Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5256—Virus expressing foreign proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island

Definitions

• the present invention relates to transgene expression in host cells using
• cassettes and vectors harbouring such cassettes The invention extends to the therapeutic uses of such cassettes and vectors for expressing transgenes for treating a range of different medical conditions.
• MAR matrix attachment region
• the inventors have previously demonstrated in unpublished work that, following gene transfer, a proportion of the AAV genome becomes heterchromatinised and is then maintained in a transcriptionally inactive state within the target cell. They therefore considered if the inclusion of MARs within an AAV vector would prevent proviral heterochromatinisation from occurring, thereby increasing transgene expression and the potency of the AAV vector system. They constructed and tested the role of the four different matrix associated regions (MARs) in the context of rAAV-mediated gene transfer in murine models.
• MARs matrix associated regions
• MARs included domains from the interferon ⁇ (IFN- ⁇ ) gene, the hypoxanthine phosphoribosyltransferase (HPRT) gene, the apolipoprotein B (ApoB) gene, and the Kaposi sarcoma herpes virus (KSHV) genome. These four MARs were selected on the basis of size ( ⁇ iooobp) and were than graded according to their in silico theoretical approximation of the "energy (GX)" of unwinding.
• the IFN- ⁇ MAR has previously been shown to enhance expression of transgenes in the context of plasmid or retroviral vectors (l, 2).
• the ApoB MAR has been extensively studied for its ability to insulate transgene expression from chromosomal position effects (3, 4).
• the interest in the HPRT and the KSHV MARs has merely been in their ability to serve as the origin of replication and support of autonomous replication respectively (5-7).
• the in silico studies suggested that the IFN- ⁇ and ApoB MARs were likely to be most efficient at enhancing gene expression with GX values of 1.55 and 0.8, respectively.
• GX (2.5) value for the HPRT MAR suggested that it was least likely to maintain the rAAV genome in an unwound state and, therefore, was most unlikely to enhance transgene expression.
• the HPRT MAR has previously been shown to exhibit all the properties of MARs except the ability to enhance transgene expression (5-7).
• Parvoviridae expression cassette comprising a transgene and a matrix attachment region (MAR) sequence or a fragment thereof from a hypoxanthine phosphoribosyltransferase (HPRT) gene.
• MAR matrix attachment region
• HPRT hypoxanthine phosphoribosyltransferase
• the inventors were surprised to observe that the HPRT matrix attachment region or fragment thereof present in the expression cassette unexpectedly enhanced transgene expression in a host cell.
• the expression was at least 10- fold higher than the level of transgene expression achieved with an identical construct containing the IFN- ⁇ MAR, and over 100-fold higher than that observed with the same construct but without any MAR sequences. This was totally unexpected.
• HPRT MAR does not have enhancer activity.
• results described herein suggest that the HPRT MAR element or a fragment thereof is a surprisingly potent enhancer of transgene expression. This has substantial implications for gene therapy of many different conditions, and will undoubtedly impact the design of future generations of Parvoviridae expression cassettes for use in the clinic.
• substantially lower doses of vector containing HPRT MAR are required to achieve therapeutic levels of transgene expression, and this has important safety implications as well as easing the burden on vector production.
• HPRT is a transferase, which catalyzes the conversion of hypoxanthine to inosine monophosphate, and guanine to guanosine monophosphate via transfer of the 5-phosphoribosyl group from 5-phosphoribosyl l-pyrophosphate.
• This enzyme plays a central role in the generation of purine nucleotides through the purine salvage pathway.
• Human HPRT gene is 40.5kb long and the HPRT protein has 218 amino acid residues. The accession number of human HPRT is
• HPRT sequence appears to function optimally in the forward orientation in the AAV-LPi-hFIXco HPRT vector as described herein.
• the MAR elements such as HPRT, can improve transgene expression when cloned into the 3' position as shown in the examples, but it is highly likely that MARs can exert enhancer effect when cloned in the 5' region of promoters, introns and within intragenic regions.
• the MAR sequence or fragment thereof maybe provided anywhere within the expression cassette, including at the 5' end of the promoter and/or within an intron of the transgene. Preferably, however, the MAR sequence or fragment thereof is provided 3' of the transgene.
• the MAR sequence or fragment thereof may be operatively linked to the transgene, i.e. the MAR sequence is capable of enhancing transgene expression.
• the MAR sequence or fragment thereof is preferably a Scaffold/Matrix Attachment Region (S/MAR) element.
• S/MAR Scaffold/Matrix Attachment Region
• the MAR region of the human HPRT gene is an AT-rich sequence and may be substantially set out as SEQ ID No:i.
• the HPRT MAR maps to the first intron of the wild-type HPRT gene between +3858 to +443obp.
• the MAR sequence or fragment thereof provided in the expression cassette of the first aspect may comprise a nucleic acid sequence substantially as set out in SEQ ID No.i, or a functional fragment or variant thereof. It will be appreciated that suitable fragments or variants of SEQ ID No.i may include truncations of this sequence while still being substantially AT- rich. Preferably, the fragment or variant comprises at least 20, 25, 30, 35, 40, 45, 50 or 55 ATs.
• a suitable fragment of the MAR sequence may comprise nucleotides 205 to 450 of SEQ ID No.i, which is referred to as Fragment 2.
• the inventors have determined that the enhancer activity of the MAR sequence is predominantly mediated by nucleotides 320 to 450 of SEQ ID No.i, which is referred to in the Figures as Fragment 2b.
• a fragment of the MAR sequence comprises nucleotides 205 to 450 of SEQ ID No.i.
• the expression cassette may comprise DNA from a member of the Dependovirus genus. It is preferred that the expression cassette comprises DNA from an adeno- associated virus (AAV) vector.
