and other plasmids, which comprise viral genes required for lentivirus packaging, are co-transduced into 293T cells. After plasmid transfection, lentivirus vector particles comprising the gene sequence of interest will be released into a culture medium. The culture medium will be harvested later and the particles will be concentrated, then formulated to the concentration ready for use and stored at -80°C.

A lentiviral vector comprises the elements necessary for infection and transduction of a host cell. Typically these are an envelope, matrix (MA), capsid (CA), ssRNA genome bound by nucleocapsid (NC), lipid membrane, reverse transcriptase (RT), integrase (IN) and protease (PR). In a specific embodiment the lentiviral vector comprises the five major lentiviral structural proteins and 3-4 non-structural proteins: gp120 surface envelope protein Sil, gp41 transmembrane envelope protein TM, P24 capsid protein CA, P17 matrix protein MA, P7/P9 capsid protein NC and also comprises reverse transcriptase, integrase, protease and dllTPase enzymes; Tat and Rev gene regulatory proteins, and Nef, Vpr, Vif, Vpu/Vpx and p6 accessory proteins.

Over the past decades, the lentivirus packaging system has developed constantly. The first-generation lentivirus packaging system at the very beginning involves three plasmids, including a transfer plasmid, a plasmid that can express an envelope protein, and a plasmid comprising both essential HIV-1 viral genes (gag, pol, tat and rev) and several accessory genes (vif , vpu, vpr and nef ). The viral gag gene encodes several viral capsid proteins, and the viral pol gene encodes reverse transcriptase, integrase and protease that are important for virus packaging and infection. As the four accessory genes are neither necessary for lentivirus packaging nor required for target cell infection, they were removed from the second-generation lentivirus packaging system. In the first- and second-generation lentivirus packaging systems, the transcription of viral genome comprising the gene of interest in the transfer plasmid is driven by both the 5' long terminal repeat (LTR), which serves as a promoter, and TAT protein, a kind of trans-activating regulatory protein that can bind to the trans-activation response (TAR) sequence in the LTR.

For the third-generation lentivirus packaging system, in order to further improve the safety of lentivirus, especially to avoid the development of replication competent lentivirus, the 5' and 3' LTR flanking the gene of interest were modified and the rev gene was moved to a fourth plasmid to further reduce the chance of sequence recombination. Accordingly, the modified LTR no longer retained the promoter activity, and the transcription of viral genome sequence on the transfer plasmid was instead driven by a Rous sarcoma virus (RSV) promoter or a cytomegalovirus (CMV) promoter positioned in front of the modified 5' LTR. Furthermore, the tat gene was removed from the third-generation lentivirus packaging system to enhance the transduction efficiency of the lentivirus packaging system.

Suitably, the vector according to the invention is packaged in a third-generation lentivirus packaging system.

Typically to achieve transduction using lentiviral vector systems, 3-4 plasmids are transfected into host cells such as A293T cells. These are:

• one transfer plasmid or ‘lentiviral transfer plasmid’

• one or two packaging plasmid(s)

• one envelope plasmid.

Lentiviral vectors are then produced from the host cells, which are used to transduce target cells with transgene (e.g. IDIIA in the context of the present invention).

The lentiviral vector of the invention may therefore include nucleic acid sequences which are typically found in plasmids used for the production of lentiviral vectors, such as those discussed above.

The lentiviral vector of the invention may be produced using a composition comprising a lentiviral transfer vector and optionally packaging genes and/or envelope gene. The various genes may be provided on separate vectors such as plasmids. The composition may be used to infect host cells (such as HEK293 cells) for production of lentiviral vectors of the invention.

The lentiviral vector may comprise an envelope, matrix (MA), capsid (CA), ssRNA genome bound by nucleocapsid (NC), lipid membrane, reverse transcriptase (RT), integrase (IN) and protease (PR). In a specific embodiment the lentiviral vector comprises the five major lentiviral structural proteins and 3-4 non-structural proteins: gp120 surface envelope protein Sil, gp41 transmembrane envelope protein TM, P24 capsid protein CA, P17 matrix protein MA, P7/P9 capsid protein NC and also comprises reverse transcriptase, integrase, protease and dllTPase enzymes; Tat and Rev gene regulatory proteins, and Nef, Vpr, Vif, Vpu/Vpx and p6 accessory proteins.

The lentiviral vector of the invention, or the transfer vector(s), packaging vector(s) and envelope vector(s) used to produce the lentiviral vector of the invention, may comprise one or more of the lentiviral vector system components as follows.

The lentiviral vector or the lentiviral transfer vector in one embodiment comprise a lentiviral packaging signal (^P). Viral packaging genes are typically provided on a packaging vector, such as a packaging plasmid. Suitably the packaging genes encode lentiviral protease, lentiviral reverse transcriptase and/or lentiviral integrase.

All elements of the lentiviral transfer vector or the lentiviral vector (e.g. lentiviral packaging signal (^P) and viral genes) are provided such that, on delivery to a cell, the elements will become operably combined. In particular, if a promoter or 5’-LTR is included in the lentiviral vector, the promoter or 5’-LTR is operably combined, such as operably linked, to the viral genes.

Each element may be derived from any suitable lentivirus, or for example from another virus. However, in a particularly suitable embodiment, the gag, pol, rev and the lentiviral genome (packaging signal, 3'-LTR and a 5'-LTR) are derived from a HIV virus, in particular from HIV-1 or HIV-2. Suitably the gag, pol, rev and the lentiviral genome (packaging signal, 3'-LTR and a 5'-LTR) are derived from the same lentivirus.

Any suitable lentiviral 5' LTR can be utilized, including an LTR obtained from any lentivirus species, sub-species, strain or clade. This includes primate and non-primate lentiviruses. Specific examples of species, etc., include, but are not limited to, e.g., HIV-1 (including subspecies, clades, or strains, such as A, B, C, D, E, F, and G, R5 and R5X4 viruses, etc.), HIV-2 (including subspecies, clades, or strains, such as, R5 and R5X4 viruses, etc.), simian immunodeficiency virus (SIV), simian/human immunodeficiency virus (SHIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine-arthritis- encephalitis virus, Jembrana disease virus, ovine lentivirus, visna virus, and equine infectious anemia virus. The 5’-LTR as used herein refers to a full length 5’-LTR or a functional fragment thereof. Similarly, the 3’-LTR as used herein refers to a full length 3’-LTR or a functional fragment thereof. A functional fragment is a truncated version of the full-length sequence which nonetheless substantially maintains function. In the case of the 5’-LTR, the functional fragment maintains activity as an RNA pol II promoter. In the case of the 3’-LTR, the functional fragment maintains the ability to terminate transcription.

The lentiviral 5' LTR comprises signals utilized in gene expression, including enhancer, promoter, transcription initiation (capping), transcription terminator, and polyadenylation. They are typically described as having U3, R, and U5 regions. The U3 region of the LTR contains enhancer, promoter and transcriptional regulatory signals, including RBEIII, NF-kB, Sp1, AP-1 and/or GABP motifs. The TATA box is located about 25 base pairs from the beginning of the R sequence, depending on the species and strain from which the 5' LTR was obtained. A completely intact 5' LTR can be utilized, or a modified copy can be utilized. Modifications preferably involve the R region, where a TAR sequence is substituted and/or deletion of all or part of a U5 region. The modified 5' LTR preferably comprises promoter and enhancer activity, e.g., preferably native U3, modified R with a substituted TAR, and native U5.

The 5' LTR can be operably linked to a polynucleotide sequence coding for lentivirus gag and pol. By the term "operably linked," it is meant that the LTR is positioned in such a way that it can drive transcription of the recited coding sequences. The gag and pol coding sequences are organized as the Gag-Pol Precursor in native lentivirus. The gag sequence codes for a 55-kD Gag precursor protein, also called p55. The p55 is cleaved by the vi rally encoded protease (a product of the pol gene) during the process of maturation into four smaller proteins designated MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved away from Gag by a vi rally encoded protease, and further digested to separate the protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities.

The standard purpose of lentiviral vector backbone plasmids is to provide a template for the synthesis of lentiviral vector genomic RNA, which can be successfully packaged into lentiviral vector virions, reverse transcribed and integrated within the cellular genome. The ^P-sequence close to the 5’-LTR is believed to be strictly required for the packaging of RNA by the Gag polyprotein. A substantial portion of the sequences within the LTRs of the lentiviral genome is required for chromosomal integration. However, some sequences within the LTRs can be removed without reduction in the integration efficiency. Wild-type retroviral and lentiviral genomes contain a promoter within their 5’-LTR to drive expression of genomic RNA. The promoter sequences can be deleted from the 5’-LTR DNA segment in the lentiviral vector backbone plasmids and a strong constitutive promoter capable of directing synthesis of the vector genomic RNA, e.g. immediate early CMV promoter, can be placed externally to the bracket of the lentiviral sequences within the plasmids. The same principles apply to the compositions of the present invention.

Native gag and pol sequences can be utilized, or modifications can be made. These modifications include, chimeric Gag-Pol, where the Gag and Pol sequences are obtained from different viruses (e.g., different species, subspecies, strains, clades, etc., and/or where the sequences have been modified to improve transcription and/or translation, and/or reduce recombination). In other embodiments, the sequences coding for the gag and pol precursors can be separated and placed on different vector constructs, where each sequence has its own expression signals. The lenti viral gag gene encodes structural proteins. The lentiviral gag gene includes a full length lentiviral gag gene, functional fragments and variants thereof. More suitably, the lentiviral gag gene is a full length lentiviral gag gene.

