EP1506019A1 - Complexes for the delivery of biologically-active material to cells - Google Patents

Complexes for the delivery of biologically-active material to cells

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Publication number
EP1506019A1
EP1506019A1 EP03730315A EP03730315A EP1506019A1 EP 1506019 A1 EP1506019 A1 EP 1506019A1 EP 03730315 A EP03730315 A EP 03730315A EP 03730315 A EP03730315 A EP 03730315A EP 1506019 A1 EP1506019 A1 EP 1506019A1
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EP
European Patent Office
Prior art keywords
complex according
same
different
complex
nucleic acid
Prior art date
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Application number
EP03730315A
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German (de)
English (en)
French (fr)
Inventor
Christopher Antony Hurley
Helen Claire Hailes
Alethea Bernice Tabor
Stephen Lewis Hart
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UCL Business Ltd
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University College London
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a complex suitable for delivery of a biologically-active material, for example nucleic acids, proteins and small molecules, to a cell.
  • a biologically-active material for example nucleic acids, proteins and small molecules
  • the invention also relates to the use of such complexes in the delivery of biologically-active material to a cell, for example in prophylaxis, treatment and vaccination.
  • the invention relates to lipids which may be used in the complexes of the invention.
  • Gene delivery for therapy or other purposes is of course well-known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers.
  • the term refers to the delivery into a cell of a gene or part of a gene to correct some deficiency.
  • the term is used also to refer to any introduction of nucleic acid material into target cells, and includes gene vaccination and the in vitro production of commercially-useful proteins in so-called cell factories.
  • Cell delivery systems fall into three broad classes, namely those that involve direct injection of naked DNA, those that make use of viruses or alternated viruses and those that make use of non- viral delivery agents. Each has its advantages and disadvantages. Although viruses as delivery agents have the advantages of high efficiency and high cell selectivity, they have the disadvantages of toxicity, production of inflammatory responses and difficulty in dealing with large DNA fragments.
  • the present invention in making use of lipids, can overcome these problems.
  • Cationic lipids for use in gene delivery were developed by Feigner in the late 1980s, and reported in Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987.
  • a recent patent to Feigner et al. that may be referred to is US 5264618. The disclosure of each of these documents is incorporated herein by reference.
  • Feigner developed the now commercially-available cationic liposome known by the trade mark "Lipofectin" which consists of the cytofectin, DOTMA and the neutral lipid DOPE in a 1 : 1 ratio.
  • Various other cationic liposome formulations have since been devised, most of which combine a synthetic cationic cytofectin and a neutral lipid.
  • LID vectors are three component vectors consisting of an integrin binding peptide, a lipid or lipid mixture such as Lipofectin and DNA. Specificity results from the targeting of the integrin, and transfection efficiencies comparable to some adenoviral vectors can be achieved (Hart et al, Hum. Gene Ther. 9, 1037-47, 1998; Harbottle et al. Hum. Gene. Ther. 9, 575-85, 1998; and Jenkins et al. Gene Therapy 7, 393-400, 2000, the disclosures of which are incorporated herein by reference).
  • Cytofectins are positively charged molecules having a cationic head group attached via some spacer to a hydrophobic tail.
  • DOTMA analogues there may be mentioned complex alkylamine/alkylamides, cholesterol derivatives, and synthetic derivatives of dipalmitol, phosphatidyl-ethanolamine, glutamate, imidazole and phosphonate.
  • a review of these materials, and of the mechanisms by which they operate, may be found in Angew. Chem. Int. Ed. 32 1768-1785, 1998, the disclosure of which is incorporated herein by reference.
  • the nucleic acid must be delivered in a form in which it will be taken up, or internalised, by the target cell and allow it to be expressed properly. Also, the nucleic acid must, in general, be protected against certain cellular enzymes such as nucleases, and for in vivo applications have suitable stability to serum. Thus, one must consider both internalization and protection when designing a lipid vector. We have devised certain new dicationic lipids and also certain new PEG- based lipids incorporating a spacer between cationic centres
  • a complex suitable for delivery of a biologically-active material to a cell which complex comprises:
  • - X 1 and X 2 are the same or different and are selected from -O-CH 2 -, and
  • R 1 and R 2 are the same or different and are straight or branched, saturated or unsaturated C 7 to C 24 hydrocarbyl groups which are unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR', wherein R' is a C x to C 6 hydrocarbyl group; - each R 3 and each R 4 is the same or different and is a straight or branched, saturated or unsaturated C j to C ⁇ 0 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, -OR', -C(O)OH, -CN, -NR'R", and -C(O)R" wherein R' and R" are the same or different and are C x to C 6 hydrocarbyl;
  • - X 1 and X 2 are the same or different and are as defined above;
  • R 1 and R 2 are the same or different and are as defined above;
  • - R 5 is -N ffi (R 3 ) 2 -R 6 wherein each R 3 is the same or different and is as defined above and R 6 is either
  • each Y is the same or different and is -N ⁇ (R 4 ) 2 -, wherein R 4 is as defined above, each A is the same or different and is a C x to C 20 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, -OR', -C(O)OH, -CN, -NR'R", and -C(O)R" wherein R' and R" are the same or different and are C to C 6 hydrocarbyl, n is from 1 to 10, and R 4 is as defined above; or
  • each B is the same or different and is a Cj to C 10 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, -OR', -C(O)OH, -CN, -NR'R" and -C(O)R" wherein R' and R" are the same or different and are to C 6 hydrocarbyl, m is from 1 to 10, and
  • Q is selected from N ⁇ (R 3 ) 3 , OH, OR', OC(O)R' and halogen, wherein R 3 and R' are as defined above; and (ii) a biologically-active material.
  • the invention also provides:
  • a process for the preparation of a complex of the invention comprises: admixing (i) a lipid of formula (I) or (II) as defined above; and (ii) a biologically-active material;
  • a method for transfecting a cell with a biologically-active material comprises contacting the cell in vivo, in vitro or ex vivo with a complex of the invention
  • - a method for the expression of a nucleic acid in a cell, which method comprises transfecting the cell with a complex of the invention using the method set out above under conditions to provide for expression of the nucleic acid component of the complex; - a method for the preparation of a polypeptide, which method comprises:
  • composition comprising a complex of the invention and a pharmaceutically-acceptable carrier, diluent or excipient;
  • - a method for the treatment of a condition caused by or related to a genetic defect or modification in a host, which method comprises administering to the host a therapeutically effective amount of a complex of the invention
  • - a method for the treatment of a condition in a host by an anti-sense nucleic acid or an iRNA, which method comprises administering to the host a therapeutically effective amount of a complex of the invention
  • a vaccine comprising a complex of the invention and a pharmaceutically- acceptable carrier, diluent or excipient; - a complex of the invention for use in a method of vaccinating a human or animal;
  • the invention relates to complexes suitable for the delivery of a biologically- active material to a cell.
  • the complexes are based on new lipids.
  • the complexes of the invention may be used, for example, in gene therapy and vaccination. Gene therapy may be carried out, for example, to correct a genetic defect of modification.
  • the orientation of the X 1 and X 2 moieties is such that the right hand side of the depicted moieties are attached to the group R 1 or R 2 .
  • the lipid of formula (I) or (II) is, of course, cationic, and will be associated with one or more pharmaceutically acceptable anion.
  • acceptable anions include anions of various mineral acids such as, for example, chloride, bromide, iodide, sulfate, nitrate, phosphate and anions of organic acids such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate.