• AAV adeno- associated virus
• the expression cassette is provided in the form of a recombinant expression vector.
• a recombinant vector comprising an expression cassette according to the first aspect.
• the recombinant vector may be a plasmid, cosmid or phage and/or be a viral vector.
• the vector may be single- or double-stranded. Such vectors are useful for
• the vector may comprise DNA from, or is a, member of the Dependovirus genus. It is preferred that the vector comprises DNA from, or is, an adeno-associated virus (AAV) vector.
• AAV adeno-associated virus
• transgene containing HPRT MAR or S/MAR may be combined with many types of backbone vector for expression purposes.
• suitable Parvoviridae backbone vectors include: single- stranded AAV vectors, self-complementary AAV vectors pseudotyped with serotype 2, 5, 8 or 9 capsid or any other capsid protein arising from wild type AAV or engineered forms of AAV capsid.
• the nucleic acid sequence of the internal terminal repeats (ITR) in the expression cassette is substantially set out as SEQ ID No:2.
• the sequence shows the two ITRs and identifies the area where expression cassettes may be inserted.
• the expression cassette or vector may comprise a nucleic acid sequence substantially as set out in SEQ ID No.2, or a functional fragment or variant thereof.
• the cassette or vector may further comprise a variety of other functional elements including a suitable promoter for initiating transgene expression upon introduction of the vector in a host cell.
• the vector is preferably capable of autonomously replicating in the nucleus of the host cell.
• elements which induce or regulate DNA replication may be required in the recombinant vector.
• the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged.
• Suitable promoters may include the SV40 promoter, CMV, EFia, PGK, viral long terminal repeats, as well as inducible promoters, such as the Tetracycline inducible system, as examples.
• the cassette or vector may also comprise a terminator, such as the Beta globin, SV40 polyadenylation sequences or synthetic polyadenylation sequences.
• the vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA.
• a selectable marker for example, ampicillin, neomycin, puromycin or chloramphenicol resistance is envisaged.
• the selectable marker gene may be in a different vector to be used simultaneously with the vector containing the transgene.
• the cassette or vector may also comprise DNA involved with regulating expression of the transgene, or for targeting the expressed polypeptide to a certain part of the host cell.
• Purified vector maybe inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake.
• the vector may be introduced directly into cells of a host subject (e.g. a eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
• vectors of the invention may be introduced directly into a host cell using a particle gun.
• the nucleic acid sequence of the expression cassette of the first aspect is substantially set out as SEQ ID No: 3.
• nucleic acid sequence of the expression cassette of the first aspect (comprising a promoter, a transgene (i.e. Factor IX), a S/MAR and a PolyA tail (without transgene) with ITRs) is substantially set out as SEQ ID No: 4.
• the expression cassette or vector of the invention may comprise a nucleic acid sequence substantially as set out in either SEQ ID No.3 or SEQ ID No.4, or a functional fragment or variant thereof.
• the transgene may be any gene encoding a protein, which may have therapeutic or industrial utility.
• the transgene may encode cystic fibrosis
• transmembrane conductance regulator CFTR
• CFTR transmembrane conductance regulator
• a blood clotting factor such as Factor VIII, Factor VII, Factor IX, or alpha-galactosidase A or glucocerebrosidase.
• Factor IX was used as the transgene in the examples.
• expression cassettes and vectors of the invention may be used to treat a wide variety of conditions.
• the invention provides an expression cassette according to the first aspect or a recombinant vector according to the second aspect, for use in therapy.
• an expression cassette according to the first aspect or a recombinant vector according to the second aspect for treating cystic fibrosis, a haematological disorder, a blood clotting disorder, haemophilia A, haemophilia B, congenital FVII and FX deficiency, a urea cycle disorder, a lysosomal disorder, a disorder affecting the liver, any form of malignant disorder or Leber's congenital amaurosis.
• a method of treating, ameliorating or preventing cystic fibrosis, a haematological disorder, a blood clotting disorder, haemophilia A, haemophilia B, congenital FVII and FX deficiency, a urea cycle disorder, a lysosomal disorder, a disorder affecting the liver, any form of malignant disorder or Leber's congenital amaurosis comprising administering, to a subject in need of such treatment, an expression cassette according to the first aspect or a recombinant vector according to the second aspect.
• the cassette or vector maybe used to treat haemophilia B.
• a host cell comprising an expression cassette according to the first aspect or a vector according to the second aspect.
• the host cell may be a bacterial cell.
• the host cell may be an animal cell, for example a mouse or rat or human cell.
• the host cell may be transformed with cassettes or vectors according to the invention, using known techniques, which will depend on the type of cell.
• a transgenic host organism comprising at least one host cell according to the sixth aspect.
• the host may be a bacterium.
• the host organism may be a multicellular organism, which is preferably non-human.
• the host organism may be a mouse or rat.
• vectors and medicaments according to the invention may be used in a monotherapy (i.e. the sole use of a recombinant vector), for use in gene therapy techniques.
• vectors and medicaments according to the invention maybe used as an adjunct to, or in combination with, known therapies or drugs.
• the vectors and medicaments according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
• the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
• the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given, and preferably enables delivery of the agents across the blood-brain barrier.
• Medicaments comprising vectors and medicaments of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid.
• Compositions comprising vectors and medicaments of the invention may be administered by inhalation (e.g. intranasally).
• Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
• Vectors and medicaments according to the invention may also be incorporated within a slow- or delayed-release device.
• Such devices may, for example, be inserted on or under the skin, and the medicament maybe released over weeks or even months.
• the device maybe located at least adjacent the treatment site.
• Such devices may be particularly advantageous when long-term treatment with vectors and medicaments according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
• vectors and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
• the amount of the vector and medicament that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the vector and medicament, and whether it is being used as a monotherapy or in a combined therapy.