The lentiviral pol gene encodes enzymes required for reverse transcription and integration into the host cell genome. The lentiviral pol gene includes a full length lentiviral pol gene, functional fragments and variants thereof. More suitably, the lentiviral pol gene is a full length lentiviral pol gene.

The lentiviral env gene encodes the viral envelope glycoprotein. The lentiviral env gene includes a full length lentiviral env gene, functional fragments and variants thereof. More suitably, the lentiviral env gene is a full length lentiviral env gene.

The lentiviral rev gene is believed to be essential for post-transcriptional transport of the unspliced and incompletely spliced viral mRNAs from nuclei to cytoplasm. The lentiviral rev gene includes a full length lentiviral rev gene, functional fragments and variants thereof. More suitably, the lentiviral rev gene is a full length lentiviral rev gene.

The Rev protein acts via binding to an RNA structural element known as the Rev responsive element (RRE). The Rev response element (RRE) is the sequence to which the Rev protein binds.

The lentiviral packaging signal (^P) is the RNA target site for packaging by nucleocapsid.

The RNA genome of HIV-1 contains an approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The critical portions of the packaging signal are between the major splice donor (SD) site and the gag initiation codon of the HIV provirus, about distal to the U5 region of the 5' LTR.

Additional promoter and enhancer sequences can be placed upstream of the 5' LTR in order to increase, improve, enhance, etc., transcription of the gag-pol precursor. Examples of useful promoters, include, mammalian promoters (e.g., constitutive, inducible, tissue-specific), CMV, RSV, LTR from other lentiviral species, and other promoters.

In addition, a vector can further comprise transcription termination signals, such as a polyA signal that is effective to terminate transcription driven by the promoter sequence. Any suitable polyA sequence can be utilized, e.g., sequences from beta globin (mammalian, human, rabbit, etc), thymidine kinase, growth hormone, SV40, and many others.

A vector can further comprise a TAR element that is obtained from a different lentiviral species, group, sub-species, sub-group, strain, or clade than the 5' LTR and/or the gag and pol sequences that are present in it, i.e. , it is heterologous to other lentiviral elements present in the plasmid construct. Such a vector may be a plasmid, and may be referred to as a helper plasmid. The TAR is preferably present in the 5' LTR in its normal location, e.g., between the U3 and U5 elements of the LTR, e.g., where the native R is replaced by R' of a heterologous lentiviral species.

The TAR element is a trans-activating response region or response element that is located in the 5'LTR (e.g., R) of the viral DNA and at the 5' terminus of the corresponding RNA. When present in the lentiviral RNA, the transcriptional transactivator, Tat, binds to it, activating transcription from the HIV LTR many-fold. Tat is an RNA binding protein that binds to a shortstem loop structure formed by the TAR element.

When a heterologous TAR element is utilized, the 5' LTR can be modified routinely by substituting its native TAR for a TAR sequence from another species. Examples of TAR regions are widely known. Such a modified lentiviral 5' LTR can comprise intact U3 and U5 regions, such that the LTR is completely functional.

As indicated above, the Tat polypeptide binds to the TAR sequence. The coding sequence for Tat can be present in the helper plasmid, or it can be on another element in the packaging system. For example, it can be present on another plasmid. Any Tat polypeptide can be utilized as long as it is capable of binding to TAR and activating transcription of the RNA. This includes native Tat sequences which are obtained from the same or different species as the cognate TAR element, as well as engineered and modified Tat sequences.

The vector may comprise a Rev response element (‘RRE’, or ‘RRE element’), optionally an RRE element which is obtained from a different lentiviral species than the 5' LTR or gag and pol sequences. The RRE element is the binding site for the rev polypeptide which is a 13-kD sequence-specific RNA binding protein. Constructs which contain the RRE sequence depend on the rev polypeptide for efficient expression. Rev binds to a 240-base region of complex RNA secondary structure of the rev response element that is located within the second intron of HIV, distal to the pol and gag coding sequences. The binding of rev to RRE facilitates the export of unspliced and incompletely spliced viral RNAs from the nucleus to the cytoplasm, thereby regulating the expression of HIV proteins. The RRE element can be in any suitable position on the construct, preferably following the Gag-Pol precursor in its approximate native position. Similarly for the Tat polypeptide, any suitable rev polypeptide can be utilized as long as it retains the ability to bind to RRE. The coding sequence for Rev can be present in any of the vectors, such as a helper plasmid, transfer plasmid or on a separate plasmid. Similarly, coding sequences for tat can be present in any of the vectors, the helper plasmid, transfer plasmid, on a separate plasmid, or integrating into the host cell line utilized for transduction vector manufacture. A viral vector may further be made to reduce immune system detection in a process termed “stealthing”. Stealthing typically involves overexpressing a “self”-antigen(s) or surface marker(s) in the viral producer cells in such a way that the antigen/surface marker becomes present at high concentrations on the resulting virion envelope. Two known surface markers used in stealthing viral vectors are clusters of differentiation 47 (CD47) and 55 (CD55). CD47 present on the viral envelope inhibits the ability of macrophages to sense and phagocytose viral particles (Schauber-Plewa, et al. 2004). CD55 present on the viral envelop inhibits complement- mediated inactivation of the viral particle (Sosale, et al. 2016).

In summary, possible components of a lentiviral vector system and their typical locations include the following in Table 1a.

Table 1a Possible Lentiviral Vector System Components

The elements described above may be present in either the lentiviral transfer vector, or the lentiviral vector of the invention.

Any of the sequences which are present in the vector(s) can be modified from their native form, e.g., to improve transcription, to improve translation, to reduce or alter secondary RNA structure, and/or to decrease recombination. Modifications include, e.g., nucleotide addition, deletion, substitution, and replacements. For example, coding sequences for gag, pol, rev, and tat can be modified by replacing naturally-occurring codons with non-naturally-occurring codons, e.g., to improve translation in a host cell by substituting them with codons which are translated more effectively in the host cell. The host cell can be referred to as a compatible cell, e.g., to indicate the sequence modification has its effect when the sequence is expressed in a particular host cell type. In addition, sequences can be modified to remove regulatory elements, such as the packaging sequence. Sequences can also be altered to eliminate recombination sites. In one embodiment, however, the sequences which are present in the vector(s) are in their native form.

In certain aspects the different genetic elements may be provided on the same vector or on a plurality of vectors.

Suitably, the vector comprises a tissue-specific promoter operably linked to the IDIIA gene sequence. Gene expression can be limited to the liver by using a hepatocyte-specific transcription factor site linked to the transthyretin promoter (TTR), referred to as ET promoter. By contrast EF1a is a ubiquitous promoter allowing efficient expression in multiple cell types.

Nucleic acids and

Sequence identity

For the purposes of comparing two closely related polypeptide sequences, the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0, using standard settings for polypeptide sequences (BLASTP). For the purposes of comparing two closely related polynucleotide sequences, the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0, using standard settings for nucleotide sequences (BLASTN).

Polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides.

A “difference” between polypeptide sequences refers to an insertion, deletion or substitution of a single amino acid residue in a position of the second sequence, compared to the first sequence. Two polypeptide sequences can contain one, two or more such amino acid differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity).

Alternatively, for the purposes of comparing a first, reference polypeptide sequence to a second, comparison polypeptide sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An “addition” is the addition of one amino acid residue into the sequence of the first polypeptide (including addition at either terminus of the first polypeptide). A “substitution” is the substitution of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. Said substitution may be conservative or non-conservative. A “deletion” is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletion at either terminus of the first polypeptide).

Using the three letter and one letter codes, the naturally occurring amino acids may be referred to as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or lie), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gin), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and Threonine (T or Thr). Where a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where a residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. References to aspartic acid include aspartate, and glutamic acid include glutamate, unless the context specifies otherwise.

A “conservative” amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group, as shown in Table 1b below.

Table 1b: Amino acids

Suitably, a hydrophobic amino acid residue is a non-polar amino acid. More suitably, a hydrophobic amino acid residue is selected from V, I, L, M, F, W or C. In some embodiments, a hydrophobic amino acid residue is selected from glycine, alanine, valine, methionine, leucine, isoleucine, phenylalanine, tyrosine, or tryptophan. Suitably, any residues in a sequence which do not correspond to the residues provided in a reference sequence are conservative substitutions with respect to the residues of the reference sequence. In one aspect of the invention there is provided a polynucleotide comprising or consisting of a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with any one of SEQ ID NOs: 2, 3 and 5. Suitably, the polynucleotide comprises or consists of the sequence of any one of SEQ ID NOs: 2, 3 and 5.

In a further aspect of the invention there is provided a lentiviral vector that comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to the sequence of any one of SEQ ID NOs: 7 to 16 and 19 to 28.

Suitably, the lentiviral vector comprises a polynucleotide which encodes IDIIA, which comprises or consists of a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with any one of SEQ ID NOs: 2, 3 and 5. Suitably, the polynucleotide comprises or consists of the sequence of any one of SEQ ID NOs: 2, 3 and 5.

