WO2008007073A2 - Method for introducing sirna into cells by photochemical internalisation - Google Patents
Method for introducing sirna into cells by photochemical internalisation Download PDFInfo
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- WO2008007073A2 WO2008007073A2 PCT/GB2007/002569 GB2007002569W WO2008007073A2 WO 2008007073 A2 WO2008007073 A2 WO 2008007073A2 GB 2007002569 W GB2007002569 W GB 2007002569W WO 2008007073 A2 WO2008007073 A2 WO 2008007073A2
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- 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
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- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Definitions
- the present invention relates to a method for introducing short interfering RNA (siRNA) into cells, preferably into the cytosol of cells, using a photosensitising agent and a carrier molecule and irradiation of the cells with light of a wavelength effective to activate the photosensitising agent, and to the use of this method for altering gene activity, e.g. gene silencing in vitro or in vivo.
- siRNA short interfering RNA
- RNA interference occurs in many organisms and in this process, double-stranded non-coding RNA silences gene expression in a sequence specific, post-transcriptional manner. In nature, the phenomenon protects an organism's genome from foreign, invading nucleic acids such as transposons, transgenes and viral genes.
- RNA silencing pathways involve the conversion of dsRNA into short interfering RNAs (siRNAs) that direct ribonucleases to homologous mRNA targets.
- the enzyme Dicer processes the dsRNA into siRNAs, which are in general 20-25 nucleotides long.
- the siRNAs then assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), which are guided to complementary RNA molecules, where they cleave and destroy the target mRNA.
- RISCs RNA-induced silencing complexes
- Small amounts of dsRNA can silence a large amount of target mRNA due to an amplification component of RNA silencing (reviewed in Hannon and Rossi (2004), Nature 431, 371-378).
- siRNA molecules are key components of the pathway led to the testing of chemically synthesised siRNA molecules of approximately 20 to 22 base pairs in length, corresponding to targeted RNA or DNA sequences. These molecules were shown to act to disrupt the expression of the targeted sequences in mammalian cells (Elbashir S.M. et al., (2001) Nature 411, 494-498).
- a 20- nucleotide siRNA is usually sufficiently long to induce gene-specific silencing, but sufficiently short to evade a host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.
- siRNA technology has thus been developed as a general technique for sequence specific gene silencing.
- Gene silencing has many applications, both in vitro and in vivo, as both a research tool and a therapeutic strategy.
- the high potency and specificity that is seen when siRNA technology is used makes this technology particularly attractive.
- siRNA molecules In all cases, delivery of the siRNA molecules to the cells represents a major challenge, as for gene silencing to occur, it is necessary for the siRNA molecules to enter the cells in sufficient concentrations to be useful.
- the strength of the silencing response and its duration is affected by the amount of siRNA that is delivered to the cell, and it has been shown that by supplying the siRNA in sufficiently high concentrations, even a relatively weak siRNA molecule can silence its target. This should however be balanced against the fact that it is known that administering large amounts of siRNA into a cell can lead to undesired effects such as "off target” effects (i.e. unwanted changes in protein expression levels) or the activation of innate immune pathways.
- siRNA has been applied to cells by using standard transfection protocols for nucleic acids, such as by using liposomes, cationic lipids, anionic lipids, and microinjection.
- siRNA is a double stranded molecule and as such, delivery and cellular uptake of the molecule is more difficult than for antisense, which binds to serum proteins to be taken up by cells.
- Various different strategies have been used, and commercially available, kits exist for this purpose.
- efficient transfection is highly desirable since the potency of gene silencing is at least in part dependent on the concentration of siRNA in the cell, but administration to cells in high concentrations may also cause undesirable side effects.
- nucleic acid molecules such as siRNA
- Viruses could be used for this purpose, however, there are safety concerns with this approach and systemic viral delivery is difficult to achieve.
- siRNA acts in the cytosol of cells, and it is necessary for the molecule to reach the cytosol for it to act.
- it would be desirable to develop an improved method of delivering siRNA to the cytosol of a cell. Desirable properties of such an improved method include i) the ability to generate time and site specific delivery of siRNA molecules to its site of action, ii) the avoidance of the use of high concentrations of transfection reagent and/or siRNA and/or iii) enhanced siRNA silencing in cell lines.
- siRNA:lipid complexes would reduce the overall number of siRNA:lipid complexes required to achieve a certain level of gene silencing or improve on it, hi such methods the siRNA:transfection reagent ratio may be altered whilst maintaining a certain amount of gene silencing or improving on it.
- Increasing the siRNA:lipid ratio is useful since it would minimize the inhibitor effects that are observed when using high concentrations of transfection reagents.
- the overall aim of the improved method can alternatively be stated as a desire to balance the need for effective and controllable siRNA delivery to the cytosol with reduction of adverse side effects caused by either high concentrations of transfection reagents, or non-specific effects, e.g. in particular cell types.