• the frequency of administration will also be influenced by the half-life of the vector within the subject being treated.
• Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular vector in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease being treated. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
• a dose of between 2 x ⁇ 3 ⁇ 4 and 2 x ⁇ of vector genome per kg of patient may be used.
• a dose of between 2 x io 10 and 2 x ⁇ of vector genome per kg of patient or between 2 x io 11 and 2 x io 12 of vector genome per kg of patient may be used.
• the vector or medicament may be administered before, during or after onset of the disease condition. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the vector or medicament may require
• vectors and medicaments may be administered as two (or more depending upon the severity of the bacterial infection being treated) daily doses of between between 2 x io 11 and 2 x io 12 of vector genome per kg of patient (i.e. assuming a body weight of 70kg).
• a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
• a slow release device may be used to provide optimal doses of vectors and medicaments according to the invention to a patient without the need to administer repeated doses.
• Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the vectors and
• medicaments according to the invention and precise therapeutic regimes (such as daily doses of the vectors and the frequency of administration).
• a pharmaceutical composition comprising an expression cassette of the first aspect or a vector according to the second aspect, and a pharmaceutically acceptable vehicle.
• the invention also provides in a ninth aspect, a process for making the composition according to the eighth aspect, the process comprising combining a therapeutically effective amount of an expression cassette of the first aspect or a vector according to the second aspect, and a pharmaceutically acceptable vehicle.
• the composition may be used for treating cystic fibrosis, a haematological disorder, a blood clotting disorder, haemophilia A, haemophilia B, congenital FVII and FX deficiency, a urea cycle disorder, a lysosomal disorder, a disorder affecting the liver, a malignant disorder or Leber's congenital amaurosis.
• the composition may be used to treat haemophilia B, i.e. it is a haemophilia B treatment composition.
• a "subject” may be a vertebrate, mammal, or domestic animal.
• medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.
• livestock e.g. a horse
• pets e.g. a human
• the subject is a human being.
• a “therapeutically effective amount” of vector is any amount which, when it is a "therapeutically effective amount" of vector is any amount which, when it is a “therapeutically effective amount" of vector is any amount which, when it is a “therapeutically effective amount" of vector is any amount which, when it is a “therapeutically effective amount" of vector is any amount which, when it is a “therapeutically effective amount" of vector is any amount which, when it is a “therapeutically effective amount" of vector is any amount which, when a
• the amount of drug that is needed to treat the infection, or produce the desired effect is the amount of drug that is needed to treat the infection, or produce the desired effect.
• the therapeutically effective amount of vector used may be from about between 2 x 10 11 and 2 x 10 12 of vector genome per kg of patient.
• a "pharmaceutically acceptable vehicle" as referred to herein is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
• the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
• a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
• the vehicle may also be an encapsulating material.
• the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
• the active agent i.e. the vector
• the powders and tablets preferably contain up to 99% of the active agents.
• Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
• the pharmaceutical vehicle maybe a gel and the composition may be in the form of a cream or the like.
• the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
• Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
• the vector according to the invention maybe dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
• the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
• suitable examples of liquid vehicles for oral and parenteral administration include water
• the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
• Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
• the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
• the vector may be prepared as a sterile solid composition that maybe dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
• compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
• solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• bile salts for example, enough saline or glucose to make the solution isotonic
• acacia gelatin
• sorbitan monoleate sorbitan monoleate
• polysorbate 8o oleate esters of sorbito
• administration include sterile solutions, emulsions, and suspensions.
• nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
• substantially the amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleotide sequence identified as SEQ ID No:i, 2, 3, or 4, and so on.
• amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
• the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
• sequence identity value may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA,
• the parameters used by the alignment method for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
• the pair-score matrix used e.g. BLOSUM62, PAM250, Gonnet etc.
• gap-penalty e.g. functional form and constants.
• percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
• acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
• a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 1-15, or their complements under stringent conditions.
• stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C.
• a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences referred to herein or encoded by the described nucleic acid molecules.
• nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
• Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
• Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
• small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
• Large non- polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
• the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
• the positively charged (basic) amino acids include lysine, arginine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
• FIG. l shows, in the top panel, a schematic of various embodiments of AAV vectors that were tested.
• a control vector (above) is shown having a Factor IX expression cassette (LPi-hFIXco) with a SV40 polyadenylation signal sequence, as well as test vectors (below) containing a MAR sequence which is positioned 3' of a hFIX transgene.
• the MAR was from either the interferon B gene, the HPRT gene, the Apolipoprotein B gene or the Kaposi sarcoma herpes virus.
• the bottom panel shows the hFIX levels in murine plasma for each of the vectors tested.
• the MAR was tested in forward (F) and reverse (R) orientations.
• Figure 3 shows that AAV vectors containing HPRT MAR mediate higher levels of hFIX expression in macaques.
• a dose of 4xio 12 vg/kg of ssAAV8-LPi-hFIXco (blue bar) or ssAAV8-LPi-hFIXco-HPRT (red bar) was injected into the peripheral vein of male rhesus macaques as a bolus infusion.
• Human FIX expression was assessed at 7 days after gene transfer. Shown are the results of repeated measurements expressed as mean ⁇ SEM;
• Figure 4 shows that single stranded AAV containing HPRT MAR have the same potency as self-complementary AAV vectors.