In one aspect of the invention there is provided a lentiviral vector which comprises a promoter region which comprises the promoter EF1a. Suitably the EF1a promoter comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to SEQ ID NO: 33. Suitably, the EF1a promoter comprises SEQ ID NO: 33. Suitably, the EF1a promoter consists of SEQ ID NO: 33.

In a further aspect of the invention there is provided a lentiviral vector which comprises a promoter region which comprises the promoter ET. Suitably the ET promoter comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to SEQ ID NO: 34. Suitably, the ET promoter comprises SEQ ID NO: 34. Suitably, the ET promoter consists of SEQ ID NO: 34.

In a further aspect of the invention there is provided a lentiviral vector that comprises a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to any one of SEQ ID NOs: 7 to 16 and 19 to 28. Suitably, there is provided a lentiviral vector comprising the sequence of any one of SEQ ID NOs: 7 to 16 and 19 to 28. Suitably, there is provided a lentiviral vector that consists of the sequence of any one of SEQ I D NOs: 7 to 16 and 19 to 28.

Mutations can be made to the DNA or cDNA that encode polypeptides which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli and S. cerevisiae, as well as mammalian, specifically human, are known. This process is referred to as codon optimisation.

Codon optimisation often results in an increase in GC content as an example: from the six codons encoding Arginine the 4 most used are fully GC. Similarly for Glycine and Alanine codons. A high GC content makes synthesis of polynucleotides less reliable and the chance of mistakes increases. Therefore, the GC content of the hIDUA had to be reduced to guarantee a fault free polynucleotide. Therefore, this optimisation of the codons and GC content enables the efficient manufacture of the IDIIA vectors as well as the efficient expression of IDIIA.

‘Reduced GC content’ as used herein refers to a nucleic acid encoding a protein which comprises fewer guanine and/or cytosine bases relative to a naturally occurring nucleic acid which encodes the same protein. Suitably, the GC content is reduced by 1%, such as by 5%, such as by 10%, such as by 20%, such as by 50% relative to the naturally occurring nucleic acid encoding the same protein.

In particular, artificial gene synthesis may be used. A gene encoding a polypeptide of the invention can be synthetically produced by, for example, solid-phase DNA synthesis. Entire genes may be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity.

IDUA

IDUA is an essential enzyme for the degradation of glycosaminoglycans (GAGs), degrading iduronate sulfatase to heparan N-sulfatase. IDUA is found in the lysosome of cells. Suitably, the IDUA of the invention is mouse or human IDUA. Suitably, the IDUA comprises the sequence of SEQ ID NO: 31 or SEQ ID NO: 32.

Disorders

Metabolic disorders are disorders that negatively alter the body’s processing and distribution of macronutrients. One cause of metabolic disorders is inherited metabolic disorders, wherein a defective gene causes an enzyme deficiency.

Mucopolysaccharidosis

The malfunction or absence of the lysosomal enzymes required to break down GAGs causes a type of metabolic disorder known as mucopolysaccharidoses. Accumulation of GAGs, such as dermatan and heparan sulfates, in lysosomes leads to dysfunction of multiple tissues and organs. This group of diseases have a range of causes and range of severity. Most mucopolysaccharidoses are autosomal recessive disorders.

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive genetic disease caused by dysfunctional variants in both alleles of the gene which encodes a-L-iduronidase (IDUA). Mutation of the gene leads to deficiency of the IDUA protein’s enzymatic activity.

The quantity of residual IDUA activity in cells determines the severity of the MPS-I disease. If the patient has between 1 and 2 % residual IDUA activity in cells they will suffer from a mild form of the disease, known as MPS-IS (Scheie syndrome, OMIM 607016). If the patient has between 0.5 and 1% residual IDUA activity in cells they will suffer from an intermediate form of the syndrome, leading to death before the age of 30. This is known as MPS-I H/S (Hurler-Scheie syndrome, OMIM 607015). If the patient only has 0 to 0.5% residual IDUA activity in cells, they will suffer from a severe disease phenotype, with death likely to occur in the first decade of life. This syndrome is categorised as MPS-IH, or Hurler Syndrome (OMIM 607014). Current methods of the treatment of MPS I include enzyme replacement therapy, wherein IDUA is administered. Additional surgeries may be required. Alternatively, bone marrow transplantation and umbilical cord blood transplantation (LICBT) can be used as a treatment. Bone marrow transplantation from siblings with identical HLA results in a significantly higher survival. LIBCT can be used from unrelated donors, however complications from this treatment includes graft versus host disease.

Treatment

According to a further aspect of the invention, there is provided a composition comprising the polynucleotide or vector as defined herein. In such embodiments, the composition may comprise the polynucleotide or vector, optionally in combination with other excipients. Also included are compositions comprising one or more additional active agents (e.g. active agents suitable for treating the diseases mentioned herein).

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising the polynucleotide or vector as defined herein, together with a pharmaceutically acceptable diluent or carrier. The polynucleotide or vector of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a polynucleotide or vector of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, salts, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide may be included.

The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions.

The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, administration is by intravenous infusion or injection. In another preferred embodiment, administration is by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.

It is within the scope of the invention to use the pharmaceutical composition of the invention in therapeutic methods for the treatment of diseases as described herein as an adjunct to, or in conjunction with, other established therapies normally used in the treatment of such diseases.

In a further aspect of the invention, the polypeptide, construct or composition is administered sequentially, simultaneously or separately with at least one active agent.

According to an aspect of the invention, there is provided a method of treating mucopolysaccharidosis using a polypeptide, construct or composition defined herein. Suitably, the mucopolysaccharidosis disease is MPS I. Suitably, the MPS I is Hurler syndrome, Hurler- Scheie syndrome or Scheie syndrome. Most suitably, the MPS I is Hurler syndrome. Suitably, the dosage regime is informed by the results of a previous study (Kobayashi et al. 2005).

According to a further aspect of the invention, there is provided a method of treating a disorder characterised by the absence or malfunction of lysosomal enzymes which break down GAGs using a polypeptide, construct or composition defined herein. Suitably, the disorder is characterised by a deficiency of a-L-iduronidase.

In a further aspect of the invention, treatment using a polypeptide, construct or composition defined herein results in an increase in IDIIA enzymatic activity by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 300%. Suitably, the increase in IDIIA enzymatic activity occurs in the plasma, liver, spleen, bone marrow or lung tissue. Most suitably, the increase in IDIIA enzymatic activity occurs in the liver.

In a further aspect of the invention, treatment using a polypeptide, construct or composition defined herein results in a decrease in GAG accumulation by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%. EXAMPLES

Methods

Tissue collection

Tissue sectioned were cryofractured for protein and nucleic acid extraction using the CP02 cryoPREP Automated Dry Pulverizer. Briefly, all tissue sections were pulverised on liquid nitrogen. The resulting pulverised tissue fragments were then processed for biochemical analysis: Protein extracts were prepared by solubilising the samples for Western blot analysis.

IDUA enzyme assay

The alpha-L-iduronidase enzyme assay was used to quantify the amount of IDUA enzyme activity present in tissue and blood samples. Cultured cells were prepared by washing the cells, centrifuging, conducting six freeze-thaw cycles and flash freezing. Cells from mouse tissue was prepared using an automated cryoPREP tissue pulveriser. Plasma from blood samples was prepared by centrifuging the EDTA collected blood sample and then removing the plasma.

Serum from blood samples was prepared by allowing the blood to clot at room temperature for

30 to 60 min prior to centrifugation as in the preparation of plasma.

The Pierce protein assay was used to calculate the protein concentration within the samples. A standard curve was produced using a stock of 4MU in absolute methanol, diluted using formate buffer.

25 pL of tissue homogenate or plasma was mixed with 25 pL of working substrate (a synthetic substrate for Iduronidase conjugated with a fluorescent dye 4-methylumbelliferyl (4- MU) at a concentration of(350 pM in a 96 well 2 mL PCT plate (Maita et al. 2013). 25 pL of working substrate (360 pM) mixed with 25 pL 0.2% BSA in PBS was used as the sample blank. The samples were placed in a thermocycler at 37 °C for 30 min, before the reaction was stopped by adding 200 pL glycine carbonate buffer. The plate was then read at 365 nm excitation and 450 nm emission. The IDUA enzyme activity was calculated in ng/h using the following equation with values from the standard curve:

Fluorescence of sample — fluorescence of blank, gradient of standard curve

Activity in ng/h = hours at 37 °C

GAG assay

DMMB reagent was prepared using 16 mg dimethylmethylene blue, 3.04 g glycine, 1.6 g NaCI and 95 mL of 0.1 M acetic acid per 1 L of reagent, at pH 3.0. Bovine chondroitin 4 sulfate was used to prepare a standard curve using a microplate spectrophotometer with a 535 nm filter. 20 pL of each sample was mixed with 200 pL of DMMB reagent before the absorbance was read at 525 nm. DMMB reacts with the sulfate group of the GAG Chain to form a DMMB- GAG complex. Protocol adapted from Farndale et al. 1982 and Whitley et al. 1989.