- the reduction of the overall number of siRNA:lipid complexes and/or an increase in the siRNA:lipid ratio would contribute to this goal.
- the inventors have combined the use of a carrier (transfection reagent) with the technique of photochemical internalisation (PCI).
- PCI photochemical internalisation
- siRNA:lipid complex such as the endosome and/or the lysosome of the cell.
- Alternative intracellular compartments into which the siRNA:lipid complex may be taken up include the Golgi apparatus and the endoplasmic reticulum.
- transfection reagents need to be used at high concentrations to optimise the delivery of siRNA to the cytosol.
- the inventors have surprisingly observed that by using low concentrations of transfection reagents (and a photochemical internaliser), the transfection step can be used to direct the siRNA to intracellular vesicles, such as the endosome, where it is contained until its release is triggered by the application of irradiation.
- the method thus allows siRNA to reach its site of action without the need to use high concentrations of transfection reagent or siRNA.
- the timing and location of the release of the siRNA molecule from intracellular vesicles such as the endosome can be controlled by using the PCI technique.
- the invention provides a method for introducing an siRNA molecule into the cytosol of a cell, comprising contacting said cell with an siRNA molecule, a carrier and a photosensitising agent, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent. Once activated, intracellular compartments within said cell containing said photosensitising agent release siRNA contained in these compartments into the cytosol.
- PCI is a technique which uses a photosensitising agent, in combination with an irradiation step to activate that agent, and achieves release of molecules coadministered to the cell into the cell's cytosol. This technique allows molecules that are taken up by the cell into organelles, such as endosomes, to be released from these organelles into the cytosol, following irradiation.
- the molecule to be internalised (which for use according to the present invention would be the siRNA molecule), in this case with a carrier molecule, and a photosensitising agent are brought into contact with a cell.
- the photosensitising agent, carrier molecule and the molecule to be internalised are taken up into a cellular membrane-bound subcompartment within the cell.
- the photosensitizing agent is activated which directly or indirectly generates reactive species which disrupt the intracellular compartment membranes. This allows the internalized molecule to be released into the cytosol.
- This method is particularly advantageous for introducing siRNA into cells because it allows the use of lower concentrations of carrier or transfection reagent and/or siRNA than is required for conventional siRNA transfection, whilst achieving effective gene inhibition. Furthermore, the timing and location of irradiation to release the siRNA molecule may be controlled such that it is released only at the time and location that is desired to achieve the required effects. As such, exposure of cells to siRNA and carrier is minimised, and undesirable side effects are minimised. This is in contrast to the standard techniques for introducing siRNA into cells, where it is not possible to control the timing and location of the release of siRNA and high concentrations of transfection reagent are needed.
- RNA is a polymer of ribonucleotides, each containing the sugar ribose in association with a phosphate group and a nitrogenous base (typically, adenine, guanine, cytosine, or uracil).
- a nitrogenous base typically, adenine, guanine, cytosine, or uracil.
- RNA can form complementary hydrogen bonds, and RNA may be double-stranded (dsRNA), single-stranded (ssRNA) or double-stranded with a single-stranded overhang.
- dsRNA double-stranded
- siRNA single-stranded RNA molecules from about 10 to about 30 nucleotides long that specifically interfere with protein expression by binding to mRNA molecules.
- siRNA molecules are 12- 28 nucleotides long, more preferably 15-25 nucleotides long, still more preferably 19- 23 nucleotides long and most preferably 21-23 nucleotides long. Therefore, preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length. The length of one strand designates the length of an siRNA molecule. For instance, an siRNA that is described as 21 ribonucleotides long (a 21-mer) could comprise two opposite strands of RNA that anneal together for 19 contiguous base pairings. The two remaining ribonucleotides on each strand would form an overhang.
- a dsRNA containing one strand that is 21 nucleotides long and a second strand that is 20 nucleotides long constitutes a 21-mer.
- siRNAs that comprise an overhang are desirable.
- the overhang maybe at the 5' or the 3' end of a strand. Preferably, it is at the 3' end of the RNA strand.
- the length of an overhang may vary, but preferably is about 1 to about 5 nucleotides, and more preferably is about 2 nucleotides long.
- the siRNA of the present invention will comprise a 3' overhang of about 2 to 4 nucleotides. More preferably, the 3' overhang is 2 ribonucleotides long. Even more preferably, the 2 ribonucleotides comprising the 3' overhang carry uracil (U) bases.
- siRNAs are designed to interact with a target ribonucleotide sequence, in other words they complement a target sequence so as to bind to the target sequence, i.e. one strand of the siRNA is complementary to a region of the target sequence.