  • Preferred anions are chloride, bromide, iodide, sulphate, nitrate, acetate, maleate, oxalate and succinate.
  • More preferred anions are bromide and iodide.
  • X 1 and X 2 are typically the same. X 1 and X 2 are preferably -O-CH 2 -.
  • unsaturated hydrocarbyl groups include alkenyl groups and alkynyl groups. Preferred unsaturated hydrocarbyl groups are alkenyl groups which contain one or more, for example one or two, double bonds, each of which may be cis or trans. Typically, unsaturated hydrocarbyl groups are alkenyl groups which contain one or two cis double bonds. Typically, a said hydrocarbyl group is unsubstituted.
  • each R' and R" is the same or different and is a C x to C 6 alkyl group.
  • R 1 and R 2 are the same or different and are straight or branched, saturated or unsaturated C 10 to C 22 hydrocarbyl groups which are unsubstituted or substituted as defined above. More preferably, R 1 and R 2 are the same or different and are straight or branched, saturated or unsaturated C 12 to C 20 hydrocarbyl groups which are unsubstituted or substituted as defined above. More preferably still, R 1 and
  • R 1 and R 2 are the same. Typically, R 1 and R 2 are unsubstituted or carry one, two or three substituents. Preferably, R 1 and R 2 are unsubstituted.
  • each R 3 and each R 4 is unsubstituted or substituted by one or more, for example one or two, substituents selected from hydroxy, -OR', -C(O)OH, -CN, - NR'R" and -C(O)R" wherein R' and R" are the same or different and are C j to C 6 hydrocarbyl.
  • each R 3 and each R 4 are the same or different and are straight or branched, saturated or unsaturated C j to C 6 hydrocarbyl groups, for example C ⁇ to C 4 hydrocarbyl groups, which are unsubstituted or substituted by one or more substituents as defined above.
  • each R 3 is the same.
  • each R 4 is the same.
  • each R 3 and each R 4 are the same.
  • R 3 and R 4 are -C K , alkyl groups, for example C r C 6 and C r C 4 alkyl groups.
  • R 3 and R 4 are methyl.
  • R 3 and R 4 are typically unsubstituted or carry one or two substituents.
  • Preferred R 3 and R 4 substituents are selected from hydroxy and -OR' wherein R' is a
  • R 3 and R 4 are unsubstituted.
  • n is from 1 to 5. More preferably, n is from 1 to 2. Typically, n is 1.
  • m is from 1 to 5. More preferably, m is from 1 to 3. Typically, m is 1 or 2.
  • A is C j to C 10 alkylene, for example C 3 , C 6 or C 10 alkylene, which is unsubstituted or substituted by one or more substituents as defined above. More preferably, A is C 2 to C 6 alkylene which is unsubstituted or substituted by one or more substituents as defined above. Yet more preferably, A is C 3 , C 4 or C 5 alkylene which is unsubstituted or substituted by one or more substituents as defined above.
  • A is propylene which is unsubstituted or substituted by one or more substituents as defined above.
  • A is unsubstituted or carries one or two substituents.
  • Preferred substituents for A are selected from hydroxy, halogen and -OR' wherein R' is a C j to C 6 alkyl group. More preferably, A is unsubstituted.
  • B is C x to C 5 alkylene which is unsubstituted or substituted by one or more substituents as defined above. More preferably, B is C 2 , C 3 or C 4 alkylene which is unsubstituted or substituted by one or more substituents as defined above. Most preferably, B is ethylene which is unsubstituted or substituted by one or more substituents as defined above.
  • B is unsubstituted or carries one or two substituents.
  • Preferred substituents for B are selected from hydroxy, halogen and -OR' wherein R' is a C j to C 6 alkyl group. More preferably, B is unsubstituted.
  • Q is preferably -N ffi (R 3 ) 3 or OH. Typically, Q is -N ⁇ Me 3 or OH.
  • the present invention also provides a composition including the structure
  • Y is NH, CH 2 , O or N(acetyl);
  • Z is O(C ⁇ to C 4 ), OC(O)R 3 , N ⁇ R 3 4 , OH, F, CI, Br or I
  • R 3 is CI to C6 alkyl
  • the R 4 s which may be the same or different, are CI to C6 chains
  • n is from 2, 3 or 4
  • m is from 1 to 200 and where it is at least 2 the resulting repeating units may be the same or different.
  • R is hydrogen and the other is group (b); or that both groups R are groups (c).
  • the lipid may be PEG- based, and in the second case it is based on erythritol.
  • m is less than or equal to 100, more preferably less than of equal to 50, especially less than or equal to 25, more especially less than or equal to 12.
  • m is at least 2.
  • formula (III) set out above is cationic, it will be accompanied by one or more appropriate non-toxic anions.
  • Suitable anions include halide, particularly iodide.
  • the two groups R are substantially identical and/or that the two groups R 1 are substantially identical.
  • the overall structure will be substantially symmetrical.
  • the groups X in the formula (in) are OC
  • the linkages will of course be ethers, and where the groups are O-C(O) the linkages will be esters.
  • the ether structures are preferred because they seem to have greater activity.
  • the chain length of the tails R 1 in the formula (III) has been investigated. Including the carbon atom of the linkage X, we prefer from CIO to C22, and preferably straight-chain. At present a value of about CI 6 or CI 8 seems to be optimum, although the precise value will depend on the application. Other possible values include C12, C14, and C20.
  • the tails may be saturated or unsaturated, and when unsaturated may contain one or more double bonds, each of which may be cis or trans, and/or one or more triple bonds.
  • Typical unsaturated structures include one or two cis double bonds. We have found higher transfection efficiencies with unsaturated lipids.
  • Groups R 2 in the formula (III) are preferably straight chain alkyl, although branched groups can be acceptable. We prefer CI, C2, C3 and C4. The same applies to group R 3 . Although (when m is two or more) the repeating units [Y-(CH 2 )-] in the formula (III) may all be the same, we prefer that Y be CH 2 in all but one of them, and therefore that it take on one of the other possibilities in only one of the repeating units. Of these other possibilities, O is at present preferred.
  • the number of methylene groups, n is preferably 2, although other values for example 1, 3, 4, 5 and 6 may be desirable in some circumstances.
  • the lipids of the invention will contain one or more chiral centre.
  • the two carbon atoms illustrated as such in the general formula given above will be chiral centres.
  • there will be four isomers unless the structure has a plane of symmetry between those carbon atoms is which case there will be three isomers.
  • the chemical structures depicted herein are intended to embrace all stereoisomers of the compounds shown, including racemic and non-racemic mixtures and pure enantiomers and/or diasteroisomers.
  • the spacer to be reasonably short, for example from C2 to C6, and preferably C3, C4 or C5.
  • the intention is that complexes be formed that are capable of entering the target cells, after which the nucleic acid can be released to be expressed in the cell nucleus or in some way to control or affect gene expression.
  • the lipids of the invention may be formulated with one or more other components as complexes which are suitable for use in the delivery of a biologically- active material to a cell.
  • complexes comprise a lipid of the invention and a biologically-active material.
  • Complexes of the invention may also comprise one or more of: an integrin-binding peptide; a polycationic component; and a neutral lipid.
  • Suitable biologically-active materials include nucleic acids, peptides and polypeptides, and small molecules.
  • a biologically-active material is one which has a biological effect when introduced into a cell or host, for example by stimulating an immune response or an inflammatory response, by exerting enzymatic activity or by complementing a mutation, etc. These particular biological activities are given merely by way of examples and are not to be taken as limiting.