• FIX levels were assessed at various time points and expressed as mean percentage of normal values ⁇ SEM;
• Figure 5 is a schematic of the 573bp HPRT MAR showing the wild type HPRT gene co-ordinates (+3858-+4430) at the top. Smaller HPRT fragments were created by PCR and their size and co-ordinates with reference to the 573bp HPRT MAR are shown ranging from 0-573DP;
• the data shows that the level of FIX expression observed with full length 573bp MAR at 4 weeks after gene transfer was comparable to that achieved with HPRT fragments 2 and 2b suggesting that the enhancer activity of HPRT MAR is mediated predominantly by sequences in its i3ibp sub-fragment;
• Figure 7 shows quantitative chromatin immunoprecipitation (qChIP). Analysis of the pattern of (active) histone lysine acetylation (acH3), dimethylation of lysine 4 (2MeK4) and (inactive) histone dimethylation of lysine 9 (2meK9H3) at the promoter region of the viral genome in mice injected with ssAAV8-LPi-hFIXco with and without HPRT SMA/R. There is a decrease in the 2meK9H3 mark in the AAV promoter in the mice injected with the construct containing the HPRT SMA/R, whilst the acH3 and 2MeK4 marks increase or remain constant. Enrichment values for the modifications studied were normalized to the Input;
• Figure 8 shows the construction and characterisation of single stranded AAV- hFIXco containing IFNB S/MAR (A) Schematic of vectors. Each vector is represented schematically as it is packaged inside the virion. Common features of all vectors include the hybrid liver specific promoter (LPi), SV40 intron, codon-optimised human FIX cDNA (hFIXco) and a truncated SV40 late polyA (tSV40pA) flanked by AAV inverted terminal repeats (ITRs represented as hairpin loops).
• LPi hybrid liver specific promoter
• hFIXco codon-optimised human FIX cDNA
• tSV40pA truncated SV40 late polyA flanked by AAV inverted terminal repeats
• ssAAV-LPi- hFIXco-Control and vector genomes containing the IFNB S/MAR in the 3' region of the expression cassette are 2.4Kb and 3.3Kb in size respectively.
• D Proviral copy numbers in liver.
• Proviral copy number was determined by a Q-PCR assay on liver samples collected 6 weeks after tail vein administration of ssAAV- hFIXco-IFN-F/R and ssAAV-hFIXco-Control vectors.
• Standards consisted of serial dilutions of ssAAV-LPi-hFIXco vector plasmid diluted in murine liver genomic DNA derived from a na ' ive animal. Results are presented as mean proviral copies/cell ⁇ SEM;
• Figure 9 shows construction of ssAAV-LPi-hFIXco containing alternative S/MARs.
• A Schematic of vectors. Each vector is represented schematically as it is packaged in the virion. Common features of each vector are as previously mentioned in Figure 8A.
• the HPRT and ApoB S/MAR elements and KSHV control element were cloned 3' of the hFIXco cDNA in forward and reverse orientation (HPRT-F/R, ApoB-F/R and KSHV- F/R respectively). All vectors depicted are approximately 3-3.3KI) in size.
• B Transgene expression in C57BL/6 mice.
• C Proviral copy numbers in liver. Proviral copy number was determined by a Q-PCR assay on liver samples collected 4 weeks after transduction and depicted as mean proviral copies/cell ⁇ SEM.
• D HFIX mRNA levels in liver.
• mRNA levels were determined by a Q-PCR assay on cDNA generated from total liver RNA collected 4 weeks after tail -vein administration of 4 x1 ⁇ 12 vg/kg of ssAAV-hFIXco-IFN-R, ssAAV-hFIXco-HPRTF/R and ssAAV-hFIXco-Control into C57BL/6 mice. Results are presented as mean mRNA levels corrected for the amount of housekeeping cDNA (GAPDH) present in each sample ⁇ SEM and then corrected for proviral copy number to take account of variation in transduction;
• Figure 10 shows lower doses of ssAAV-LPi-hFrXco-HPRT-F mediate therapeutic transgene expression in male C57BL/ 6 mice.
• B Proviral copy numbers in liver.
• Proviral copy number was determined by a Q-PCR assay on liver samples collected 4 weeks after tail -vein administration of 4x1 ⁇ 10 vg/kg of ssAAV-hFIXco-HPRT-F and ssAAV-hFIXco-Control vectors. Results are depicted as mean proviral copies/cell ⁇ SEM;
• Figure 11 shows higher potency of HPRT S/MAR containing vector in rhesus macaques.
• Human FIX levels in rhesus plasma were determined at the indicated time points after peripheral vein administration of 4x1 ⁇ 12 vg/kg of ssAAV-LPi- hFIXco-HPRT-F and ssAAV-LPi-hFIXco-Control vectors. Results are depicted as averages of repeat ELISA results ⁇ SEM;
• Figure 12 shows deletion analysis of HPRT S/MAR.
• A Schematic: Smaller HPRT S/MAR fragments were generated by a standard PCR method and cloned 3' of the hFIXco cDNA in forward orientation (FRi, FR2, FR2a and FR2b).
• FIG. 13 shows HPRT S/MAR improves potency of self complementary LPi- hFIXco vector.
• Figure 14 shows epigenetic modification of ssAAV-LPi-hFIXco proviral DNA (A) ⁇ S/MAR ChlP.
• ChIP analysis was carried out on chromatin extracted from liver samples collected 6 weeks after tail-vein administration of 4x1 ⁇ 12 vg/kg of ssAAV- LPi-hFIXco-IFN-R and ssAAV-LPi-hFIXco-Control vectors into 4 to 6 week old male C57BL/6 mice.
• Vector genome regions amplified by Q-PCR included proximal and distal regions of the LPi promoter (152 and 366 respectively).
• Antibodies used for ChIP included anti H3K9me2, anti ⁇ , anti H3K4me2 and anti H3AC.
• Figure 15 shows cytosine methylation profiles of the AAV LPi promoter revealed by bisulphite sequencing.