Bone marrow mRNA analysis

The femur was dissected from the mouse and flushed with PBS. The extracted cells were centrifuged (300 g for 5 min at 4 °C) and the pellet was resuspended with 2 ml of red blood cell lysis buffer for 3 minutes at room temperature. The reaction was then stopped by adding an excess of ice cold PBC. This was centrifuged to pellet the cells (300 g for 5 min at 4 °C). The supernatant was removed and replaced with 375ul of Qiazol. This was frozen and RNA was extracted from flash-frozen tissue with Qiazol Lysis Reagent (Qiagen). Briefly, following centrifugation to separate the aqueous phase from the organic trizol phase, the sample was mixed with 100 % ethanol and cleaned with the RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen). Complementary DNA (cDNA) was synthesized using the QuantiNova Reverse Transcription kit according to the manufacturer’s protocol with 1000 ng RNA in a 40 pL reaction (Qiagen). Quantitative PCR (qPCR) was performed with SYBR Green Master Mix (Life Technologies) on a QuantStudio 5000 (Life Technologies) according to the manufacturer’s protocol.

Example 1: Design of vectors

The cDNA sequence for Homo sapiens alpha-L-iduronidase (SEQ ID NO: 1 , encoding the polypeptide of SEQ ID NO: 31) was used as a starting gene. The cDNA sequence was codon optimised for improved expression (SEQ ID NO: 2). Further optimisation was conducted to reduce the GC content of the cDNA sequence (SEQ ID NO: 3).

Alternatively, the cDNA sequence for Mus musculus alpha-L-iduronidase (SEQ ID NO: 4, encoding the polypeptide of SEQ ID NO: 32) was used as a starting gene. The cDNA sequence was codon optimised for improved expression (SEQ ID NO: 5).

Vectors were prepared using a 3rd generation lentiviral vector system using plasmids pMDLg/pRRE, pRSV-REV and pMD2.G. Lentiviral transfer vectors were produced in vectors that had previously been used to express effective CAR constructs in T cells. The plasmid backbone of each vector contains the kanamycin resistance gene to allow bacterial selection. The lentiviral RNA is expressed from a CMV promoter upstream of a truncated 5’ LTR. The vector also contains a truncated WPRE with the residual woodchuck hepatitis virus gene X removed, in order to avoid the risk of oncogenic potential described for woodchuck hepatitis virus gene X gene (Donello et al. 1998, Choil et al. 2014).

Elongation factor 1a promoter (EF1a) was used as a ubiquitous promoter, which causes expression in most cells and tissues in humans (Uetsuki et al. 1989; Kim et al. 1990, SEQ ID NO: 33). Alternatively, mouse transthyretin (TTR) promoter was used in combination with hepatocyte nuclear factors to result in liver-specific expression (Costa & Grayson 1991, Vigna et al. 2005, SEQ ID NO: 34), referred to as ET promoter.

Vectors were created as shown in Table 1c. Vector maps are shown in Figure 1.

Table 1c: Vector design

Additional vectors were designed which do not include microRNA-142 binding sites. These vectors were created as shown in Table 2.

Table 2: Vector design without microRNA-142 binding sites

Example 2: Validation of transgene expression

Cells were transformed by infection with the lentiviral vectors produced above in vitro to confirm the viability of the cells, expression of the protein and to establish the viral titre for optimum expression of the protein. Example 2A: Viability assessment

The following transfections were conducted:

Vector 11 was transformed into 293T cells (kidney cells). Vector 12 was transformed into HEPG2 cells (liver cells).

The cells were assessed at 24 and 48 hours using both phase and GFP microscopy (Figure 1 - Vector maps of designed vectors with microRNA 142 binding sites

Figure 2 - Vector maps of designed vectors

Figure 3). The transformed cells maintained viability after 48 hours. No GFP expression was detected.

Example 2B: Transformation of kidney cells

293T kidney cells were transformed with Vector 17. The cells were assessed at 24 and 48 hours using both phase and GFP microscopy (Figure 4). Cell death was apparent at higher doses. GFP was detected at both 24 and 48 hours.

Viral titre was determined using FACS analysis at 72 hours post transfection (Figure 5 and Table 3). A high transformation efficiency was confirmed.

Table 3: Viral titre at 72 hours post transformation in 293T kidney cells transformed with Vector 17

Example 2C: Transformation of liver cells

HepG2 hepatocellular carcinoma cells were transformed with Vector 18. The cells were assessed at 24 and 48 hours using both phase and GFP microscopy (Figure 6). Cell death was apparent at higher doses. GFP was detected at both 24 and 48 hours.

Viral titre was determined using FACS analysis at 72 hours post transfection (Figure 7 and Table 4). A high transformation efficiency was confirmed.

Table 4: Viral titre at 72 hours post transformation in HepG2 cells transformed with Vector 18

Example 3: IDUA protein expression and activity

Cell lines were transformed by infection with lentiviral vectors encoding IDUA genes as described in Table 5. IDUA protein expression was determined using a Western blot (Figure 8A). The left lane of each combination shows the baseline levels of IDIIA expression in nontransformed cells (depicted

Each IDIIA band was quantified, as shown in Figure 8B. Increased levels of IDIIA protein expression were found in each combination.

Table 5: Cell line and transformation vector combinations of Figure 8 and Figure 9

The IDIIA enzyme activity of each cell line was determined using the IDIIA enzyme assay described above. The results are shown in Figure 9. Increased enzymatic IDIIA activity was shown in all cell lines with increased IDIIA protein expression.

Example 4: Cross correction study

293T cells transformed with either a Vector 17 (GFP) or Vector 11 (Human IDIIA), each driven by an EF1a specific promoter, were used to cross-correct MPS1 /Hurler (GM00798) fibroblasts.

GM00798 cells were incubated with the transformed cells for cross correction over 6 hours and 24 hours.

The GAG assay described above was performed in order to quantify intracellular GAG amounts in the GM00798 cells. The results are shown in Figure 10 which shows the % reduction of intracellular GAGs relative to GM00798 cells cross corrected with untransformed 293T cells (white bar). The data shows two repeat plots. GM00798 cells showed only a 9% decrease in intracellular GAG levels when cross corrected with control 293T cells transformed with GFP (Vector 17). GM00798 cells showed an 18% decrease in intracellular GAG levels after 6 hours of cross correction with 293T cells transformed with codon optimised human IDIIA (Vector 11). GM00798 cells showed an 36% decrease in intracellular GAG levels after 24 hours of cross correction with 293T cells transformed with codon optimised human IDIIA (Vector 11). Both sets of graphs demonstrates repeatability of GAG reduction in the cross corrected Hurler GM0798) cells

Example 5: Intravenous administration in mice Study/treatment groups

A single injection of the test item was administered intravenously via the tail vein. Animals were sacrificed one week after injection (Table 6).

Table 6: Treatment groups of wildtype C57/BL6 mice

There were no observable deterioration of the animals one week following treatment of the LV-IDUA vectors. The body weight of all animals did not deviate from the normal range (Figure 11).

IDUA expression

IDUA mRNA expression significantly increased in the lung tissues of animals which had been treated with IDUA targeted to all tissues (Group 2, see Figure 12A).

In the liver, there appeared to be an increase in IDUA mRNA levels in animals treated with IDUA targeted to liver tissues (Group 3, see Figure 12B).

No changes in IDUA expression were observed in the spleen (Figure 12C) and in the bone marrow (Figure 12D).

IDUA activity

The IDUA enzyme activity of plasma from each mouse was analysed using the protocol above. There was no change in the IDUA activity found in plasma from the treatment groups when compared to control (Figure 13).

Example 6: Second intravenous administration in mice

Study/treatment groups

A single injection of the test items or PBS was administered intravenously via the tail vein. The animal studies were performed in compliance with approval from the Institutional Animal Care and Use Committee of College of Medicine, National Taiwan University (No.20220032). the MPS I mice (B6.129S4-lduatm1.1 Kmke) with IDUA gene knock-in mutation on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and were bred and maintained by the National Laboratory Animal Center. At the time of treatment, the MPS I mice had a body weight of 15.2-24.7 g. The age is 4-8 weeks old. Mice were treated with PBS, Vector 13 (mIDUA EF1a) or Vector 14 (mIDUA ET).

Tissue samples were collected from the mice 30 days after administration.

IDUA activity

IDUA enzyme activity was analysed in plasma, liver, spleen, bone marrow and lung tissue harvested from the mice (Figure 14).

In plasma (Figure 13) and spleen (Figure 14C) significantly enhanced IDUA enzymatic activity was seen in animals treated with IDUA targeted to all tissue compared to treatment with the vector comprising the liver specific promoter.

Significantly enhanced IDUA enzymatic activity was found in liver (Figure 14B) and the lungs (Figure 14E) in animals treated with the vector comprising the liver specific ET promoter. Minor enhancement was seen in the liver in animals treated with the vector comprising EF1a promoter.

Enhanced IDUA enzymatic activity was found in bone marrow for animals treated with both vectors (Figure 14D).