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, cosmids, vectors, artificial chromosomes, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides branched polynucleotides
  • plasmids plasmids
  • cosmids vectors
  • artificial chromosomes isolated DNA of any sequence
  • isolated RNA of any sequence nucleic acid probes, and primers.
  • Polynucleotides of the invention may include within them synthetic or modified nucleotides.
  • a number of different types of modification to polynucleotides are known in the art. Such modifications may be carried out in order to enhance the in vivo activity, lifespan, nuclease resistance or ability to enter cells.
  • phosphorothioate oligonucleotides may be used.
  • Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates.
  • MBOs Mixed backbone oligonucleotides
  • MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy- or oligoribonucleotides.
  • MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'- O-alkyloligoribonucleotides .
  • a DNA in a complex of the invention may be in the form of a linear molecule or a circular molecule, for example a plasmid or cosmid.
  • linear DNA molecules include DNA in the form of a chromosome or a mini chromosome.
  • An RNA used in a complex of the invention may be polycistronic, i.e. may comprise more than one coding sequence, and therefore may comprise an internal ribosome entry site (IRES).
  • a DNA for use in the invention comprises more than one DNA coding sequence
  • those coding sequences may be operably linked to independent control sequences.
  • the coding sequences may be operably linked to common control sequences, in which case the coding sequences may be separated by an (IRES).
  • RNA in complex of the invention may be linear or circular, for example a replicon, in particular an alpha virus replicon.
  • the RNA may be single stranded or double stranded.
  • the RNA may be an mRNA.
  • Suitable mRNAs will typically comprise a 5' cap and/or a 3' polyA tail.
  • the length of the polyA tail may be modulated to regulate the stability of the mRNA within target cells and hence control the duration of transgene expression from the mRNA.
  • a polyA tail of will be up to about 300 residues in length, preferably from about 50 to about 90 residues in length.
  • a “gene” as used in the context of the present invention is a sequence of nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a genetic function is associated.
  • a gene is a hereditary unit, for example of an organism, comprising a polynucleotide sequence (e. g., a DNA sequence for mammals) that occupies a specific physical location (a "gene locus” or “genetic locus”) within the genome of an organism.
  • a gene can encode an expressed product, such as a polypeptide or a polynucleotide (e. g., tRNA).
  • a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids (e. g., phage attachment sites), wherein the gene does not encode an expressed product.
  • a gene comprises coding sequences, such as polypeptide encoding sequences, and non-coding sequences, such as promoter sequences, poly-adenlyation sequences, transcriptional regulatory sequences (e. g., enhancer sequences).
  • Many eukaryotic genes have "exons" (coding sequences) interrupted by "introns" (non-coding sequences).
  • a gene may share sequences with another gene (s) (e. g., overlapping genes).
  • a "coding sequence” or a sequence which "encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements").
  • the boundaries of a coding sequence are determined by a start codon at the 5'
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located ' to the coding sequence. Transcription and translation of coding sequences are typically regulated by "control elements", including, but not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences.
  • a nucleic acid for use in the invention which comprises a coding sequence may be contained in an expression vector.
  • a suitable expression vector comprises nucleotide sequences, for example a coding sequence encoding a desired peptide or polypeptide.
  • Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid or cosmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression.
  • Other suitable vectors would be apparent to persons skilled in the art.
  • a nucleic acid suitable for use in the invention may also be inserted into a vector in an antisense orientation in order to provide for the production of antisense RNA.
  • a “promoter” is a nucleotide sequence which initiates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters
  • promoter or "control element” includes full-length promoter regions and functional (e. g., controls transcription or translation) segments of these regions.
  • a polynucleotide, especially a coding, for use in a complex of the invention is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.
  • the control sequence will typically comprise a promoter and optionally also comprise other types of control sequence, for example an enhancer and/or terminator.
  • An enhancer is any polynucleotide sequence capable of increasing the level of transcription initiating from a promoter and may act on a cis or trans basis.
  • a terminator is any polynucleotide sequence capable of promoting dissociation of an RNA polymerase from the said sequence.
  • a control sequence may be positioned 5', 3' or internal to (for example in an intron) a coding sequence.
  • a coding sequence may be operably linked to more than one control sequence, for example two, three, four or five control sequences. Such multiple control sequences may be positioned, for example, entirely 5' to the coding sequence. However, more typically control sequences will be located both 5' and 3' to the coding sequence, with optional internal control sequences. Control sequences may be derived from any suitable source and may be generated by recombinant techniques or synthetic means.
  • the vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the desired polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector.
  • Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell.
  • the vectors may also be adapted to be used in vivo, for example in a method of gene therapy or vaccination.
  • Suitable nucleic acids for use in a complex of the invention may be obtained from natural sources, or may be produced recombinantly or by chemical synthesis. They may be modified, for example, to comprise a sequence encoding a specific function, for example, a nuclear localisation sequence.
  • a nucleic acid in a complex of the invention may be selected for use in gene therapy, in gene vaccination, in anti-sense therapy or in therapy by interfering RNA.
  • the nucleic acid component is generally presented in the form of a nucleic acid insert in a plasmid or vector. In some cases, however, it is not necessary to incorporate the nucleic acid component in a vector in order to achieve expression.
  • gene vaccination and anti-sense therapy can be achieved using a naked nucleic acid.
  • the nucleic acid is generally DNA but RNA may be used in some cases, for example, in cancer vaccination.
  • the nucleic acid in a complex of the invention may be or may relate to a gene that is the target for particular gene therapy or may be a molecule that can function as a gene vaccine or as an anti-sense therapeutic agent.
  • the nucleic acid may be or correspond to a complete full-length coding sequence or may be part of a coding sequence.
  • a nucleic acid may be selected to act via an antisense mechanism or via an
  • RNA interference mechanism An antisense RNA may comprise a polynucleotide which has substantial complementarity to all or part of its target mRNA.
  • a polynucleotide which has substantial sequence complementarity to all or part of its target mRNA is typically one which is capable of hybridizing to that mRNA. If the RNA has substantial complementarity to a part of its target mRNA of, it generally has substantial complementarity to a contiguous set of nucleotides within that mRNA.
  • a vector which allows for the expression of a polynucleotide which has substantial sequence complementarity to all or part of the target mRNA (i.e. a polynucleotide which can hybridize to that mRNA). This results in the formation of an RNA-RNA duplex which may result in the direct inhibition of translation and/or the destabilization of the target message, by rendering it susceptibility to nucleases, for example.
  • the vector will typically allow the expression of a polynucleotide which hybridizes to the ribosome binding region and/or the coding region of the target mRNA.
  • an oligonucleotide may be delivered which is capable of hybridizing to the target mRNA.
  • Antisense oligonucleotides are postulated to inhibit target gene expression by interfering with one or more aspects of RNA metabolism, for example processing, translation or metabolic turnover.
  • Chemically modified oligonucleotides may be used and may enhance resistance to nucleases and/or cell permeability.
  • the vector is capable of expressing a polynucleotide which has substantial sequence complementarity to all of part of the target mRNA.
  • Such a polynucleotide will be capable of hybridizing to the target mRNA.
  • such a polynucleotide will be an RNA molecule.
  • Such a polynucleotide may hybridize to all or part of the target mRNA.
  • the polynucleotide will be complementary to all of or part of such an mRNA.