• Bisulphite sequencing was carried out on DNA extracted from rhesus liver samples 1 week, 2 months and 1 year after peripheral vein
• scAAV8-LPi-hFIXco vector at a dose of 2x 10 12 vg/kg.
• the vector genome is represented schematically (top). Boxes represent CpG regions of the LPi promoter (bottom). Unfilled boxes depict non-methylated CpG regions.
• MAR sequences were synthesised in a PUC57 plasmid vector with flanking Noti restriction sites (Genescript), allowing for all new MARs to be cloned into Noti digested pAV-LPi-hFIXco-mcs-SV40pA plasmid DNA in both forward and reverse orientation.
• pAV-LPi-hFIXco-MCS-SV40pA is a single stranded AAV expression cassette in which the codon optimised hFIX gene is under the control of the LPi liver specific promoter.
• Primers for the generation of short HPRT MAR fragments were designed to incorporated Kpni (forward primers) and Noti (reverse primers) restriction sites for cloning into the pAV-LPi-hFIXco-mcs-SV40pA plasmid in forward orientation.
• PCR products and pAV-LPi-hFIXco-mcs-SV40pA were first digested with Kpni followed by purification using the PCR purification kit (Qiagen) and subsequent Noti digestion.
• DH50 competent cells were thawed briefly on ice and divided into 25 ⁇ 1 aliquots. 5 ⁇ 1 of each ligation mixture including negative control was then added to one individual aliquot of competent cells and left for 30 min on ice.
• positive control ⁇ of PUC19 plasmid DNA (NEB) was also added to 25 ⁇ 1 of DH50 cells and incubated on ice for the same period of time. Next, to allow for the uptake of DNA, all samples and controls were heat shocked at 42°C for 20 sec and immediately placed on ice for a further 5 minutes.
• the extracted DNA was used in a diagnostic restriction digest whereby the enzymes selected would generate fragments of sizes that enabled correct constructs to be differentiated from the incorrect. All diagnostic digests were carried out at 37°C for 3 hrs with the following components per sample: 5 ⁇ 1 DNA, ⁇ buffer, o.iul BSA, 0.25 ⁇ 1 enzyme and 3 ⁇ 65 ⁇ 1 molecular biology water. Samples were run on a 1% agarose gel at 120V.
• HEK293T cells For each virus 40 ⁇ 15cm plates of HEK293T cells were used at approximately 70% confluence on the day of transfection. Cells were cultured at 37 C, 5% C0 2 in DMEM (PAA) with 10% FBS (GIBCO) and enzymatically passaged every 2-3 days. For the transfection mixture 6ml of Polyethyleneimine (PEI) (Polysciences) was added to 54ml serum free DMEM.
• PEI Polyethyleneimine
• helper plasmid containing essential genes from the adenoviral genome to support rescue and replication of the viral genome
• o.6mg 2/8 plasmid containing rep and cap genes
• o.6mg of transgene containing plasmid 4C ⁇ g of PCLio.i-EFia-GFP plasmid was added to serum free DMEM to a final volume of 62ml.
• the mixture containing DNA was then filtered into the PEI containing mixture through a 0.2 ⁇ syringe filter.
• the solution was mixed and incubated at room temperature for 15mm. 3ml of transfection solution was then added to each 15cm dish.
• AAV2/8 Purification of AAV2/8 was carried out using a 5ml AVB Sepharose (GE Healthcare) packed column. Briefly, following line washes with the appropriate buffers, the affinity medium was equilibrated with filtered PBS pH 7.5 for io-20min (or until pH and absorbance readings were stable) at a flow rate of 2ml/min. The cell lysates obtained from 40 plate preparations was then loaded onto the column at 5ml/min (traceable as an increase in absorbance at 260 and 28onm as unbound protein and DNA exits the column). Residual unbound protein and DNA was washed off the column in filtered PBS allowing for viral particles exclusively to be eluted in 5omM Glycine pH 2.7 (5ml/min).
• the eluate was collected in lml fractions into tubes containing 3 ⁇ 1 Tris pH 8.8 to neutralise the glycine.
• the virus containing fractions were identified by a peak in absorbance at 260 and 28onm.
• fractions were pooled together and dialysed overnight at 4°C in PBS using a loKDa cut-off dialysis cassette (Slide-A-Lyzer Thermo Scientific). Titration of AAV8 ( OPCR based method)
• the titre of all AAV2/8 viruses was determined from a standard generated from LPi containing plasmid DNA. Each virus was quantified in three different dilutions (1:100, 1:1000 and 1:10000 in molecular biology water), ⁇ of virus samples, negative control samples (Water) and standards were added to a 96 well plate in triplicate. To each well the following master mix was added: ⁇ 2.5 ⁇ 1 SYBR Green PCR master mix (Qiagen), ⁇ forward primer, ⁇ reverse primer, 0.5 ⁇ 1 molecular biology water ( ⁇ 5 ⁇ 1 master mix/well). Plates were then sealed, briefly spun by centrifugation and placed in a thermal cycler (Eppendorf ).
• AAV8 capsid was assessed by relative band intensity of capsid proteins (VPi, VP2 and VP3).
• SIGMA Laemmli buffer
• Viruses were run on denaturing alkaline gels and band intensity was quantified by comparison against a standard (DNA ladder).
• 8.5 ⁇ 1 of alkaline sample loading buffer 200 ⁇ 1 glycerol, 8 ⁇ 1 50X alkaline running buffer, 6 ⁇ 1 20% SDS, Xylene cyanol, up to lml deionised water) was added to each 25 ⁇ aliquot of virus to be titred.
• Viral titre could then be calculated by determining the number of AAV genomes in the given amount of DNA (based on the length of the viral genome in question).
• mice used in the study were male C57BL/ 6 aged between 4 and 8 weeks.