Example 7 - Production of liver-directed lentiviral vectors

Towards the goal of generating a liver-directed lentiviral vector system that is able to avoid off-target issues with antigen presenting cells, the miR-142 target site was cloned into the 3’ UTR of a GFP-luciferase transgene (Figure 15A) and screened for cell-specific expression. Figure 15A demonstrates that the addition of miR-142 restricts the expression of transgenes in monocytes. APCs, monocytes, and macrophages express high amounts of miR-142, whereas liver cells do not (Brown et al. 2007). A miR-142 target site was cloned and its ability to be susceptible to miR-142 targeting was assessed. It was found that 4 repeat concatemers of the miR-142 target site were required in the 3’ UTR to restrict transgene expression in monocytes and macrophages in vitro and this sequence was incorporated into the 3’UTR of a lentiviral vector GFP-luciferase transgene (Figure 15A). The incorporation of this miR-142 site had little effect on transgene expression in HEK293 cells, which lack miR-142 expression, but abolished expression in transduced RAW264.7 macrophage cells (Figures 15B-C). Figure 15C shows graphically the data in Figure 15B and that expression is restricted to non-immune cells. To further restrict the expression of the GFP-luciferase transgene to liver cells, a murine TTR liverspecific promoter-reporter system was developed and assessed in human liver cells. It was found that TTR restricts GFP expression to liver-specific cells and that the incorporation of the miR-142 target site had little effect on transgene expression in these cells (Figure 15D). Figure 15D demonstrates that the liver specific promoter only expressed the transgene in liver cells (e.g. HEPG2), and not HEK cells.

Lastly, to make stealth lentiviruses whereby the over-expression of CD47 and CD55 on the vector particle has been shown to reduce the susceptibility of VSV-G pseudotyped IV administered particles to complement-mediated inactivation and phagocytosis (Milani et al. 2019), a dual-CD47/CD55 expressing lentiviral vector was generated and tested. These vectors acted as positive controls, comprising the envgene. The envelope plasmid pCMV-G (VSV-G) was used in the stealth lentiviral vector controls.

It was found that both CD47 and CD55 can be expressed in high levels on the cell surface (Figures 16A-B) and in the same cell (Figure 16C). Figures 16A and B show that overexpression of CD47 and CD55 receptors in cells is possible and that those viral particles made from these cells will be shielded from being engulfed by those cells in the immune system that engulf particles. Figure 16C demonstrates that both CD47 and CD55 may be expressed in the same cell. So, it is possible to produce stable over-expressing CD47/CD55 cell lines and these can be used to make stealth lentivirus vectors.

These data demonstrate that liver directed phagocytosis shielded lentiviral vectors can be generated and may prove advantageous in the development of a direct injectable liver targeted lentiviral vector approach.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part of may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims which follow.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. Clauses of the invention

A series of clauses setting out embodiments of the invention are as follows.

1. A lentiviral vector comprising (i) a nucleic acid encoding the polypeptide sequence for a- L-iduronidase (IDIIA), optionally (ii) a nucleic acid sequence encoding truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, (iii) a nucleic acid sequence encoding multiple microRNA-142 (miR-142) target sites, optionally (iv) a nucleic acid sequence encoding a genetic insulator sequence, and (v) a modified viral envelope whereby cluster of differentiation 47 and 55 (CD47/55) are present on the envelope surface.

2. The lentiviral vector of clause 1 wherein the lentiviral vector is replication incompetent.

3. The lentiviral vector of clause 1 or clause 2 wherein the lentiviral vector is selfinactivating.

4. A lentiviral transfer vector comprising (i) a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA), optionally (ii) a nucleic acid sequence encoding a truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, (iii) a nucleic acid sequence encoding multiple microRNA-142-3p (miR-142) target sites, and optionally (iv) a nucleic acid sequence encoding a genetic insulator sequence.

5. The lentiviral transfer vector of clause 4 wherein the lentiviral transfer vector is a plasmid.

6. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 5 wherein the IDIIA sequence is substituted for nucleic acid sequence encoding a fusion polypeptide sequence of transferrin protein and IDIIA, fused directly through any one of the last 25 amino acids within the C-terminus of transferrin and any one of the first 25 amino acids within the N-terminus of IDIIA.

7. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 6 wherein the IDIIA sequence is substituted for nucleic acid sequence encoding a fusion polypeptide sequence of transferrin protein and IDIIA fused through a flexible, rigid, and/or cleavable amino acid linker sequence to any one of the last 25 amino acids within the C-terminus of transferrin and any one of the first 25 amino acids within the N-terminus of IDIIA.

8. A method of producing a lentiviral vector comprising the steps of transfecting a host cell with a lentiviral vector system, wherein the lentiviral vector system comprises a lentiviral transfer vector, packaging genes and envelope genes and wherein the lentiviral transfer vector comprises a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDUA) optionally linked to nucleic acid encoding the polypeptide sequence for truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, and a nucleic acid encoding multiple miR-142 target sites. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 8 wherein the IDUA is mouse or human IDUA. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 9 wherein the IDUA is human IDUA. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 10 wherein the IDUA comprises a polypeptide with at least 90%, such as at least 93%, such as at least 95%, such as at least 97%, such as at least 99% identity to the sequence of SEQ ID NO: 31 or 32. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 11 wherein the IDUA comprises the polypeptide sequence of SEQ ID NO: 31 or 32. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 12 wherein the IDUA comprises the polypeptide sequence of SEQ ID NO: 31. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 13 wherein the nucleic acid encoding the polypeptide sequence for IDUA is codon optimised. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 14 wherein the nucleic acid encoding the polypeptide sequence for IDUA is codon optimised for expression in human cells. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 15 wherein the codon optimised nucleic acid has reduced GC content relative to a fully codon optimised sequence. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 16 wherein the nucleic acid encoding IDUA comprises at least 90%, such as at least 93%, such as at least 95%, such as at least 97%, such as at least 99% identity to the sequence of SEQ ID NO: 1 to 5. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 17 wherein the nucleic acid encoding IDUA comprises the sequence of SEQ ID NO: 1 to 5. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 18 wherein the nucleic acid encoding IDUA consists of the sequence of SEQ ID NO: 1 to 5. 20. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 19 wherein the expression of I DU A is controlled by a ubiquitous promoter.

21. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 20 wherein the promoter is elongation factor 1a (EF1a).

22. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 21 wherein the promoter comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to the sequence of SEQ ID NO: 33.

23. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 22 wherein the promoter comprises the sequence SEQ ID NO: 33.

24. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 23 wherein the promoter consists of the sequence SEQ ID NO: 33.

25. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 20 wherein the expression of IDUA is substantially restricted to the liver.

26. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 25 wherein the expression of IDUA is controlled by a hepatocyte-specific transcription factor site linked to a transthyretin (ET) promoter.

27. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 26 wherein the promoter comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to the sequence of SEQ ID NO: 34.

28. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 27 wherein the promoter comprises the sequence SEQ ID NO: 34.

29. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 28 wherein the promoter consists of the sequence SEQ ID NO: 34

30. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 29 wherein transduction of cells with the lentiviral vector or nucleic acid causes upregulation in IDUA activity.

31. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 30 wherein the lentiviral vector or lentiviral transfer vector comprises any number from one to fifty microRNA-142 binding sites.

32. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 31 wherein the lentiviral vector or lentiviral transfer vector comprises a CMV enhancer. 33. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 to 32 wherein the lentiviral vector or lentiviral transfer vector comprises a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to any one of SEQ ID NOs: 7 to 16 and 19 to 28.

34. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 33 wherein the lentiviral vector or lentiviral transfer vector comprises the sequence of any one of SEQ I D NOs: 7 to 16 and 19 to 28.

35. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 34 wherein the lentiviral transfer vector consists of the sequence of any one of SEQ ID NOs: 7 to 16 and 19 to 28.

36. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 35, wherein the lentiviral vector or lentiviral transfer vector comprises a lentiviral packaging signal (^P).

37. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 36, wherein the lentiviral transfer vector comprises a genetic insulator sequence within the 3’-LTR.

38. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 37, wherein the lentiviral vector or lentiviral transfer vector comprises a lentiviral 5'-LTR.

39. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 38 wherein the nucleic acid encoding the polypeptide sequence for IDIIA is positioned downstream of the 5'-LTR.

40. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 39, wherein the lentiviral vector or lentiviral transfer vector comprises a lentiviral 3'-LTR.

41. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 40 wherein the nucleic acid encoding the polypeptide sequence for IDIIA is positioned upstream of the 3'-LTR.

42. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to either clause 40 or 41 wherein the nucleic acid encoding the polypeptide sequence for IDIIA is positioned between the 5’-LTR and the 3'-LTR. 43. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38 to 42, wherein the 5’-LTR and/or 3’-LTR comprise U3, R, TAR and/or U5.

44. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38 to 43 wherein the 5’-LTR sequence is partially deleted.

45. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38 to 44 wherein the 5’-LTR sequence is fused to a heterologous enhancer and/or promoter.

46. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 40 to 45 wherein the 3’-LTR sequence is partially deleted.

47. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 46 wherein the lentiviral vector or lentiviral transfer vector comprises a nucleic acid sequence encoding a Rev response element (RRE).

48. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 47 wherein the lentiviral vector or lentiviral transfer vector further comprises a nucleic acid sequence encoding a central polypurine tract (cPPT or CPPT Tract).

49. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 48 wherein the lentiviral vector or lentiviral transfer vector further comprises a nucleic acid sequence encoding a WPRE.

50. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 49 wherein the lentiviral vector or lentiviral transfer vector further comprises a nucleic acid Flap sequence.

51. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 50 wherein the lentiviral vector or lentiviral transfer vector comprises a selection marker.

52. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 51 wherein the lentiviral vector or lentiviral transfer vector comprises a suicide gene.

53. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to either clause 51 or 52 wherein a sequence encoding the selection marker and/or suicide gene is substituted in place of the sequence encoding tEGFR.

54. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 53 wherein the lentiviral vector or lentiviral transfer vector comprises the following components in the following order: 5’-LTR-Psi-cPPT-CTS/Flap- promoter-transgene-WPRE-3’-LTR; wherein the transgene is the nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

55. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 53 wherein the lentiviral vector or lentiviral transfer vector comprises the following components in the following order: 5’-LTR-Psi-RRE-cPPT- CTS/Flap-promoter-transgene-WPRE-3’-LTR; wherein the transgene is the nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

56. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 to 55 wherein the polynucleotide sequence of the 5’-LTR shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 35.

57. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38 to 56 wherein the polynucleotide sequence of the 5’-LTR comprises or consists of SEQ ID NO: 35.

58. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 36 to 57 wherein the polynucleotide sequence of the Psi shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 36.

59. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 58 wherein the polynucleotide sequence of the Psi comprises or consists of SEQ ID NO: 36.

60. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 37 to 59 wherein a polynucleotide sequence encoding a genomic insulator, is introduced into the 3’-LTR and shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with the sequence TACTACATCTGTCCACAGAAGGGCTGGGGAGCAGCTTTCCTGTCCCTCCTGTGAG TGGCCACCAGGGGGAGCGTGGACACAGCTGCCCGTGCAGTGACCACCTGCCCCC CACTCCCGCTACTCCAGCGTA (SEQ ID NO: 45) if DNA, or UACUACAUCUGUCCACAGAAGGGCUGGGGAGCAGCUUUCCUGUCCCUCCUGUG AGUGGCCACCAGGGGGAGCGUGGACACAGCUGCCCGUGCAGUGACCACCUGCC CCCCACUCCCGCUACUCCAGCGUA (SEQ ID NO: 46) if RNA.

61. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 60 wherein the polynucleotide sequence of the genomic insulator comprises or consists of the sequence TACTACATCTGTCCACAGAAGGGCTGGGGAGCAGCTTTCCTGTCCCTCCTGTGAG TGGCCACCAGGGGGAGCGTGGACACAGCTGCCCGTGCAGTGACCACCTGCCCCC CACTCCCGCTACTCCAGCGTA (SEQ ID NO: 45) if DNA, or UACUACAUCUGUCCACAGAAGGGCUGGGGAGCAGCUUUCCUGUCCCUCCUGUG AGUGGCCACCAGGGGGAGCGUGGACACAGCUGCCCGUGCAGUGACCACCUGCC CCCCACUCCCGCUACUCCAGCGUA (SEQ ID NO: 46) if RNA.

62. The lentiviral vector, nucleic acid, use or method according to any one of clauses 37 to 61 wherein during the production of virion particles, CD47 and CD55 are expressed from an additional plasmid(s) in viral producer cells in such a way that CD47 and CD55 are incorporated onto the surface of the viral envelope.

63. The lentiviral vector, nucleic acid, use or method according to clause 62 wherein the polynucleotide sequence of CD47 and/or CD55 encodes a protein(s) comprising or consisting of a sequence(s) encoded by amino acid sequence(s) encoding a protein(s) sharing at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with a protein(s) encoded by human CD47 and/or CD55.

64. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 48 to 63 wherein the polynucleotide sequence of the cPPT shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 38.

65. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 64 wherein the polynucleotide sequence of the cPPT comprises or consists of SEQ ID NO: 38.

66. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 50 to 65 wherein the polynucleotide sequence of the Flap shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 39. 67. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 66 wherein the polynucleotide sequence of the Flap comprises or consists of SEQ ID NO: 39.

68. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 49 to 67 wherein the polynucleotide sequence of the WPRE shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 40.

69. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 68 wherein the polynucleotide sequence of the WPRE comprises or consists of SEQ ID NO: 40.

70. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 40 to 69 wherein the polynucleotide sequence of the 3’-LTR shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 41.

71. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 70 wherein the polynucleotide sequence of the 3’-LTR comprises or consists of SEQ ID NO: 41.

72. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 47 to 71 wherein the RRE polynucleotide sequence shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 42.

73. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 72 wherein the RRE polynucleotide sequence comprises or consists of SEQ ID NO: 42.

74. A composition comprising the lentiviral vector of any one of clauses 1 to 73.

75. The composition of clause 74 wherein the composition comprises additional excipients.

76. The composition of clause 74 or 75 wherein the composition comprises one or more additional active agents.

77. The composition of either clause 75 or 76 wherein the composition comprises a pharmaceutically acceptable diluent or carrier. 78. The composition of any one of clauses 74 to 77 wherein the composition is in liquid, semi-solid or solid dosage form.

79. The composition of any one of clauses 74 to 78 wherein the composition is in the form of injectable or infusible solutions.

80. The lentiviral vector or nucleic acid of any one of clauses 1 to 73 or composition of any one of clauses 74 to 79 for use as a medicament.

81. The lentiviral vector or nucleic acid of any one of clauses 1 to 73 or composition of any one of clauses 74 to 80 for use in the treatment of metabolic disorder.

82. A method of treating metabolic disorder comprising administration of the lentiviral vector or nucleic acid of any one of clauses 1 to 73 or composition of any one of clauses 74 to 80.

83. Use of the lentiviral vector or nucleic acid of any one of clauses 1 to 73 or composition of any one of clauses 74 to 82 for the manufacture of a medicament for the treatment of metabolic disorder.

84. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 to 83 wherein the metabolic disorder is mucopolysaccharidoses type I (MPS I).

85. The lentiviral vector or nucleic acid or composition for use, method or use of clause 84 wherein the MPS I is of subtype MPS-IH, MPS-IH/S or MPS-IS.

86. The lentiviral vector or nucleic acid or composition for use, method or use of clause 85 wherein the MPS I is MPS-IH (Hurler syndrome).

87. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 to 86 wherein the subject receiving treatment is human.

88. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 to 87 wherein the mode of administration of the lentiviral vector, nucleic acid or composition is parenteral.

89. The lentiviral vector or nucleic acid or composition for use, method or use of clause 88 wherein administration is by intravenous infusion, intravenous injection, intramuscular injection or subcutaneous injection.

90. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 to 89 wherein the metabolic disorder is characterised by the absence or malfunction of lysosomal enzymes which break down GAGs.

91. The lentiviral vector or nucleic acid or composition for use, method or use of clause 90 wherein the disorder is characterised by a deficiency of a-L-iduronidase. 92. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 to 91 wherein the treatment results in an increase in IDIIA enzymatic activity, such as by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 300%.

93. The lentiviral vector or nucleic acid or composition for use, method or use of clause 92 wherein the increase in IDIIA enzymatic activity occurs in the plasma, liver, spleen, bone marrow or lung tissue.

94. The lentiviral vector or nucleic acid or composition for use, method or use of clause 93 wherein the increase in IDIIA enzymatic activity occurs in the liver.

95. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 to 94 wherein the treatment results in a decrease in GAG accumulation, for example by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%.

96. The lentiviral vector or nucleic acid or composition for use or use of any one of clauses 1 to 95 wherein the lentiviral vector comprises (i) a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA), (ii) a nucleic acid sequence encoding truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, (iii) a nucleic acid sequence encoding multiple microRNA-142 (miR- 142) target sites, (iv) a nucleic acid sequence encoding a genetic insulator sequence, and (v) a modified viral envelope whereby cluster of differentiation 47 and 55 (CD47/55) are present on the envelope surface.

97. The lentiviral vector or nucleic acid or composition for use or use of any one of clauses 1 to 95 wherein comprising (i) a nucleic acid encoding the polypeptide sequence for a- L-iduronidase (IDIIA), (ii) a nucleic acid sequence encoding a truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, (iii) a nucleic acid sequence encoding multiple microRNA-142-3p (miR-142) target sites, and (iv) a nucleic acid sequence encoding a genetic insulator sequence.

98. The method of any one of clauses 8 to 95, wherein the lentiviral vector system comprises a lentiviral transfer vector, packaging genes and envelope genes and wherein the lentiviral transfer vector comprises a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA) linked to nucleic acid encoding the polypeptide sequence for truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, and a nucleic acid encoding multiple miR-142 target sites.

A series of clauses setting out further embodiments of the invention is as follows.

1 B. A lentiviral vector comprising a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

2B. The lentiviral vector of clause 1 B wherein the lentiviral vector is replication incompetent.

3B. The lentiviral vector of clause 1 B or clause 2B wherein the lentiviral vector is selfinactivating.

4B. A lentiviral transfer vector comprising a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

5B. The lentiviral transfer vector of clause 4B wherein the lentiviral transfer vector is a plasmid.

6B. A nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDUA).

7B. Use of a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDUA) in the production of a lentiviral vector comprising a nucleic acid encoding the polypeptide sequence for IDUA.

8B. A method of producing a lentiviral vector comprising the steps of transfecting a host cell with a lentiviral vector system, wherein the lentiviral vector system comprises a lentiviral transfer vector, packaging genes and envelope genes and wherein the lentiviral transfer vector comprises a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDUA).

9B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 8B wherein the IDUA is mouse or human IDUA.

10B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 9B wherein the IDUA is human IDUA.

11 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 10B wherein the IDUA comprises a polypeptide with at least 90%, such as at least 93%, such as at least 95%, such as at least 97%, such as at least 99% identity to the sequence of SEQ ID NO: 31 or 32.