  • the polynucleotide may be the exact complement of such an mRNA.
  • absolute complementarity is not required and preferred polynucleotides which have sufficient complementarity (i.e. substantial complementarity) to form a duplex having a melting temperature of greater than 40°C under physiological conditions are particularly suitable for use in the present invention.
  • the polynucleotide may be a polynucleotide which hybridises to the target mRNA under conditions of medium to high stringency, such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50 to about 60 degrees centigrade.
  • the polynucleotide hybridizes to a coding region of the target mRNA.
  • a polynucleotide may be employed which hybridises to all or part of the 5'- or 3 '-untranslated region of such an mRNA.
  • the polynucleotide will typically be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400, 500, 600 or 700 nucleotides in length or even up to a few nucleotides, such as five or ten nucleotides, shorter than the full-length mRNA.
  • the polynucleotide, i.e.
  • the "antisense" polynucleotide may be expressed in a cell from a suitable vector.
  • a suitable vector is typically a recombinant replicable vector comprising a sequence which, when transcribed, gives rise to the polynucleotide (typically an RNA).
  • the sequence encoding the polynucleotide is operably linked to a control sequence which is capable of providing for the transcription of the sequence giving rise to the polynucleotide.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a sequence giving rise to an antisense RNA is ligated in such a way that transcription of the sequence is achieved under conditions compatible with the control sequences.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for transcription to occur and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • Vectors may be used in vitro, for example for the production of antisense RNA, or used to transfect or transform a host cell.
  • the vector will typically be adapted for use in vivo, for example in a method of gene therapy.
  • Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
  • mammalian promoters such as ⁇ -actin promoters
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long te ⁇ ninal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adeno virus promoters. All these promoters are readily available in the art.
  • Preferred promoters are tissue specific promoters, for example promoters driving expression specifically within vascular tissue.
  • a suitable oligonucleotide will typically have a sequence such that it will bind to the target mRNA. Therefore, it will typically have a sequence which has substantial complementarity to a part of such an mRNA.
  • a suitable oligonucleotide will typically have substantial complementarity to a contiguous set of nucleotides within the target mRNA.
  • An antisense oligonucleotide will generally be from about 6 to about 40 nucleotides in length. Preferably it will be from 12 to 20 nucleotides in length.
  • the oligonucleotide used will have a sequence that is absolutely complementary to the sequence. However, absolute complementarity may not be required and in general any oligonucleotide having sufficient complementarity (i.e. substantial complementarity) to form a stable duplex (or triple helix as the case may be) with the target nucleic acid is considered to be suitable.
  • the stability of a duplex (or triplex) will depend inter alia on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity between the antisense oligonucleotide and the target sequence. The system can tolerate less complementarity when longer oligonucleotides are used.
  • oligonucleotides especially oligonucleotides of from 6 to 40 nucleotides in length, which have sufficient complementarity to from a duplex having a melting temperature of greater than 40°C under physiological conditions are particularly suitable for use in the present invention.
  • the polynucleotide may be a polynucleotide which hybridises to under conditions of medium to high stringency such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50 to about 60 degrees centigrade.
  • Antisense oligonucleotides may be chemically modified.
  • phosphorothioate oligonucleotides may be used.
  • Other deoxynucleotide analogs include methylphosphonates, phosphora idates, phosphorodithioates, N3T5'- phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates.
  • MBOs Mixed backbone oligonucleotides
  • MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy- or oligoribonucleotides.
  • MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'- O-alkyloligoribonucleotides .
  • An nucleic acid suitable for use in the invention may act via an RNA interference (RNAi) mechanism.
  • RNAi RNA interference
  • Such a nucleic acid is typically a double-stranded RNA and has a sequence substantially similar to part of the target mRNA.
  • Preferred nucleic acids of this type are typically short, for example 15mers to 25mers, in particular 18mers to 23mers. These short nucleic acids may be referred to as interfering RNAs (iRNAs).
  • iRNAs interfering RNAs
  • the use of short nucleic acids of the type described above is preferred because such inhibitors do not appear to trigger viral defence mechanisms of higher organisms.
  • Such nucleic acids can be used to inhibit translation of the mRNA.
  • small fragments of sequence encoding the target gene product may be provided, cloned back to back in a suitable vector.
  • the vectors described above are suitable for expression of such back to back sequences. Expression of the sequence leads to production of the desired double-stranded RNA.
  • a nucleic acid suitable for use in a complex of the invention may comprise a sequence which encodes an antigen.
  • An antigen is a molecule which contains one or more epitopes that will stimulate a host's immune system to make a cellular antigen- specific immune response, or a humoral antibody response.
  • a suitable nucleic acid sequence encoding an antigen can be derived from any known organism or pathogen, e.g. a virus, a bacterium, a parasite, a plant, a protozoan, or a fungus. The term also includes tumor antigens.
  • the antigen typically comprises one or more T cell epitopes. "T cell epitopes"are generally those features of a peptide structure capable of inducing a T cell response.
  • T cell epitopes comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC molecules, (Unanue et al. Science 236, 551- 557, 1987).
  • a T cell epitope is generally a peptide having about 8-15, preferably 5-10 or more amino acid residues.
  • a nucleic acid suitable for use in a complex of the invention may encode such a T cell epitope.
  • a nucleic acid suitable for use in a complex of the invention may encode a protein that is commercially useful, for example industrially or scientifically useful, for example an enzyme; pharmaceutically useful, for example, a protein that can be used therapeutically or prophylactically as a medicament or vaccine; or diagnostically useful, for example, an antigen for use in an ELISA.
  • a biologically-active material for use in a complex of the invention may be a peptide or polypeptide. Suitable peptides/polypeptides are those encoded by one of the nucleic acids set out above.
  • the peptide/polypeptide may be, for example, one that is absent or deficient in a genetic disease or an antigen or immunogen.
  • the peptide/polypeptide may be, for example, a natural hormone such as tissue insulin, calcitonin and human growth hormone, or a synthetic analogue of such a natural hormone.
  • peptides/polypeptides which may be used in a complex of the invention include interleukin-2, tumor necrosis factor, tissue plasminogen activator, factor VIII, erythropoietin, growth factors such as epidermal growth factor, growth hormone releasing factor, neural growth factor and toxic peptides such as ricin, diphtheria toxin, or cobra venom factor, capable of eliminating diseased or malignant cells. Fragments of any of the polypeptides mentioned above may also be used in a complex of the invention.
  • Peptides/polypeptides suitable for use in a complex of the invention may be chemically modified, e.g. post-translationally modified.
  • they may be glycosylated or comprise modified amino acid residues.
  • They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane.
  • a biologically-active material for use in a complex of the invention may be a small molecule.
  • Preferred small molecules are therapeutic agents, for example steroids such as hydrocortisone, fluocinolone acetonide, fluocinonide and dexamethasone, non-steroidal anti-inflammatory agents such as 1-acetylsalicyclic acid, antiviral nucleosides such as AZT, acyclovir and gancyclovir, or phospholipid derivatives of such antiviral nucleosides, antibiotics, anaesthetic agents, cytostatic agent or immunomodulators.
  • a complex ofthe invention may also contain a sunscreen or a cosmetic.
  • a complex of he invention will typically comprise a ratio of from 0.25 to 12: 1 by weight of a lipid ofthe invention: a biologically-active material (such as a nucleic acid), for example a ratio of from 0.5 to 8: 1 by weight of a lipid ofthe invention: a biologically-active material, such as from 0.75 to 4: 1 weight of a lipid of the invention: a biologically-active material, for example from 1 to 2: 1 by weight of a lipid ofthe invention: a biologically-active material.