• AAV2/8 viruses were prepared in X-VTVO 10 media and administered via tail vein injection with 3-4 mice per group. Mice were bled at regular intervals and plasma was collected by centrifugation (4000 xg for 15 min at 4°C) of blood samples in tri- sodium citrate. 4 weeks after the injection date mice were sacrificed and livers were taken for further investigations.
• hFIX levels of hFIX in mouse plasma and supernatants from transduced cell cultures were determined by FIX ELISA.
• Standards for analysis on cell cultures and mouse plasma were prepared by serial dilutions of 1000% recombinant hFIX coagulation reference (Technoclone) in X-Vivo 10 (GIBCO) or C57BL/6 plasma respectively.
• Huh7 supernatants and corresponding standards were diluted 1:2 in PBST +2% bovine serum albumin (dilution buffer).
• Mouse plasma samples and corresponding standards were diluted 1:100 in dilution buffer (where required, plasma samples were initially diluted in naive C57BL/6 plasma to ensure values fell within the range of the standard curve).
• Secondary antibody was prepared by adding 5 ⁇ 1 Goat antihuman FIX-HRP peroxidise labelled antibody (Affinity Biological Inc) to 10ml of dilution buffer, ⁇ of diluted secondary antibody was added to each well. Plates were incubated with secondary antibody for ruling at 37°C followed by 5 washes with PBST. 200 ⁇ 1 SIGMA FASTTM OPD peroxidase substrate was then added to each well and colour was allowed to develop for 3-4 minutes. To stop the reaction soul/well of 3M HCL was added. Plates were read at 490nm on an ELISA plate reader. Viral Genome copy number analysis
• genomic DNA was extracted from mouse liver samples using the DNeasy® Blood and Tissue kit (Qiagen) as per the manufacturer's instructions. For each sample DNA was diluted to an approximate concentration of 7-5ng/ ⁇ and loaded in 5 1 aliquots in duplicate to a 96 well QPCR plate. 2 ⁇ 1 of QPCR master mix ( ⁇ 2.5 ⁇ 1 SYBR Green master mix, ⁇ forward LPi primer, ⁇ reverse LPi primer, 5 ⁇ 5 ⁇ 1 molecular biology water per well) was then added to each well and cycling conditions were carried out as described previously.
• QPCR master mix ⁇ 2.5 ⁇ 1 SYBR Green master mix, ⁇ forward LPi primer, ⁇ reverse LPi primer, 5 ⁇ 5 ⁇ 1 molecular biology water per well
• Copy number was determined from an LPi plasmid DNA standard (using LPi primers) and DNA concentration for each sample was determined from levels of housekeeping gene expression (derived from a mouse genomic DNA standard using Gapdh primers in a separate reaction). To calculate copies per cell LPi copy number results were divided by the amount of DNA in each sample (ng); these figures were then multiplied by 37.5 (the theoretical amount of DNA loaded in each well) and divided by 6528.5 (the approximate number of cells from which 37.5ng of DNA is derived). To normalise FIX levels for viral genome copy number (FIX/ copy/ cell) values of % normal FIX were divided by copy number from the corresponding liver DNA sample. FIX RNA copy number analysis
• RNAlater® Qiagen stabilised mouse liver samples were placed in liquid nitrogen and disrupted with a mortar and pestle. Samples were homogenised using Qiashredder® (Qiagen) columns and RNA was extracted using the RNeasy Plus® kit (Qiagen) as per the manufacturer's instructions. RNA was then converted to cDNA by using the cDNA synthesis kit (Bioline). 2 ⁇ 1 of cDNA samples, cDNA standards and negative controls (-RT and water) were then loaded onto a 96well QPCR plate in duplicate.
• Liver tissue (about 200mg) was chopped into small pieces (sesame seed size), transfered to a 40 um nylon cell strainer and washed in PBS with protease inhibitors. Livers were fixed with 1% formaldehyde at RT for 10 min, lysed and sonicated using a Bioruptor (Diagenode) for 45 minutes. The sonicated extract was
• MARs matrix associated regions
• IFN- ⁇ interferon ⁇
• HPRT hypoxanthine-guanine phosphoribosyltransferase
• ApoB apolipoprotein B
• FIG 3 there is shown that AAV vectors containing HPRT MAR mediate higher levels of hFIX expression in macaques.
• Figure 4 shows that single stranded AAV containing HPRT MAR have the same potency as self-complementary AAV vectors.
• Figure 5 is a schematic of the 573bp HPRT MAR showing the wild type HPRT gene co-ordinates (+3858-+4430) at the top
• the data shows that the level of FIX expression observed with full length 573bp MAR at 4 weeks after gene transfer was comparable to that achieved with HPRT fragments 2 and 2b suggesting that the enhancer activity of HPRT MAR is mediated
• HPRT MAR reduces the interaction of histones with inactive marks with the LPi promoter whilst facilitating an association with histones containing active marks thus enhancing transcription from the promoter.
• the LPi-hFIXco AAV expression cassette encoding hFIX has been previously described (Nathwani et al., 2006) Key elements of this cassette include a hybrid liver-specific promoter (LPi) driving the expression of a codon optimized hFIX cDNA.
• LPi hybrid liver-specific promoter
• this expression cassette was modified by inserting a Bbsl-Hpal oligonucleotide encoding a multiple cloning site (MCS) followed by a truncated SV40 polyadenylation (tSV4opA) sequence downstream of the hFIX cDNA (AAV-LPi-hFIXco-Control) (Hart et al., 1985).