- siRNA molecules having modified backbones so as to increase their half life have also been generated (e.g. as described in Chiu et al, (2003), RNA. 9(9), 1034-48 and Czauderna et al., (2003), Nucleic Acids Research 31, 2705-2716).
- the term "siRNA” thus also includes such modified molecules.
- Reference to siRNA thus encompasses derivatives or variants of siRNA which exhibit the same function, i.e. interaction with a target mRNA sequence.
- Preferred variants include those in which a modified backbone has been used (as above) or one or more non-naturally occurring bases is used.
- the method may be used to introduce more than one type of siRNA molecule into a cell; In other words, siRNA molecules having different sequences can be introduced simultaneously into a cell. If multiple siRNA molecules are to be introduced, this can be achieved by simultaneously associating more than one siRNA molecule with the carrier. Alternatively, each type of siRNA molecule can separately be associated with a carrier.
- siRNAs There are several methods for preparing siRNAs, such as chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes. Such techniques are well known in the art. See for example Pon et al, (2005) Nucleosides Nucleotides Nucleic Acids. 24(5-7): 777-81, Du et al, (2006), Biochem. Biophys. Res. Commun. 345(l):99-105 and Katoh et al, (2003), Nucleic Acids Res Suppl. (3): 249-50.
- siRNA target site must first be chosen. This can be carried out by using various techniques (see e.g. Jagla et al, (2005), RNA. 1 l'(6):864-72 and Takasaki et al, (2006), Comput. Biol. Chem. 30(3): 169- 78).
- the method of the invention achieves translocation of the siRNA molecule into the cytosol. It will be appreciated however that uptake of each and every molecule contacted with the cell is not achievable. Significant and improved uptake relative to background levels in which no PCI or carrier is used is however achievable.
- Preferably methods of the invention allow the uptake of siRNA molecules at sufficient levels that their effect is evident in the expressed products of those cells.
- the appropriate concentration of siRNA to be contacted with the cell may be adjusted to achieve this aim, e.g. to achieve a reduction in expression of a target gene of at least 10%, e.g. at least 20, 30, 40 50, 60, 70, 80 or 90% reduction (e.g. in the expression of one or more proteins encoded by the target gene) after incubation with cells for e.g. 24, 48, 72 or 96 hours (e.g. 24 to 48 hours).
- the carrier type and/or concentration, the photosensitising agent type and/or concentration and the irradiation time can be adjusted to achieve the reduction set out above.
- the level of reduction of the protein is dependent on the half-life of the protein, i.e. pre-existing protein will be removed in accordance with its half-life.
- a reduction in expression of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% is achieved relative to expression at the same time point without siRNA so that half-life is taken into account.
- the method can be carried out to achieve a reduction in mRNA levels of at least 10%, e.g. at least 20, 30, 40, 50, 60, 70, 80 or 90% reduction after incubation with cells for e.g. 24, 48, 72 or 96 hours e.g. 24 to 48 hours) relative to mRNA levels of the target sequence at the same time point without siRNA.
- This can also be measured using standard techniques known in the art such as hybridisation or blotting techniques and RT-PCR.
- the present methods require the use of significantly less carrier or transfection agent (and/or less siRNA, depending on whether the overall number of complexes is to be reduced, or the siRNAxarrier or transfection agent ratio is to be modified, or both) than standard methods for transfecting siRNA molecules, it is also possible to express the improvement in transfection using the method of the invention in terms of the amount of carrier or transfection agent that is required to achieve a certain amount of reduction in protein expression or mRNA levels.
- the method of the invention preferably allows a certain reduction in target protein expression or mRNA levels (e.g. of at least 10%, e.g.
- a carrier concentration and/or siRNA concentration that is e.g. at least 10, 20, 30, 40, 50 or 60% lower than the amount of carrier that is required to achieve the same level of reduction in target protein expression or mRNA levels without PCI.
- Comparisons can also be made between the levels of reduction in protein expression or mRNA levels that are seen at a certain siRNA and carrier concentration, in the presence and absence of PCI.
- the method of the invention preferably allows a reduction in target protein expression or mRNA levels of at least 10%, e.g. at least 20, 30, 40, 50, 60, 70, 80 or 90%, as described above, compared to protein expression or mRNA levels achieved by carrying out the method in the absence of the irradiation step of the PCI technique.
- cell is used herein to include all eukaryotic cells (including insect cells and fungal cells).
- Representative “cells” thus include all types of mammalian and non-mammalian animal cells, plant cells, insect cells, fungal cells and protozoa.
- the cells are mammalian, for example cells from cats, dogs, horses, donkeys, sheep, pigs, goats, cows, mice, rats, rabbits, guinea pigs, but most preferably from humans.
- contacting refers to bringing the cells and the photosensitizing agent and/or siRNA and carrier into physical contact with one another under conditions appropriate for internalization into the cells, e.g. preferably at 37°C in an appropriate nutritional medium, e.g. from 25-39°C.