  • a biologically-active material such as a nucleic acid
  • a complex ofthe invention may comprise an integrin-binding component, for example an integrin-binding peptide.
  • An integrin-binding component suitable for use in a complex ofthe invention is any such component which is capable of binding specifically to integrins found on the surface of cells.
  • the integrin-binding component may be a naturally occurring integrin-binding ligand, for example, an extra-cellular matrix protein, a viral capsid protein, 'the bacterial protein invasin, a snake venom disintegrin protein, or an integrin-binding fragment of any such protein.
  • Such integrin-binding proteins and fragments thereof may be obtained from natural sources or by recombinant techniques, but they are difficult to synthesise and purify in large amounts, they require conjugation directly to DNA or RNA or to poly cationic elements for DNA or RNA binding, and are immunogenic in vivo.
  • integrin-binding peptides it is thus preferable to use integrin-binding peptides, in particular because of their ease of synthesis, purification and storage, their potential for chemical modification, and their potentially low immunogenicity in vivo.
  • Examples of integrin-binding peptides are given in Verfaille, 1994 #635; Wang, 1995 #645; Wunschz, 1991 #539; Pierschbacher, 1984 #314; Massia, 1992 #86; Clements et al. J. Cell Science 107, 2127-2135, 1994; Lu et al, Biochemistry J.
  • peptides containing the conserved amino acid sequence arginine-glycine-aspartic acid bind with high affinity to integrins.
  • peptides comprising the RGD sequence are particularly useful.
  • the affinity between integrin and peptide ligands is influenced by the amino acid sequence flanking the RGD domain.
  • Peptides having a cyclic region in which the conformational freedom ofthe RGD sequence is restricted generally have a higher affinity for integrin receptors than do their linear counterparts.
  • Such cyclic peptides are particularly preferred.
  • Cyclic peptides may be formed by the provision of two cysteine residues in the peptide, thus enabling the formation of a disulphide bond.
  • a cysteine residue may be separated from the RGD sequence by one or more residues, for example, up to six residues, or may be immediately adjacent to the RGD sequence, although preferably both cysteines are not immediately adjacent to the ends ofthe RGD sequence.
  • CRGDMFGC An example of an amino acid sequence that will permit cyclisation by disulphide bond formation is CRGDMFGC.
  • a peptide that consists of or comprises the sequence CRGDMFGC may advantageously be used as an integrin-binding peptide according to the present invention.
  • Examples of peptides that comprises the sequence CRGDMFGC and that are effective integrin-binding ligands are the peptides GGCRGDMFGC, GGCRGDMFGCG, GGCRGDMFGCA and GACRGDMFGCA.
  • the peptide GACDCRGDCFCA has the potential to form two disulphide bonds for stabilising the RGD loop. That peptide and others having the potential to form two RGD-stabilising disulphide bonds, may be particularly useful as integrin-binding ligands according to the present invention.
  • a further useful peptide is GACATRWARECG.
  • not all integrin-binding peptides contain the conserved RGD sequence.
  • the peptides GACRRETAWACA, GACRRETAWACG and XSXGACRRETAWACG are integrin-specific peptides.
  • peptides comprising the sequence CRRETTAWAC or CRRETAWAC may be used, as may other non- RGD peptides, particularly those that have the potential for disulphide bond formation.
  • Peptide sequences may be designed on the basis of known ligands, for example, on the basis of integrin-binding domains of naturally-occurring integrin- binding ligands, or on the basis of known peptides that bind to integrins.
  • integrins are a family of heterodimeric proteins found on the surface of cells. They consist of several different ⁇ and ⁇ subunits. Some integrins are found on many types of cells, others are more specific, for example, ⁇ 5 and ⁇ v integrins are widespread and are found on a diverse range of cells. Integrin-binding ligands can vary in their affinity for different integrins. For example, GACRGDMFGCA (peptide 1) has affinity for ⁇ 5 and av integrins but is non-specific (O'Neil et al. 1992, supra; Hart et al. 1997, supra).
  • GACDCRGDCFCA (peptide 5) has high affinity for integrin ⁇ v but is not ⁇ v-specific (Koivunen et al. 1995, supra; Hart et al. 1997, supra).
  • GACRRETAWACG which does not contain the conserved RGD region, is ⁇ 5 ⁇ l-specific (Koivunen et al. 1995, supra).
  • Various integrin-binding peptides and their integrin specificity are set out below:
  • GA-CXCG where X is SERSMNF, YGLPHKF, PSGAARA, VKSMVTH or LQHKSMP.
  • Alternative oligopeptides may be used in conjunction with or instead of an integrin-binding peptide, for example alternative targetting ligands, such as oligopeptides identified by panning with phage-based peptide-display libraries; membrane-active peptides such as melittin; fragments ofthe HIV tat protein; single chain Fv regions of antibodies; VP22; peptides containing nuclear localisation sequences; mitochondrial localisation sequences; and peptides based on the influenza virus haemagglutinin protein.
  • alternative targetting ligands such as oligopeptides identified by panning with phage-based peptide-display libraries; membrane-active peptides such as melittin; fragments ofthe HIV tat protein; single chain Fv regions of antibodies; VP22; peptides containing nuclear localisation sequences; mitochondrial localisation sequences; and peptides based on the influenza virus haemagglutinin protein.
  • a complex ofthe invention will typically comprise a ratio of from 0.25 to 4: 4 by weight of a lipid ofthe invention: an integrin-binding peptide, for example a ratio of from 0.5 to 2: 4 by weight of a lipid ofthe invention: an integrin-binding peptide, such as from 0.75 to 1 : 4 by weight of a lipid ofthe invention: an integrin- binding peptide.
  • complexes ofthe invention will comprise a cationic component, such as a cationic polymer. This is particularly the case for complexes where the biologically-active material is a nucleic acid.
  • Cationic polymers suitable for use in the invention are typically capable of binding to a nucleic acid.
  • Especially preferred cationic polymers are capable of condensing nucleic acids into a particle with a diameter of from about 50nm to about 150nm.
  • suitable cationic polymers are low molecular weight polymers and the number of positive charges per polymer molecule is typically from about 7 to about 50, preferably from about 7 to about 25 or more preferably from about 12 to about 16.
  • Suitable cationic polymers may have any number of cationic monomers, although generally the polymer must retain the ability to bind nucleic acids. For example, from 3 to 100 cationic monomers may be present, for example, from 10 to 20.
  • Suitable polymers include oligolysine, for example having from 10 to 20 lysine residues, for example, from 15 to 17 residues, especially 16 residues, i.e. [K] 16 .
  • poly-L-lysine which has a molecular weight of 3.4kDa (and which has an average of 16 positive charges per molecule) is preferred.
  • suitable polymers include polyethylenimine (PEI) which has a molecular weight of 2kDa (with an average of 12 positive charges per molecule at neutral pH) and polyamidoamine dendrimers.
  • PEI polyethylenimine
  • Suitable peptides are disclosed in USSN 09/424656 and 08/836786, the disclosures of which are incorporated herein by reference. The polymers pLL and pEI will condense RNA and DNA in water or 1 OmM
  • cationic polymers with a pKa of greater than 9.0 generally do not possess endosomolytic activity and require the presence of an endosome-disrupting agent, for example chloroquine, to enable them to gain access into the cytosol.