• the IFNB S/MAR was derived from MP4253.SFGmSR-preMSV.eGFP plasmid (a gift from Dr Martin Pule, UCL Cancer Institute, UK) and ligated as a blunt fragment into the EcoR-V site of the MCS in LPi-hFIXco-Control plasmid. HPRT, ApoB
• All vectors were made by the adenovirus-free transient transfection method previously described, using a chimeric AAV2 Rep-8Cap packaging plasmid (pAAV2- 8) and an adenoviral helper plasmid (Nathwani et al., 2001). Vectors were purified as described before (Davidoff et al., 2004) and vector genome (vg) titers determined by standard alkaline gel or quantitative real-time PCR (Q-PCR) based methods (Fagone et al., 2011).
• Captive-bred male Macaca mulatto approximately 4 years of age and weighing between 4 and 5 kg were purchased from Charles River Laboratories (Sierra, NV).
• Single stranded AAV8-LPi-hFIXco vectors containing S/MAR or control sequences were administered into the peripheral vein as a bolus infusion as previously described (Nathwani et al., 2007).
• Human FIX antigen levels in murine and rhesus samples were determined by enzyme-linked immunosorbent assay (ELISA) as previously described (Nathwani et al., 2002).
• AAV transgene copy number in liver was quantified using quantitative real-time PCR (Q-PCR) with the following primers designed to amplify a 283bp region of the LPi promoter: 5' primer, 5'-GGA GAG GAG CAG AGG TTG TC-3' [SEQ ID No: 6]; 3' primer, 5' - TGG TGG TGC CTG AAG CTG AG -3' [SEQ ID No: 7].
• the Q-PCR reaction was performed using the QuantiFast SYBR Green kit as per the
• RNA from each sample was subjected to reverse transcription using the cDNA Synthesis Kit (Bioline, London, UK).
• 5 ⁇ cDNA was then used in a Q-PCR reaction as described previously with primers designed to amplify a I27bp region of the hFIXco cDNA: 5' primer, 5'- GGG CAA GTA TGG CAT CTA CA -3' [SEQ ID No: 10]; 3' primer, 5'- AAA GCA TCG AGT ACGTAACT-3' [SEQ ID No: 11].
• GAPDH primers used to establish equivalent cDNA loading were sourced from the Mm_Gapdh _3_SG QuantiTect primer assay (Qiagen, Crawley, UK). Chromatin Immunoprecipitation
• immunoprecipitation was carried out using the EZ-Magna ChIP A-Chromatin Immunoprecipitation Kit (Millipore, Feltham, UK) as per the manufacturer's instructions.
• Antibodies used included anti-H3K4me2, anti-H3ac, anti- ⁇ (from Millipore, Feltham, UK) anti H3K9me2 and Rabbit control IgG (from Abeam, Cambridge, UK). IPs were carried out at 4°C overnight with either 4 ⁇ g of specific antibody or with no antibody as control.
• DNA pull down was quantified by Q-PCR using the following primer pairs: 152 (proximal LPi promoter) 5' primer, 5'- GGAGTCGTGACCCCTAAAATG-3' [SEQ ID No: 12]; 3' primer, 5'- CTCTGACCTCTGCCCCAGCTC-3' [SEQ ID No: 13]. 366 (distal LPi promoter) 5' primer, 5 ' -TTCGGTAAGTGCAGTGGAAG-3 ' [SEQ ID No: 14]; 3' primer, 5'- GTTATCGGAGGAGCAAACAG-3' [SEQ ID No: 15].
• transgene expression was assessed by determining plasma hFIX levels at varying time points over a period of 8 weeks.
• the kinetics of hFIX expression with or without the S/MAR element was similar, with hFIX being detectable at 2 weeks and reaching steady state levels by 4 weeks (see Figure 8C), consistent with previous observations. Highest levels of hFIX expression were observed in mice transduced with ssAAV-LPi-hFIXco-IFN-R with steady state hFIX levels of i88 ⁇ i8% of normal.
• the inventors cloned a 587bp S/MAR element derived from the human HPRT gene and a 486bp S/MAR from the human ApoB gene into the ssAAV-LPi-hFIXco vector plasmid downstream of the hFIXco cDNA in forward and reverse orientation (ssAAV8-LPi-hFIXco-HPRT-F, ssAAV8-LPi-hFIXco-HPRT-R, ssAAV8-LPi-hFIXco- ApoB-F and ssAAV8-LPi-hFIXco-ApoB-R, respectively).
• a 52obp non-S/MAR control element derived from a region of the Kaposi sarcoma herpes virus (KSHV) genome known to promote autonomous replication (a property attributed to some S/MARs) was cloned into the same position of the vector genome in forward and reverse orientation (ssAAV8-LPi- hFIXco-KSHV-F and ssAAV8-LPi-hFIXco-KSHV-R respectively) (see Figure 9A).
• KSHV Kaposi sarcoma herpes virus
• a group of mice was also transduced with the same dose of ssAAV8-LPi-hFIXco-IFN-R.
• the highest levels of transgene expression were observed in the ssAAV8-LPi-hFIXco-HPRT-F and ssAAV8-LPi-hFIXco-ApoB-R transduced cohorts with hFIX levels of 1401 ⁇ 87% and 1540 ⁇ 149% of normal, respectively.
• mice transduced with ssAAV8-LPi-hFIXco-HPRT- F had at least four times higher levels of hFIX/transgene copy when compared to the cohorts transduced with other S/MAR containing AAV vectors including ssAAV8- LPi-hFIXco-ApoB-R (see Figure 1, lower panel).
• the LPi promoter has previously been shown to restrict transcription to hepatocytes.
• the inventors assessed hFIX mRNA levels in the liver at 4 weeks after gene transfer in a selected number of cohorts.