- the photosensitising agent is an agent which is activated on illumination at an appropriate wavelength and intensity to generate an activated species.
- an agent may be one which localises to intracellular compartments, particularly endosomes or lysosomes.
- a range of such photosensitising agents are known in the art and are described in the literature, including in WO96/07432, which is incorporated herein by reference. Mention may be made in this respect of di- and tetrasulfonated aluminium phthalocyanine (e.g.
- the photosensitizing agent is preferably an agent which is taken up into the internal compartments of the cell, e.g. lysosomes and/or endosomes.
- photosensitizers for use in the invention are described in WO03/020309, which is also incorporated herein by reference, namely sulphonated meso-tetraphenyl chlorins, preferably TPCS 2a -
- the siRNA molecule a mechanism in which photochemical effects on such molecules leads to reduced transport of the molecule to be internalized (i.e. the siRNA molecule) to degradative vesicles such as lysosomes, so that the molecule to be internalized can escape to the cytosol before being degraded.
- molecules not necessarily located in membrane restricted compartments are several molecules of the microtubular transport system such as dynein and components of dynactin; and for example rab5, rab7, N-ethylmaleimde sensitive factor (NSF), soluble NSF attachment protein (SNAP) and so on.
- dynein and components of dynactin and for example rab5, rab7, N-ethylmaleimde sensitive factor (NSF), soluble NSF attachment protein (SNAP) and so on.
- NSF N-ethylmaleimde sensitive factor
- SNAP soluble NSF attachment protein
- Classes of suitable photosensitising agents which may be mentioned thus include porphyrins, phthalocyanines, purpurins, chlorins, (particularly chlorin derivatives of the prophyrins described below) benzoporphyrins, lysomotropic weak bases, naphthalocyanines, cationic dyes and tetracyclines or derivatives thereof (Berg et al, (1997), J. Photochemistry and Photobiology, 65, 403-409).
- Suitable photosensitising agents include texaphyrins, pheophorbides, porphycenes, bacteriochlorins, ketochlorins, hematoporphyrin derivatives, and derivatives thereof, endogenous photosensitizers induced by 5-aminolevulinic acid and derivatives thereof, dimers or other conjugates between photosensitizers.
- Preferred photosensitising agents include TPPS 4 , TPPS 23 , AlPcS 23 , TPCS 2a and other amphophilic photosensitizers.
- Other suitable photosensitizing agents include the compound 5-aminolevulinic acid or esters of 5-aminolevulinic acids or pharmaceutically acceptable salts thereof.
- “Irradiation" of the cell to activate the photosensitising agent refers to the administration of light directly or indirectly as described hereinafter.
- cells may be illuminated with a light source for example directly (e.g. on single cells in vitro) or indirectly, e.g. in vivo when the cells are below the surface of the skin or are in the form of a layer of cells not all of which are directly illuminated, i.e. without the screen of other cells.
- the siRNA molecule to be introduced into the cell is attached to or associated with or conjugated to one or more carrier molecules or transfection agents which act to facilitate or increase the uptake of the photosensitizing agent or the siRNA molecule into the cell.
- This attachment, association or conjugation may be performed prior to contacting the siRNA molecule and its carrier with the cell or at the time of said contact by virtue of bringing these molecules into contact.
- carrier and transfection agent are used interchangeably herein.
- the carrier molecule may be associated, bound or conjugated to the siRNA molecule or to both the siRNA and the photosensitizing agent.
- the siRNA may be attached to the carrier via chargexharge interactions.
- more than one carrier may be used simultaneously, and the carrier may be associated, bound or conjugated to more than one siRNA molecule, or more than one type of siRNA molecule.
- the carrier comprises a compound, preferably in a non-liposomal formulation that contains two or more amine groups i.e. is a polyamine and which is cationic and preferably protonatable (i.e. may be protonated to carry one or more additional hydrogen atoms under suitable reaction conditions) at different pH values.
- the different pH values applies to different values for protonatable atoms within a single molecules and/or within different molecules.
- protonatable is used herein to mean that a group is capable of accepting a hydrogen atom, i.e. a protonatable group is a hydrogen accepting group. It is clear that the ability of a group to accept hydrogen depends not only on the nature of the group, but also on the pH to which the group is exposed. Preferably said protonatable group contains a nitrogen atom and it is this atom which accepts the hydrogen atom.
- cationic denotes that the overall, or net, charge of the molecule is +1 or higher. This is preferably measured at physiological pH, i.e. pH 7.2.
- the molecule may have a higher charge, e.g.
- +2 or higher +3 or higher, +4 or higher, +5 or higher, +6 or higher, +7 or higher, +8 or higher, +9 or higher, +10 or higher, +11 or higher, +12 or higher, +13 or higher, +14 or higher, +15 or higher, +20 or higher, +25 or higher, +50 or higher, +75 or higher, +100 or higher, +150 or higher, +200 or higher, +250 or higher, +300 or higher, +400 or higher, +500 or higher, +750 or higher or +1000 or higher.