12B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 11B wherein the IDUA comprises the polypeptide sequence of SEQ ID NO: 31 or 32. 13B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 12B wherein the IDIIA comprises the polypeptide sequence of SEQ ID NO: 31.

14B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 13B wherein the nucleic acid encoding the polypeptide sequence for I DUA is codon optimised.

15B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 14B wherein the nucleic acid encoding the polypeptide sequence for I DUA is codon optimised for expression in human cells.

16B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 15B wherein the codon optimised nucleic acid has reduced GC content relative to a fully codon optimised sequence.

17B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 16B wherein the nucleic acid encoding IDUA comprises at least 90%, such as at least 93%, such as at least 95%, such as at least 97%, such as at least 99% identity to the sequence of SEQ ID NO: 1 to 5.

18B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 17B wherein the nucleic acid encoding IDUA comprises the sequence of SEQ ID NO: 1 to 5.

19B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 18B wherein the nucleic acid encoding IDUA consists of the sequence of SEQ ID NO: 1 to 5.

20B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 19B wherein the expression of IDUA is controlled by a ubiquitous promoter.

21 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 20B wherein the promoter is elongation factor 1a (EF1a).

22B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 21 B wherein the promoter comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to the sequence of SEQ ID NO: 33.

23B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 22B wherein the promoter comprises the sequence SEQ ID NO: 33.

24B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 23B wherein the promoter consists of the sequence SEQ ID NO: 33. 25B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 20B wherein the expression of IDIIA is substantially restricted to the liver.

26B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 25B wherein the expression of IDIIA is controlled by a hepatocyte-specific transcription factor site linked to a transthyretin (ET) promoter.

27B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 26B wherein the promoter comprises at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% such as at least 99% identity to the sequence of SEQ ID NO: 34.

28B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 27B wherein the promoter comprises the sequence SEQ ID NO: 34.

29B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 28B wherein the promoter consists of the sequence SEQ ID NO: 34

30B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 29B wherein transformation of cells with the lentiviral vector or nucleic acid causes upregulation in IDIIA activity.

31 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 30B wherein the lentiviral vector or lentiviral transfer vector comprises microRNA-142 binding sites.

32B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 31 B wherein the lentiviral vector or lentiviral transfer vector comprises a CMV enhancer.

33B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of any one of clauses 1 B to 32B wherein the lentiviral vector or lentiviral transfer vector comprises a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to any one of SEQ ID NOs: 7 to 16 and 19 to 28.

34B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 33B wherein the lentiviral vector or lentiviral transfer vector comprises the sequence of any one of SEQ ID NOs: 7 to 16 and 19 to 28.

35B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method of clause 34B wherein the lentiviral transfer vector consists of the sequence of any one of SEQ ID NOs: 7 to 16 and 19 to 28. 36B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 35B, wherein the lentiviral vector or lentiviral transfer vector comprises a lentiviral packaging signal (^P).

37B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 36B, wherein the lentiviral vector or lentiviral transfer vector comprises a gag gene.

38B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 37B, wherein the lentiviral vector or lentiviral transfer vector comprises a lentiviral 5'-LTR.

39B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 38B wherein the nucleic acid encoding the polypeptide sequence for IDIIA is positioned downstream of the 5'-LTR.

40B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 39B, wherein the lentiviral vector or lentiviral transfer vector comprises a lentiviral 3'-LTR.

41 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 40B wherein the nucleic acid encoding the polypeptide sequence for IDIIA is positioned upstream of the 3'-LTR.

42B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to either clause 40B or 41 B wherein the nucleic acid encoding the polypeptide sequence for IDIIA is positioned between the 5’-LTR and the 3'-LTR.

43B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38B to 42B, wherein the 5’-LTR and/or 3’-LTR comprise U3, R, TAR and/or U5.

44B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38B to 43B wherein the 5’-LTR sequence is partially deleted.

45B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38B to 44B wherein the 5’-LTR sequence is fused to a heterologous enhancer and/or promoter.

46B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 40B to 45B wherein the 3’-LTR sequence is partially deleted.

47B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 46B wherein the lentiviral vector or lentiviral transfer vector comprises a nucleic acid sequence encoding a Rev response element (RRE). 48B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 47B wherein the lentiviral vector or lentiviral transfer vector further comprises a nucleic acid sequence encoding a central polypurine tract (cPPT or CPPT Tract).

49B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 48B wherein the lentiviral vector or lentiviral transfer vector further comprises a nucleic acid sequence encoding a WPRE.

50B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 49B wherein the lentiviral vector or lentiviral transfer vector further comprises a nucleic acid Flap sequence.

51 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 50B wherein the lentiviral vector or lentiviral transfer vector comprises a selection marker.

52B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 51 B wherein the lentiviral vector or lentiviral transfer vector comprises a suicide gene.

53B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to either clause 51 B or 52B wherein the selection marker and/or suicide gene is truncated epidermal growth factor receptor (tEGFR).

54B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 53B wherein the lentiviral vector or lentiviral transfer vector comprises the following components in the following order: 5’-LTR-Psi-gag-Flap- promoter-transgene-WPRE-3’-LTR; wherein the transgene is the nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

55B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 53B wherein the lentiviral vector or lentiviral transfer vector comprises the following components in the following order: 5’-LTR-Psi-gag-RRE-Flap- promoter-transgene-WPRE-3’-LTR; wherein the transgene is the nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

56B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 1 B to 55B wherein the polynucleotide sequence of the 5’-LTR shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 35. 57B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 38B to 56B wherein the polynucleotide sequence of the 5’-LTR comprises or consists of SEQ ID NO: 35.

58B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 36B to 57B wherein the polynucleotide sequence of the Psi shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 36.

59B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 58B wherein the polynucleotide sequence of the Psi comprises or consists of SEQ ID NO: 36.

60B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 37B to 59B wherein the polynucleotide sequence of gag shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 37.

61 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 60B wherein the polynucleotide sequence of gag comprises or consists of SEQ ID NO: 37.

62B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 37B to 61 B wherein the polynucleotide sequence of gag encodes a protein sharing at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with a protein encoded by SEQ ID NO: 37.

63B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 62B wherein the polynucleotide sequence of gag encodes a protein comprising or consisting of a sequence encoded by SEQ ID NO: 37; or wherein the polynucleotide sequence of gag encodes a protein comprising or consisting of a sequence encoded by SEQ ID NO: 43.

64B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 48B to 63B wherein the polynucleotide sequence of the cPPT shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 38. 65B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 64B wherein the polynucleotide sequence of the cPPT comprises or consists of SEQ ID NO: 38.

66B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 50B to 65B wherein the polynucleotide sequence of the Flap shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 39.

67B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 66B wherein the polynucleotide sequence of the Flap comprises or consists of SEQ ID NO: 39.

68B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 49B to 67B wherein the polynucleotide sequence of the WPRE shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 40.

69B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 68B wherein the polynucleotide sequence of the WPRE comprises or consists of SEQ ID NO: 40.

70B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 40B to 69B wherein the polynucleotide sequence of the 3’-LTR shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 41.

71 B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 70B wherein the polynucleotide sequence of the 3’-LTR comprises or consists of SEQ ID NO: 41.

72B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to any one of clauses 47B to 71 B wherein the RRE polynucleotide sequence shares at least 60% identity, such as at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 99% identity with SEQ ID NO: 42. 73B. The lentiviral vector, lentiviral transfer vector, nucleic acid, use or method according to clause 72B wherein the RRE polynucleotide sequence comprises or consists of SEQ ID NO: 42.

74B. A composition comprising the lentiviral vector of any one of clauses 1 B to 73B.

75B. The composition of clause 74B wherein the composition comprises additional excipients.

76B. The composition of clause 74B or 75B wherein the composition comprises one or more additional active agents.

77B. The composition of either clause 75B or 76B wherein the composition comprises a pharmaceutically acceptable diluent or carrier.

78B. The composition of any one of clauses 74B to 77B wherein the composition is in liquid, semi-solid or solid dosage form.

79B. The composition of any one of clauses 74B to 78B wherein the composition is in the form of injectable or infusible solutions.

80B. The lentiviral vector or nucleic acid of any one of clauses 1 B to 73B or composition of any one of clauses 74B to 79B for use as a medicament.

81 B. The lentiviral vector or nucleic acid of any one of clauses 1 B to 73B or composition of any one of clauses 74B to 80B for use in the treatment of metabolic disorder.

82B. A method of treating metabolic disorder comprising administration of the lentiviral vector or nucleic acid of any one of clauses 1 B to 73B or composition of any one of clauses 74B to 80B.

83B. Use of the lentiviral vector or nucleic acid of any one of clauses 1 B to 73B or composition of any one of clauses 74B to 82B for the manufacture of a medicament for the treatment of metabolic disorder.

84B. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 B to 83B wherein the metabolic disorder is mucopolysaccharidoses type I (MPS I).

85B. The lentiviral vector or nucleic acid or composition for use, method or use of clause 84B wherein the MPS I is of subtype MPS-IH, MPS-IH/S or MPS-IS.

86B. The lentiviral vector or nucleic acid or composition for use, method or use of clause 85B wherein the MPS I is MPS-IH (Hurler syndrome).