• the mRNA levels/copy of the transgene were greatest in the ssAAV8-LPi-hFIXco-HPRT- F cohort at 433 ⁇ 132RU (see Figure 9D), which was approximately 2-fold higher than mRNA levels observed in the ssAAV8-LPi-hFIXco-HPRT-R (204 ⁇ 57RU) and ssAAV8-LPi-hFIXco-IFN-R (i76 ⁇ 8iRU) cohorts but an over 20-fold increase in transcript levels when compared to the ssAAV8-LPi-hFIXco-Control animals (i8 ⁇ 6RU).
• the non-human primate model provides an opportunity to validate observation in murine models in a context relevant to humans (Herzog at al., 2011).
• Peripheral vein administration of 4xio 12 vg/kg of ssAAV8-LPi-hFIXco-Control or ssAAV8-LPi- hFIXco-HPRT-F AAV vector into male adolescent rhesus monkeys was well- tolerated with no perturbation of vital signs (pulse, respiration and temperature) or liver transaminases (alanine aminotransferase ⁇ 45U/L) over a period of 37 weeks after gene transfer.
• Human FIX was detectable in monkey plasma within 72 hours of vector
• the mean transgene copy number in the liver by Q-PCR was 32 ⁇ 9 and 13 ⁇ 7 proviral copies/cells respectively for the monkeys transduced with ssAAV8-LPi-hFIXco-Control and ssAAV8-LPi-hFIXco- HPRT-F.
• single stranded AAV vector containing HPRT-S/MAR mediated higher FIX expression/ copy of proviral DNA when compared to control vector in NHPs.
• HPRT S/MAR was divided into smaller fragments as outlined in Figure 12A and then cloned into ssAAV-LPi-hFIXco in order to identify the minimum sequences required for enhancement of AAV transgene expression.
• an equivalent dose of ssAAV8- LPi-hFIXco-HPRT-F vector containing the full 587bp HPRT S/MAR was assessed 2 weeks after vector administration.
• AAV8 capsid pseudotyped vector was next administered into the tail vein of 6 to 8 week old male C57BL/ 6 mice at a dose of 4x 10 10 vg/kg and plasma hFIX levels and proviral copy number in the liver were assessed at 4 weeks after gene transfer.
• mice transduced with scAAV8- LPi-hFIXco-HPRT-FR2b expressed hFIX at approximately 105 ⁇ 6% (see Figure 13), which was 35-fold higher (P 0.0038, student T-test) than that observed in scAAV8- LPi-hFIXco transduced animals (3 ⁇ i%) suggesting that S/MAR elements are also effective in a self-complementary format.
• S/MARs confer higher transgene expression through epigenetic modification of the ssAAV-LPi-hFIXco genome
• heterochro matin markers (dimethylation of lysine 9 on histone 3 [H3K9me2] and heterochromatic adaptor proteins ⁇ ) in the LPi promoter region (see Figure 14A). Both H3K9me2 and ⁇ marks are associated with inactive promoters (Lienert et al., 2011; Wreggett et al., 1994).
• H3K42me histone H3 with dimethylation on lysine 4
• H3AC acetylation of H3 lysines
• Both H3K4me2 and H3AC marks correlate with transcriptionally active genes (Yan et al., 2006).
• ChIP analysis of ssAAV-LPi-hFIXco-IFN-F showed the same in-vivo chromatinization pattern as the control vector (data not shown).
• S/MARs improve AAV transgene expression by maintaining AAV concatamers in an open chromatin, transcriptionally active, configuration.
• S/MARs have other biological properties that could potentially influence AAV transgene expression.
• some S/MARs have enhancer properties capable of upregulating the expression of heterologous reporters (Stief et al., 1989). Additionally, they have the ability to bring the AAV proviral DNA in close proximity to key nuclear regulatory elements within the nucleus by forming "anchor points" with nuclear matrix.
• S/MARs may provide significant enhancement of transcription by providing topological separation of individual expression cassettes within large AAV concatamers, thus reducing read-through transcription and/or "insulating" the proviral DNA from the inhibitory influences of other host genes.
• Previous studies show that some or all of these mechanisms are in play in the context of gene transfer.
• the human ⁇ gene S/MAR element has been shown to prevent epigenetic silencing of episomally maintained plasmid vectors as well as retroviral vectors.
• the ApoB S/MAR used in this study was reported to insulate associated genes from positional silencing effects.
• the HPRT S/MAR is primarily documented to have a role in supporting autonomous replication. As such, the observation that highest enhancement of AAV transgene expression was achieved with the HPRT vector was totally unexpected.
• S/MARs do not have an obvious consensus sequence although prototypic elements are AT-rich.
• the structure of S/MAR in relation to the transgene appears to have a greater influence on activity than the overall nucleotide base composition. This may explain there may be an orientation-dependent effect in the context of rAAV mediated gene transfer.
• Deletion analysis of the HPRT S/MAR showed that a i3obp region (Fragment 2b) was able to enhance FIX expression in the context of self- complementary vectors.
• the only unique nucleotide consensus sequence found in HPRT fragment 2b was TYRTTT, which occurs twice in this sequence and may represent a core enhancer.
• the potency of fragment 2b may be purely structural due to the large quantity of poly-pyrimidine and poly-purine tracts present in the sequence. These enable the formation of secondary non-B DNA structures, which facilitate base-unpairing and strand-separation, thereby catalyzing transcription.
• S/MAR containing rAAV vectors provides a novel, safe, inbuilt molecular strategy for augmenting rAAV transgene expression by overcoming some of the limitation of AAV biology.
• Adeno-associated virus vector encoding codon optimized human factor IX shows great promise in severe hemophilia B patients. However, hepatocellular toxicity was observed in some patients treated at the high dose levels.
• various scaffold/ matrix attachment regions S/MARs were cloned at the 3' end of a modified single- stranded (ss) AAV-LPi-hFIXco expression cassette.
• Hpi Drosophila Heterochromatin Protein-i

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