- Cationic polyamines for use in accordance with methods of the invention are as defined hereinafter and include
- PEI polyethyleneimine
- X is an integer from 1 to 100 inclusive and n is an integer from 4 to 10 inclusive,
- the polyamine as referred to herein contains primary or secondary amine groups, or a mixture thereof (e.g. at least two primary amine groups).
- the polyamine region has at least 2,3, 4, 5 or 6 nitrogen atoms and a charge of at least +1, +2, +3, +4, or +5 (or at least +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +20, +25, +50, +75, +100, +150, +200, +250, +300, +400, +500, +750 or +1000) at physiological pH, e.g. some or all of the amine groups are charged.
- at least one e.g.
- nitrogen containing group e.g. NH is uncharged at physiological pH.
- the pKa at which the last amine of the polyamine is protonated, e.g. the lipopolyamine is preferably approximately 5.5, i.e. jon decreasing the pH, or adding acidic compounds, the last amine to be protonated is protonated at a pH less than or equal to 5.5.
- the carrier comprises a lipopolyamine in a non-liposomal formulation.
- lipopolyamine it is meant an amphiphilic molecule comprising at least one hydrophilic polyamine region (i.e. which contains two or more amine groups) and one lipophilic region.
- the lipophilic region may contain one or more lipophilic chains.
- the polyamine region of the lipopolyamine preferably has the formula (I)
- each (CH) m -NH group may have a different m value, and m may be the same or different where it appears in said formula.
- R 1 is a hydrogen or a linking group to the lipo portion of the lipopolyamine or the lipo portion itself as described hereinafter, and may be the same of different at each carbon atom.
- R 2 is a hydrogen or a linking group to the lipo potion of the lipopolyamine or the lipo portion itself as described hereinafter.
- m is between 2 and 6 inclusive, more preferably 3 or 4 and n is between 1 and 5 inclusive, more preferably 3.
- R and R is a linker, a linker attached to the lipo portion of the polyamine, or the lipo portion of the polyamine.
- R 1 group is a linker, the linker attached to the lipo portion of the polyamine or the lipo portion of the polyamine.
- R 2 is preferably H.
- formula (I) is the lipopolyamine.
- formula (I) is the polyamine region only when R 1 or R 2 is not the lipo portion or a linking group to which a lipo portion is attached.
- polyamine region is represented by the following formula
- R 2 and R Ia to R 1 -* are as R 1 defined above, and preferably R la is a linker and the remaining R 1 groups and R 2 are hydrogen.
- R 1 or R 2 represents a hydrogen atom or a radical of general formula II:
- R 3 and R 4 which may be identical or different, each represent a saturated aliphatic radical C p H 2p+2 or unsaturated aliphatic radical C p H 2p or C p H 2P-2 , p being an integer between 12 and 22 inclusive, and R 5 represents a hydrogen atom or an alkyl radical containing 1 to 4 carbon atoms optionally substituted with a phenyl radical.
- R 1 or R 2 may each be a radical of general formula III:
- R 6 and R 7 which may be identical or different, each represent a saturated aliphatic radical C p - H 2p - +2 or unsaturated aliphatic radical C p - H 2p - or C p - H 2 ⁇ -2 , p' being an integer between 11 and 21 inclusive.
- R and R can represent a radical of general formula (II) or (III),
- n is equal to 3 and the values of m in the fragments
- R 1 or R 2 represents: either a radical of general formula (II) in which R 3 and R 4 each represent an alkyl radical containing 12 to 22 carbon atoms and R 5 represents a hydrogen atom, or either R 1 or R 2 represent a radical of general formula (III) in which R 6 -X- and R 7 -X- each represent an alkanoyl radical containing 12 to 22 carbon atoms.
- DOGS 5-carboxyspermylglycinedioctadecylarnide
- DPES dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide
- the lipophilic region can be as defined for R 3 , R 4 , R 5 or R 6 above or any saturated or unsaturated hydrocarbon chain, cholesterol or other steroid, a natural lipid or a synthetic lipid capable of forming lamellar or hexagonal phases.
- the length of the hydrocarbon chain may be from 10 to 30 carbons in length, e.g. 12-28, 14-26, 16-24, 18-22 carbons in length.
- the carrier is preferably JetSF M or JetSI-ENDOTM, both of which are available from Polyplus transfection.
- the carrier may be Transfectam®, available from Promega.
- liposomal formulation it is meant that the cationic charged amphiphile (i.e. the lipopolyamine) is combined with a neutral helper lipid such as DOPE (dioleoyl phosphatidylethanolamine), so as to form liposomes.