  • the poly cationic component may advantageously be linked or otherwise attached to the integrin-binding component.
  • a polycationic polymer may be chemically bonded to an integrin-binding component, for example, by a peptide bond in the case of an oligolysine. Other types of suitable bonds are thioether and disulphide bonds.
  • the polycationic component may be linked at any position ofthe integrin-binding component.
  • Preferred combinations of integrin- binding component and polycationic polymer are an oligolysine, especially [K] 16 , linked via a peptide bond to an intergin-binding peptide peptide, for example any one ofthe peptides set out described above.
  • a complex ofthe invention will typically comprise a ratio of from 0.25 to 4: 4 by weight of a lipid of the invention: a polycationic component (such a polycationic peptide), for example a ratio of from 0.5 to 2: 4 by weight of a lipid ofthe invention: a polycationic component, such as from 0.75 to 1 : 4 by weight of a lipid ofthe invention: a polycationic component.
  • a polycationic component such a polycationic peptide
  • the ratio of a lipid ofthe invention to the combined peptide is as given above for an integrin-binding peptide and polycationic component individually.
  • Agents other than peptides may also be introduced onto the condensing cationic polymer, for example saccharide residues or lipids, to modulate solubility and interaction of formulations with biological proteins, fluids, membranes and cells, including to specific receptors.
  • a neutral lipid may be used in a complex ofthe invention.
  • a complex ofthe invention may be free of a neutral lipid.
  • Any neutral lipid may be used in a complex ofthe invention, although typically those that have membrane destabilising properties are preferred.
  • An example of a suitable neutral lipid which has membrane destabilising properties is dioleyl phosphatidylethanolamine (DOPE).
  • DOPE dioleyl phosphatidylethanolamine
  • DOPE has membrane destabilising properties sometimes referred to as "fusogenic" properties.
  • the ratio of neutral lipid: a lipid ofthe invention is from about 0.5 to 2: 1 for example 1: 1.
  • a complex ofthe invention may be prepared by a process which comprises admixing the components ofthe complex. Although, the components may be admixed in any order, it is generally preferable that the lipid component is not added last. In the case where there is a combined integrin-binding peptide/polycationic component, it is generally preferable to combine the components in the following order: lipid; combined integrin-binding peptide/polycationic component; biologically-active material.
  • the components of a complex of the invention are preferably admixed in the amounts set out above.
  • a typical complex ofthe invention may be prepared by admixing a lipid ofthe invention combined integrin-binding peptide-cationic component (such as
  • the invention also provides a mixture which comprises: a lipid; and one or more of an integrin-binding peptide, a polycationic component and a neutral lipid. All ofthe components of such a mixture may be as set out above.
  • a mixture may be used to produce a biologically-active material containing complex ofthe invention by the incorporation of a biologically-active material with the mixture, for example by admixture.
  • the present invention further provides a process for the production of a complex ofthe present invention, which comprises admixing a biologically-active material with a mixture ofthe invention.
  • a process for the production of a complex ofthe present invention which comprises admixing a biologically-active material with a mixture ofthe invention.
  • the preferred components, preferred combinations of components, preferred ratios of components and preferred order of mixing, both with regard to the mixture and to the production of a complex are as described above in relation to the complex ofthe invention.
  • a complex ofthe invention may be used in a process for transfecting a cell with a biologically-active material.
  • a host cell is contacted with a complex ofthe invention.
  • a complex ofthe invention may also be used in a process for expressing a nucleic acid in a host cell.
  • Such a process comprises contacting the host cell with a complex ofthe invention which comprises a nucleic acid.
  • the host cell is then subjected to conditions that enable the cell to express the nucleic acid, for example it is cultured in a medium which allows expression ofthe nucleic acid.
  • a complex ofthe invention may be further used in a process for the production of a polypeptide.
  • a host cell is transfected with a nucleic acid using the method set out above.
  • the transfection is carried out under conditions suitable for expression ofthe polypeptide encoded by the nucleic acid or, alternatively, the transfected cell is transferred to conditions suitable for expression ofthe polypeptide encoded by the nucleic acid.
  • the polypeptide may then be recovered from the host cell or from the culture medium.
  • the host cell may be any host cell.
  • the cell may be a prokaryotic cell or a eukaryotic cell.
  • the cell may be from a bacterium, a mycobacterium, a protozoan, a parasite, a fungus a plant or an animal. Suitable animal cells are mammalian cells, for example human cells. All ofthe methods may be carried out in vivo, in vitro or ex vivo. Also, cells may be obtained from a host, transfected according to the method set out above, and then returned to the host.
  • the invention also provides a cell transfected with a complex ofthe invention and progeny cells derived from such a transfected cell.
  • kits for delivery of a biologically-active material to a cell which kit comprises a mixture ofthe invention or components suitable for the preparation of a mixture of the invention.
  • a kit may comprise a lipid of formula (I) or (II) as set out above and one or more of an integrin-binding peptide, a polycationic component and a neutral lipid (all as described above).
  • the kit may also comprise a biologically- active material.
  • the kit may comprise a nucleic acid, optionally in the form of a plasmid or vector which may be empty or comprise a coding sequence.
  • a kit ofthe invention may comprise appropriate buffers and/or control cells. Also, a kit ofthe invention may comprise appropriate packaging and instructions for using the kit in one ofthe methods set out above. Thus, the instructions may indicate the preferred ratios ofthe components and the preferred order of admixing the components, for example as described above.
  • a kit may be used for producing a complex suitable for use in gene therapy, vaccination, antisense of iRNA therapy. Alternatively, it may be used for producing a complex suitable for transfecting a host cell with a nucleic acid encoding a commercially useful protein, i.e. to produce a so- called "cell" factory.
  • a complex ofthe invention may be used in a method of treatment or vaccination.
  • Treatment according to the invention may be prophylactic or therapeutic.
  • a complex ofthe invention may be used in a method for nucleic acid transfer, for example in a method of treatment ofthe human or animal body by therapy.
  • a complex ofthe invention may also be used for the manufacture of a medicament for use in nucleic acid transfer, for example in treatment ofthe human or animal body by therapy, especially in the treatment of a condition caused by or related to a genetic defect or modification.
  • the condition of a patient suffering from such a condition can be improved by administration of a complex ofthe invention.
  • a therapeutically effective amount of a complex ofthe invention may be given to a host in need thereof.
  • the host may be a human or non- human animal.
  • Targets for gene therapy are well known and include monogenic disorders, for example, cystic fibrosis, various cancers, and infections, for example, viral infections, for example, with HIV.
  • transfection with the p53 gene offers great potential for cancer treatment.
  • Targets for gene vaccination are also well known, and include vaccination against pathogens for which vaccines derived from natural sources are too dangerous for human use and recombinant vaccines are not always effective, for example, hepatitis B virus, HIV, HCV and herpes simplex virus.
  • Targets for anti-sense therapy are also known. Further targets for gene therapy and anti-sense therapy are being proposed as knowledge ofthe genetic basis of disease increases, as are further targets for gene vaccination.
  • a complex ofthe invention may be used in vaccination.
  • a complex of the invention may be used to deliver an antigen or a nucleic acid encoding an antigen. That is to say, a complex ofthe invention may be used in gene vaccination.
  • a complex ofthe invention may be used to elicit an immune response against a wide variety of antigens for the treatment and/or prevention of a number of conditions mcluding, but not limited to, cancer, allergies, toxicity and infection by pathogens such as viruses, bacteria, fungi, and other pathogenic organisms.