87B. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 B to 86B wherein the subject receiving treatment is human. 88B. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 B to 87B wherein the mode of administration of the lentiviral vector, nucleic acid or composition is parenteral.

89B. The lentiviral vector or nucleic acid or composition for use, method or use of clause 88B wherein administration is by intravenous infusion, intravenous injection, intramuscular injection or subcutaneous injection.

90B. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 B to 89B wherein the metabolic disorder is characterised by the absence or malfunction of lysosomal enzymes which break down GAGs.

91 B. The lentiviral vector or nucleic acid or composition for use, method or use of clause 90B wherein the disorder is characterised by a deficiency of a-L-iduronidase.

92B. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 B to 91 B wherein the treatment results in an increase in IDIIA enzymatic activity, such as by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 300%.

93B. The lentiviral vector or nucleic acid or composition for use, method or use of clause 92B wherein the increase in IDIIA enzymatic activity occurs in the plasma, liver, spleen, bone marrow or lung tissue.

94B. The lentiviral vector or nucleic acid or composition for use, method or use of clause 93B wherein the increase in IDIIA enzymatic activity occurs in the liver.

95B. The lentiviral vector or nucleic acid or composition for use, method or use of any one of clauses 81 B to 94B wherein the treatment results in a decrease in GAG accumulation, for example by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%.

A series of clauses setting out further embodiments of the invention is as follows.

1C. A lentiviral vector comprising a nucleic acid encoding the polypeptide sequence for a-L-iduronidase (IDIIA).

2C. The lentiviral vector of clause 1C wherein the vector is replication incompetent.

3C. The lentiviral vector of clause 1C or clause 2C wherein the vector is selfinactivating. 4C. The lentiviral vector of any one of clauses 1C to 3C wherein the IDIIA is mouse or human IDIIA.

5C. The lentiviral vector of clause 4C wherein the IDIIA is human IDIIA.

6C. The lentiviral vector of clause 4C or clause 5C wherein the IDIIA comprises a polypeptide with at least 95% identity to the sequence of SEQ ID NO: 31 or 32.

7C. The lentiviral vector of clause 6C wherein the IDIIA comprises the polypeptide sequence of SEQ ID NO: 31 or 32.

8C. The lentiviral vector of any one of clauses 1C to 7C wherein the nucleic acid encoding the polypeptide sequence for IDIIA is codon optimised.

9C. The lentiviral vector of clause 8C wherein the codon optimised nucleic acid has reduced GC content.

10C. The lentiviral vector of any one of clauses 1C to 9C wherein the nucleic acid comprises the sequence of SEQ ID NO: 2, 3 or 5.

11C. The lentiviral vector of any one of clauses 1C to 10C wherein the expression of IDIIA is controlled by a ubiquitous promoter.

12C. The lentiviral vector of clause 11C wherein the promoter is elongation factor 1a (EF1a).

13C. The lentiviral vector of any one of clauses 1C to 10C wherein the expression of IDIIA is substantially restricted to the liver.

14C. The lentiviral vector of clause 13C wherein the expression of IDIIA is controlled by a hepatocyte-specific transcription factor site linked to a transthyretin (ET) promoter.

15C. The lentiviral vector of any one of clauses 1C to 14C wherein transformation of cells with the vector causes upregulation in IDIIA activity.

16C. The vector of any one of clauses 1C to 15C for use as a medicament.

17C. The vector of any one of clauses 1C to 15C for use in the treatment of metabolic disorder.

18C. The vector for use of clause 17C wherein the metabolic disorder is mucopolysaccharidoses type I (MPS I).

19C. The vector for use of clause 18C wherein the MPS I is of subtype MPS-IH, MPS- IH/S or MPS-IS.

20C. The vector for use of clause 19C wherein the MPS I is MPS-IH (Hurler syndrome).

21 C. The lentiviral vector of clause 1C wherein the lentiviral vector is replication incompetent, the nucleic acid encoding IDIIA comprises the sequence of SEQ ID NO: 2 or 3, and the expression of IDIIA is controlled by a ubiquitous promoter which is EF1a, for use in the treatment of MPS I.

22C. The lentiviral vector of clause 1C wherein the lentiviral vector is replication incompetent, the nucleic acid encoding IDIIA comprises the sequence of SEQ ID NO: 2 or 3, the expression of IDIIA is substantially localised to the liver, and wherein the promoter controlling the expression is an ET promoter, for use in the treatment of MPS I.

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Patent Citations

Publication number: WO2015138852A1; Priority date: 2015-03-13; Publication date: 2015- 09-17; Assignee: University of Washington; Title: Genomic insulator elements and uses thereof

Claims

Claims

1. A lentiviral vector comprising a nucleic acid encoding the polypeptide sequence for a-L- iduronidase (IDIIA), a nucleic acid sequence encoding truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, a nucleic acid sequence encoding multiple microRNA-142-3p (miR-142) target sites, a nucleic acid sequence encoding a genetic insulator sequence, and a modified viral envelope whereby cluster of differentiation 47 and 55 (CD47/55) are present on the envelope surface.

2. The lentiviral vector of claim 1 wherein the vector is replication incompetent.

3. The lentiviral vector of claim 1 or claim 2 wherein the vector is self-inactivating.

4. The lentiviral vector of claims 1 to 3 wherein a genomic insulator sequence is present in the 3’-LTR of the lentiviral transfer vector utilized in the manufacturing of the lentiviral vector.

5. The lentiviral vector of any one of claims 1 to 4 wherein the IDIIA is mouse or human IDUA.

6. The lentiviral vector of claim 5 wherein the IDUA is human IDUA.

7. The lentiviral vector of claim 5 or claim 6 wherein the IDUA comprises a polypeptide with at least 95% identity to the sequence of SEQ ID NO: 31 or 32.

8. The lentiviral vector of claim 7 wherein the IDUA comprises the polypeptide sequence of SEQ ID NO: 31 or 32.

9. The lentiviral vector of any one of claims 1 to 8 wherein the nucleic acid encoding the polypeptide sequence for IDUA is codon optimised.

10. The lentiviral vector of claim 9 wherein the codon optimised nucleic acid has reduced GC content.

11. The lentiviral vector of any one of claims 1 to 10 wherein the nucleic acid comprises the sequence of SEQ ID NO: 2, 3 or 5.

12. The lentiviral vector of any one of claims 1 to 11 wherein the expression of IDUA is controlled by a ubiquitous promoter.

13. The lentiviral vector of claim 12 wherein the promoter is elongation factor 1a (EF1a).

14. The lentiviral vector of any one of claims 1 to 11 wherein the expression of IDUA is substantially restricted to the liver.

15. The lentiviral vector of claim 14 wherein the expression of IDUA is controlled by a hepatocyte-specific transcription factor site linked to a transthyretin (ET) promoter.

16. The lentiviral vector of any one of claims 1 to 15 wherein transformation of cells with the vector causes upregulation in I DU A activity.

17. The vector of any one of claims 1 to 16 for use as a medicament.

18. The vector of any one of claims 1 to 16 for use in the treatment of metabolic disorder.

19. The vector for use of claim 18 wherein the metabolic disorder is mucopolysaccharidoses type I (MPS I).

20. The vector for use of claim 19 wherein the MPS I is of subtype MPS-IH, MPS-IH/S or MPS-IS.

21. The vector for use of claim 20 wherein the MPS I is MPS-IH (Hurler syndrome).

22. The lentiviral vector of claim 1 wherein the lentiviral vector is replication incompetent, the nucleic acid encoding IDUA comprises the sequence of SEQ ID NO: 2 or 3, and the expression of IDUA is controlled by a ubiquitous promoter which is EF1a, for use in the treatment of MPS I.

23. The lentiviral vector of claim 1 wherein the lentiviral vector is replication incompetent, the nucleic acid encoding IDUA comprises the sequence of SEQ ID NO: 2 or 3, the expression of IDUA is substantially localised to the liver, and wherein the promoter controlling the expression is an ET promoter, for use in the treatment of MPS I.

24. The lentiviral vector of either claim 22 or claim 23 wherein the nucleic acid encoding the polypeptide sequence for IDUA is comprised in a nucleic acid sequence encoding a fusion polypeptide sequence of transferrin protein and IDUA, fused directly through any one of the last 25 amino acids within the C-terminus of transferrin and any one of the first 25 amino acids within the N-terminus of IDUA.

25. The lentiviral vector of either claim 22 or claim 23 wherein the nucleic acid encoding the polypeptide sequence for IDUA is comprised in a nucleic acid sequence encoding a fusion polypeptide sequence of transferrin protein and IDUA fused through a flexible, rigid, and/or cleavable amino acid linker sequence to any one of the last 25 amino acids within the C-terminus of transferrin and any one of the first 25 amino acids within the N- terminus of IDUA.

26. A lentiviral vector comprising (i) a nucleic acid encoding the polypeptide sequence for a- L-iduronidase (IDUA), optionally (ii) a nucleic acid sequence encoding truncated epidermal growth factor receptor (tEGFR) via an internal ribosomal entry site (IRES) sequence, (iii) a nucleic acid sequence encoding multiple microRNA-142 (miR-142) target sites, optionally (iv) a nucleic acid sequence encoding a genetic insulator sequence, and (v) a modified viral envelope whereby cluster of differentiation 47 and 55

(CD47/55) are present on the envelope surface