- a non-liposomal formulation of a lipopolyamine is a lipopolyamine containing formulation in which the lipopolyamine is not present in the form of a liposome.
- such formulations do not contain, in addition to the lipopolyamine, any neutral helper lipids.
- helper lipids are neutral phospholipids, cholesterol, glycerophosphoethanolamines and diacylglycerol.
- the lipopolyamine formulation contains solely the lipopolyamine described herein. It is known that in general combining transfection agents with helper lipids such as DOPE will increase transfection efficiency, and hence it is surprising that an improved, and more selective degree of inhibition can be achieved by omitting these compounds from the formulation when used in methods of the invention.
- the carrier is preferably not lipofectamine 2000, lipofectin, jet PEI, or a carrier having the composition of these commercially available transfection reagents.
- the composition of lipofectamine 2000 is a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3 -dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1-propanaminium trifluoroacetate (DOSPA) (Chemical Abstracts Registry name: N- [2-(2,5-bis[(3-aminopropyl)amino]-l-oxpentyl-amino) ethyl]-N,N-dimethyl-2,3- bis(9-octadecenyloxy)-l-propanaminium trifluoroacetate), and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE).
- DOSPA Polycationic lipid 2,3
- composition of lipofectin is a 1:1 mix of DOTMA (l,2-dioleoyloxypropyl-3- trimethyl ammonium bromide) and DOPE (dioleoylphosphatidylethanolamine).
- DOTMA l,2-dioleoyloxypropyl-3- trimethyl ammonium bromide
- DOPE dioleoylphosphatidylethanolamine
- the carrier is also preferably not siPORT or a carrier having the composition of this commercially available transfection reagent.
- the carrier is a polyamine compound which is cationic (and preferably protonatable) at physiological pH, e.g. polyethyleneimine (PEI).
- PEI polyethyleneimine
- M n value number average molecular weight
- GPC Gel Permeation Chromatography
- the PEI may have an M n value of 500-700, 500-750, 750-1000, 100-1250, 1000-1250, 1250-1500, 1000-20000, 1100-15000, 1200-12500, 1250-10000, 1500-7500, 1750-5000, 2000-4000 or 2500- 3500.
- the number average molecular weight is a way of determining the molecular weight of a polymer. Polymer molecules, even ones of the same type, come in different sizes (chain lengths, for linear polymers), so the average molecular weight will depend on the method of averaging.
- the number average molecular weight is the common, mean, average of the molecular weights of the individual polymers. It is determined by measuring the molecular weight of n polymer molecules, summing the weights, and dividing by n.
- Linear forms of PEI or non-linear, e.g. brainched PEI (which can for example be of low molecular weight of the Mw values described herein), may be used.
- branched PEI it is meant PEI which contains tertiary amine groups, as well as primary and secondary amine groups.
- the number of tertiary amine groups, relative to the primary and/or secondary amine groups is indicative of the amount or degree of branching in the polymer.
- branched PEI contains primary, secondary and tertiary amine groups in a ratio of 0.5-1.5:1.5-2;5:0.5-l.5 e.g. 1:2:1 (i.e. a ratio of 2:1 for the secondary and tertiary amine groups), but branched PEIs with a branching structure such that they contain relatively more or fewer tertiary amine groups also exist and can be used in the present invention.
- Examples of alternative ratios are 1:1 to 3:1 (secondary to tertiary amine groups) e.g. 1.2:1 to 2.8:1, 1.4:1 to 2.6:1, 1.6:1 to 2.4:1, 1.8:1 to 2.2:1.
- the molecular weight of the PEI is preferably lower than 3OkDa or 25kDa, e.g. lower than 15, 10, 5 or 2 kDa.
- PEI polyethylenimine
- GPC GPC
- Suitable PEI is available from Sigma (408719 Polyethylenimine (average Mw ⁇ 800Daby LS, average Mn ⁇ 600 by GPC, Low molecular weight, water-free).
- Other commercially available PEI based regents include Poly Sciences, Inc PEI (branched, Mw 10,000), US Biological Exgen 500, Polyplus transfection jetPEITM, Sigma ESCORTTMV Transfection reagent, and Mirus TransIT-TKO®,
- one or more betacyclodextrin amine polymers can be used as the carrier molecule, i.e. the polyamine compound is a betacyclodextrin amine polymer.
- the polyamine compound is a betacyclodextrin amine polymer.
- Suitable betacyclodextrin amine polymers and methods of synthesising such molecules are described in Hwang et at, (2001) Bioconjugate Chem, 12, 280-90.
- Suitable betacyclodextrin amine polymers and a schematic showing their synthesis from appropriate monomers are set out below:
- n can be an integer from 4 to 10, inclusive, preferably 5 to 8 inclusive, or 6 to 7 inclusive. It is most preferably 4, 6 or 8.