  • Suitable viral antigens and nucleic acids encoding such antigens for use in the complexes of the invention include, but are not limited to, those obtained or derived from the hepatitis family of viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV).
  • HCV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HDV delta hepatitis virus
  • HEV hepatitis E virus
  • HGV hepatitis G virus
  • proteins, and nucleic acids encoding such proteins, from the herpesvirus family can be used as antigens in the present invention, including proteins derived from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens from varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; and antigens from other human herpesviruses such as HHV6 and
  • HHV7 See, e. g. Chee et al. (1990) Cytomegaloviruses (J. K. McDougall, ed., Springer Verlag, pp. 125-169; McGeoch et al. (1988) J. Gen. Virol. 69: 1531-1574; U. S. Patent No. 5,171,568; Baer et al. (1984) Nature 310: 207-211; and Davison et al. (1986) J. Gen. Virol.
  • HIV antigens such as gpl20 molecules for a multitude of HIV- 1 and HIV-2 isolates, including members ofthe various genetic subtypes of HIV, are known and reported (see, e. g., Myers et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, New Mexico (1992); and Modrow et al. (1987) J. Virol. 61: 570-578) and antigen-containing nucleic acid sequences derived or obtained from any of these isolates will find use in the present invention.
  • HIV Human immunodeficiency virus
  • immunogenic proteins derived or obtained from any ofthe various HIV isolates will find use herein, including nucleic acid sequences encoding one or more ofthe various envelope proteins such as gap 160 and gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol, env, tat, vif, rev, nef, vpr, vpu and LTR regions of HIV.
  • Antigens derived or obtained from other viruses will also find use herein, such as without limitation, antigens from members ofthe families Picornaviridae (e. g., polioviruses, rhinoviruses, etc.); Caliciviridae; Togaviridae (e. g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae (e. g., rotavirus, etc.);
  • Birnaviridae Rhabodoviridae (e. g., rabies virus, etc.); Orthomyxoviridae (e. g., influenza virus types A, B and C, etc.); Filoviridae; Paramyxoviridae (e. g., mumps virus, measles virus, respiratory syncytial virus, parainfluenza virus, etc.); Bunyaviridae; Arenaviridae; Retroviradae (e.
  • HTLV-I HTLV-11
  • HIV-1 also known as HTLN-111, LAV, ARV, hTLR, etc.
  • HIV-1CM235 HIV-1
  • HIV-2 among others
  • simian immunodeficiency virus (SIV) Papillomavirus, the tick-bourne encephalitis viruses; and the like.
  • SIV simian immunodeficiency virus
  • Papillomavirus the tick-bourne encephalitis viruses; and the like. See, e. gNirology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2 nd Edition (B. ⁇ . Fields and D. M. Knipe, eds. 1991), for a description of these and other viruses.
  • Nucleic acid sequences encoding such antigens may of course also be used in a complex of the invention.
  • a selected antigen is obtained or derived from a viral pathogen that typically enters the body via a mucosal surface and is known to cause or is associated with human disease, such as, but not limited to, HIN (AIDS), influenza viruses (Flu), herpes simplex viruses (genital infection, cold sores, STDs), rotaviruses (diarrhea), parainfluenza viruses (respiratory infections), poliovirus (poliomyelitis), respiratory syncytial virus (respiratory infections), measles and mumps viruses (measles, mumps), rubella virus (rubella), and rhinoviruses (common cold).
  • HIN HIV
  • influenza viruses Felu
  • herpes simplex viruses geneital infection, cold sores, STDs
  • rotaviruses diarrhea
  • parainfluenza viruses respiratory infections
  • poliovirus poliomyelitis
  • respiratory syncytial virus respiratory
  • Suitable bacterial and parasitic antigens can be obtained or derived from known causative agents responsible for diseases including, but not limited to, Diptheria, Pertussis, Tetanus, Tuberculosis, Bacterial or Fungal Pneumonia, Otitis Media, Gonnorhea, Cholera, Typhoid, Meningitis, Mononucleosis, Plague,
  • antigens can be obtained or derived from unconventional pathogens such as the causative agents of kuru, Creutzfeldt- Jakob disease (CJD), scrapie, transmissible mink encephalopathy, and chronic wasting diseases, or from proteinaceous infectious particles such as prions that are associated with mad cow disease.
  • CJD Creutzfeldt- Jakob disease
  • scrapie transmissible mink encephalopathy
  • chronic wasting diseases or from proteinaceous infectious particles such as prions that are associated with mad cow disease.
  • nucleic acid sequences encoding such antigens may be used in a complex ofthe invention.
  • pathogens from which antigens can be derived include M. tuberculosis, Chlamydia, ⁇ . gonorrhoeae, Shigella, Salmonella, Vibrio Cholera, Treponema pallidua, Pseudomonas, Bordetella pertussis, Brucella, Franciscella tulorensis, Helicobacter pylori, Leptospria interrogaus, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and B), Pneumococcus, Meningococcus, Hemophilus influenza (type b), Toxoplasma gondic, Complylobacteriosis, Moraxella catarrhalis, Donovanosis, and Actinomycosis ; fungal pathogens including Candidiasis and Aspergillosis; parasitic pathogens including Taenia, Flukes, Roundworms, Amebiasis, Gi
  • the present invention can also be used to provide a suitable immune response against numerous veterinary diseases, such as Foot and Mouth diseases, Coronavirus, Pasteurella multocida, Helicobacter, Strongylus vulgaris, Actinobacillus pleuropneumonia, Bovine viral diarrhea virus (BVDV), Klebsiella pneumoniae, E. coli, Bordetella pertussis, Bordetella parapertussis and brochiseptica.
  • BVDV Bovine viral diarrhea virus
  • Klebsiella pneumoniae E. coli
  • Bordetella pertussis Bordetella parapertussis and brochiseptica
  • nucleic acid sequences encoding an antigen as set out above may be used in a complex ofthe invention.
  • a nucleotide sequence corresponding to (encoding) one or more of the above-listed antigen(s) is used in a complex ofthe invention.
  • a complex ofthe invention may be in the form of a pharmaceutical composition which additionally comprises a pharmaceutically-acceptable carrier, diluent or excipient for example water or a physiologically-acceptable buffer.
  • a vaccine composition comprises a complex ofthe invention and a pharmaceutically-acceptable carrier, diluent or excipient for example water or a physiologically-acceptable buffer.
  • Such pharmaceutical and vaccine compositions may be supplied in any suitable dispenser, for example a puffer.
  • a complex ofthe invention may be administered in a variety of dosage forms.
  • the complexes can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the complexes may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • the complexes may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
  • a complex of the invention may be formulated for simultaneous, separate or sequential use.
  • a complex ofthe invention is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective amount of a complex is administered to a patient.
  • the dose of a complex may be determined according to various parameters, especially according to the substance used; the age, weight and condition ofthe patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • the amount of nucleic acid delivered in a complex ofthe invention will be in the range of from l ⁇ g to lg, preferably from lOO ⁇ g to lOmg, according to the activity ofthe specific formulation, the age, weight and conditions ofthe subject to be treated, the type and severity ofthe degeneration and the frequency and route of administration.
  • a single dose may be administered daily or alternatively, may multiple doses, for example two, three, four or five doses may be administered daily.
  • the amount of nucleic acid referred to above may represent the total amount administered in the treatment regime or may represent each separate administration in the regime.