- X can be any integer. X is preferably 1 to 100, 10 to 50, 15 to 25, 1 to 20, e.g. 2 to 15, 3 to 12, 4 to 10, 5 to 8 or 6 to 7 inclusive. X is most preferably 4 or 5.
- one or more amine group containing dendrimers can be used as the carrier molecule, i.e. the polyamine compound is an amine group containing dendrimer.
- Dendrimers represent a class of macromolecular architecture called "dense star" polymers. Unlike classical polymers, dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific size and shape characteristics, and a highly- functionalized terminal surface. Dendrimers are thus artificially manufactured or synthesized molecules, which are built up from branched units or monomers to make a monodisperse, tree-like or generational structure.
- Synthesizing monodisperse polymers demands a high level of synthetic control which is achieved through stepwise reactions, building the dendrimer up one monomer layer, or "generation,” at a time.
- Each dendrimer consists of a multifunctional core molecule with a dendritic wedge attached to each functional site. The core molecule is referred to as "generation 0."
- Each successive repeat unit along all branches forms the next generation, “generation 1,” “generation 2,” and so on until the terminating generation.
- the manufacturing process is thus a series of repetitive steps starting with a central initiator core.
- Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately doubles the molecular weight of the preceding generation.
- the generation of the PAMAM dendrimer is described in Esfand et al, (2001) Drug Discovery Today, 6(8), 427-36 and in Kukowska-Latallo et al, (1996), 93(10), 4897-902.
- Suitable dendrimers include all dendrimers containing amine groups, e.g; dendrimers with triethanolamine, NH 3 , or ethylenediame cores to which amine containing monomers are attached. Particularly preferred are PAMAM dendrimers.
- the dendrimer is made up to polyamine monomers, e.g. having the general formula H 2 N-(CH 2 ) m -NH-(CO) n -(CH 2 ) o wherein m and o are integers from 1 to 10, preferably 1 or 2 and n is 0 or 1.
- PAMAM dendrimers are shown in Figure 14. Each "generation” represents the addition of two new H 2 N-CH 2 -CH 2 -NH-CO-CH 2 -CH 2 - groups to each of the terminal amino groups of the preceding generation; as also illustrated in the Figure.
- the Table below shows the calculated properties of amine surface functional PAMAM dendrimers by generation.
- the PAMAM dendrimer is of molecular weight 1000-235000 or 3000- 117000, e.g. 6000-60000 or 14000-30000 Da.
- Dendrimers can also be defined with reference to their generation, and as such the dendrimer (e.g. the PAMAM dendrimer) is preferably of generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, particularly generation 2-6.
- the dendrimer e.g. the PAMAM dendrimer
- the PAMAM dendrimer is preferably of generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, particularly generation 2-6.
- one or more cationic polypeptides can be used as the carrier molecule, i.e. the polyamine compound is a cationic polypeptide.
- the polyamine compound is a cationic polypeptide.
- Various cationic peptides are known in the art.
- a "peptide" as defined herein includes any molecule containing any number of amino acids, i.e. one or more amino acids. Preferably however the peptide is a polymer of consecutive amino acids.
- the peptides may be prepared by any convenient means, e.g. direct chemical synthesis or by recombinant means by expressing a nucleic acid molecule of the appropriate sequence in a cell.
- cationic denotes that the overall, or net, charge of the peptide is +1 or higher at physiological pH, i.e. pH 7.2.
- An amino acid is considered +1 if the predominant species at physiological pH is positively charged when present in the context of the peptide.
- Each such amino acid in a peptide contributes a further positive charge to calculate the final charge of the peptide.
- the peptide may contain one or more negatively charged amino acid residues, as well as neutral residues, as long as the net charge of the peptide (calculated by adding together the charge attributed to each amino acid) is positive.
- the charge of the peptide therefore depends on its amino acid composition. Certain amino acids are charged at normal physiological pH. Positively charged amino acids are lysine (K), arginine (R) and histidine (H) and are considered to be +1 on the above-described scale. Aspartic acid (D) and glutamic acid (E) carry a negative charge at most physiological pHs and are considered -1 on the above scale. Other naturally occurring amino acids are considered to carry no charge. Any number of positively charged or negatively charged amino acids may be present, as long as the overall charge of the peptide is +1 or more.
- amino acids used in peptides for use in the invention need not necessarily be naturally occurring amino acids.
- One of more of the amino acids in the peptide may be substituted for a non-naturally occurring, e.g. a derivatized amino acid.
- Such amino acids would similarly be assessed on the basis of their contribution to the charge of the peptide.
- the predominant species is positive at physiological pH, whether or not that charge is derived from the derivatized portion (e.g. an introduced amine group) or a portion also present in the natural amino acid is irrelevant as long as the overall charge is +1 or more.