  • the complex may be administered to the host in one or more administrations. Typically after the initial administration a "booster" can be given. Typically the host is given 1, 2, 3 or more separate administrations, each of which is separated by at least 12 hours, 1 day, 2 days, 7 days, 14 days, 1 month or more.
  • lipids ofthe invention can be prepared by analogy with known methods.
  • compounds of formula (I) can be prepared from 1,4- dibromobutanediol, using dimetl ylamine in methanol, according to the following reaction scheme.
  • R in the above reaction scheme is, of course, defined as R 3 and R 4 above.
  • X in the above reaction scheme is defined as X 1 and X 2 above.
  • R in the above reaction scheme is defined as R 1 and R 2 above.
  • step (1) is typically conducted stepwise.
  • a first -N(R ) 2 moiety is added in a first step and a second -N(R ) 2 moiety is added in a second step.
  • one ofthe hydroxy groups on compound (1) can be protected by a protecting group, prior to such a two stage reaction.
  • the protecting group would, of course, then be removed after introduction ofthe first N(R ) 2 moiety and before the introduction ofthe second N(R ) 2 moiety.
  • step 2 introduction ofthe XR moieties
  • L-R wherein L is a leaving group such as a mesylate
  • X is -O-CO-
  • the XR moieties can be introduced by reaction with RCO 2 H, in the presence of EDCl, DMAP, ttiethylamine and DCM.
  • the reaction is effected in a dark environment at room temperature.
  • one XR moiety is introduced in a first step and a second XR moiety is introduced in a second step.
  • One ofthe hydroxy groups on compound (2) can, if necessary, be protected prior to such a two stage reaction step by a standard hydroxy protecting group.
  • the protecting group would of course then be removed after introduction ofthe first XR moiety and before introduction ofthe second XR moiety.
  • R 7 in the above reaction scheme is, of course, defined as R 3 and R 4 above.
  • X in the above reaction scheme is defined as X 1 and X 2 above.
  • R in the above reaction scheme is defined as R 1 and R 2 above.
  • Z in the above reaction scheme corresponds to the moiety -[A-Y] n R 4 except for the absence ofthe terminal -N + (R 4 ) 3 .
  • the Z group can comprise carbamate protected amino moieties in place of quaternary ammonium moieties.
  • the carbamate protected amino moieties in compound (8) can be deprotected and converted to quaternary ammonium moieties by standard methods. Quaternisation can, for example, be effected by reaction with R 4 -I.
  • the compounds (8) are generally purified by recrystallisation.
  • Q can be effected by standard techniques.
  • Q when Q is -N + (R 3 ) 3 , it can be introduced by reacting compound (11) with a corresponding amino compound.
  • the moiety N + (CH 3 ) 3 can be introduced by reaction with trimethylamine (45 wt% in H 2 O) in the presence of methanol in a sealed tube at 90°C for 24 hours.
  • Example 1 Reaction Scheme I was followed to produce l,4-Di(trimethylammonium)-2,3-dioleoyloxy-butane; diiodide.
  • the first step was the production of l,4-Di(dimethylamino)-2,3-butanediol
  • Powdered sodium hydroxide (1.28 g, 32.0 mmol) was stirred in methanol (7 ml) at 0 °C.
  • Dimethylamine hydrochloride (1.96 g, 24.0 mmol) was added followed by 1,4- Dibromo-2,3-butanediol (1.00 g, 4.00 mmol).
  • the mixture was then heated to 80 °C in a sealed tube for 24 hr.
  • the mixture was then concenttated in vacuo.
  • the residue was re-dissolved in chloroform (25 ml) and the resulting mixture was filtered.
  • the filtrate was concentrated in vacuo to yield the titled product as clear oil which solidifies to a colourless waxy solid upon refrigeration (0.69 g, 98%).
  • Example 2 also illustrates Reaction Scheme 1.
  • the first step of Example 1 was repeated, but in the second step the resulting butane diol was then used to synthesise an ether, rather than ester based lipid, namely l,4-Di(dimethylamino)-2,3-dioleyloxy- butane.
  • the third step produced l,4-Di(ttime1hylammomum)-2,3-dioleyloxy-butane; diiodide
  • Example 3 illustrates the use of Reaction Scheme 3. [2,3-Di-(oleyloxy)-propyl]-(3- bromo-propyl)-dimethyl-ammonium; bromide was prepared in a first step
  • Example 5 illustrates Reaction Scheme 5. 2-[2-(2-Bromo-ethoxy)-ethoxy]-ethanol was produced in a first step.
  • Tri (ethylene) glycol (4.50 g, 30.0 mmol) and hydrobromic acid solution (48%>, 5.09 ml, 45.0 mmol) were stirred in toluene (70 ml) at reflux for 72 hr. After cooling the solution was neutralized by the addition of saturated sodium hydrogencarbonate solution. Water (50 ml) was added and the resulting mixture was extracted with DCM (3 x 50 ml). The chlorinated extract was dried over anhydrous magnesium sulfate and concentrated in vacuo. No further purification was required and the above product was obtained as a yellow oil (2.29 g, 36 %).
  • the activity of various compositions ofthe invention were measured in ⁇ AE cells using a Luciferase assay.
  • RLU/mg refers to relative light units per mg of protein.
  • the assay was carried out as follows: Lipid (10 mg/mL; 100 ⁇ L [1 mg of lipid]) in chloroform was placed into a glass vial (sterile). The solvents were removed in vacuo and further traces of chloroform were removed on the high vacuum for 24 h. Cationic Lipids were either formulated alone or with DOPE (mole ratio, 1 :1). Deionized water (1 mL; MilliQ) was added to the film, to generate 1 mg/mL solution of lipid in water. The mixture was allowed to hydrate at 4 °C for 24 h. After warming to 40 °C the mixture was sonicated to generate a clear solution (5 min approx). The resulting liposome formulations were stable for up to 3months.
  • the components ofthe complex were mixed in a the required weight ratio.
  • the lipid was initially mixed with the peptide and the resulting mixture was added to the plasmid DNA.
  • Human airway epithelial cells were seeded for 24 h at 37 °C in complete growth medium in a 96 well plate. Transfection complexes were left to aggregate for 30 min and diluted to a concentration of 1 ⁇ g of DNA per 0.5 ml in OptiMEM (Life technologies). The medium was removed from each well and replaced with 0.5 ml of transfection complex and was allow to incubate for a further 4 h. The transfection complexes were removed and then replace with growth medium and the cells were allowed to incubate for 48 h.
  • the transfected cells were washed with phosphate-buffered saline (PBS). Reporter lysis buffer (100 ⁇ l) (Promega) was added to each well and cooled to 4 ° C for 15 min. The cells were then assayed with a luciferase assay kit. The total light emission was measured for each well for 60 sec. The protein concentration of each sample was determined using a protein assay reagent and the activity expressed as relative light units per milligram of protein (RLU/mg).
  • RLU/mg relative light units per milligram of protein

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EP03730315A 2002-05-08 2003-05-08 Complexes for the delivery of biologically-active material to cells Withdrawn EP1506019A1 (en)

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GBGB0210538.5A GB0210538D0 (en) 2002-05-08 2002-05-08 Lipids and gene delivery
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AU7078396A (en) * 1995-09-27 1997-04-30 Regents Of The University Of California, The Polyfunctional cationic cytofectins, formulations and methods for generating active cytofectin:polynucleotide transfection complexes
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