- the charge of the peptide is >1, preferably from +1 to +1000, from +1 to +500, from +1 to 250 or from +1 to +100, e.g. +2 to +80, such as +3 to +60 or +4 to +50, +5 to +30, +6 to +20 e.g. +10 or +15.
- the cationic peptide comprises L or D lysine, L or D arginine, L or D histidine and/or ornithine residues.
- the peptide is rich in one or more of these residues e.g. comprises 10-100%, 20-80%, 30-70%, 40-60% or 50% positively charged residues.
- Examples of such peptides include Poly-L-Lysine, Poly-D-Lysine, Poly-Histidine, Histidylated poly-lysine and Poly-ornithine or copolymers of L or D lysine, L or D arginine, L or D histidine and/or ornithine residues with other amino acids e.g.
- Said cationic polypeptides can be defined in terms of their molecular weight. As such they are preferably at least 1000 Da, 1500Da, 2000Da, 2500Da, 5000Da, 7500Da, 10000Da, 15000Da, 20000Da, 25000Da, 30000Da, 40000Da, 50000Da, 60000Da, 70000Da, 80000Da, 90000Da or lOOOOODa in weight.
- the cationic polypeptides can be defined in terms of their length.
- Preferred cationic polypeptides are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500 amino acids in length.
- a highly preferred cationic peptides is polyarginine, particularly polyarginine with a Mw of at least 1500OkDa.
- siRNA-carrier complexes interact with anionic proteoglycans at the cell surface and are taken up by endocytosis.
- the association, binding or conjugation of the carrier with the siRNA molecule or both the siRNA and the photosensitizing agent is achieved by simply mixing the two components under appropriate conditions and concentrations and allowing the components to interact.
- the method comprises the additional step of contacting said siRNA with said carrier.
- the conditions under which this contacting step is carried out, and appropriate concentrations for each of the carrier and the siRNA molecule can readily be determined by the person skilled in the art by carrying out routine testing. Examples of suitable conditions are as set out in the Examples.
- the siRNA molecule and the carrier molecule may be mixed together, e.g. by vortexing, and allowed to stand, e.g. at room temperature. The siRNA molecule and the carrier molecule may then be left for approximately 10-20, 10-30 or 20-40 minutes before being contacted with the cell. '
- 10nM-200nM e.g. 15-15OnM, 20-10OnM, 20 ⁇ 10OnM, 30-9OnM, 40- 8OnM, or 50-7OnM siRNA is used for transfection, e.g. in a well of a standard 6 well plate, although different concentrations may be tested. It is a matter of routine to determine the optimum concentration of siRNA to be used in the method.
- the cells to which the siRNA and carrier are applied are prepared using standard cell culture techniques. If the cells are adherent cells they are preferably at 50-70% or 25-50% confluence.
- the carrier and the siRNA molecule may be mixed in various ratios, based on standard protocols. For example, as set out in Examples 1 to 6, 8.4 ⁇ l of 2mM carrier made up in an aqueous solution can be mixed with 2.8 ⁇ g siRNA in 2ml medium for application to a 6 well plate. Similarly, 4.2 ⁇ l of 2mM carrier made up in an aqueous solution can be mixed with 1.4 ⁇ g siRNA in ImI medium for application to a 6 well plate.
- the carrier need not always be at 2mM, suitable ranges include 0.5-5mM, l-3mM, 1.5-2.5mM (e.g. made up in an aqueous solution). For every ng of siRNA, an amount equivalent to approximately 6 nanomoles of carrier (e.g. 1-10, 2-8) total in the solution with the photosensitiser can be used.
- concentrations are l ⁇ g/ml and lO ⁇ g/ml (e.g. 0.5-20 or 1- 15 ⁇ g/ml) with 10OnM siRNA, e.g. in ImI medium for application to a 6 well plate.
- This has been shown to be particularly effective for PEI with a molecular weight of 1200-25000 Daltons (e.g. 1300-2000 Daltons) as set out in Example 9.
- the concentration of PEI used can be modified to achieve the charge ratio and/or N/P ratio that is achieved using the conditions set out in Example 9.
- betacyclodextrin amine polymers For betacyclodextrin amine polymers, a suitable concentration is lOO ⁇ g/ml (e.g. 10- 1000, 20-800, 30-600, 40-400, 50-200 ⁇ g/ml) with 5OnM siRNA, e.g. in ImI medium for application to a 6 well plate. This is based on the use of the polymer as described in Example 11. The values of n and X will influence the charge of the molecule and as such different concentrations may be appropriate. The concentration of betacyclodextrin amine polymer used can be modified to achieve the charge ratio and/or N/P ratio that is achieved using the conditions set out in Example 11.
- a suitable concentration is lOO ⁇ g/ml (e.g. 10-1000, 20-800, 30-600, 40-400, 50-200 ⁇ g/ml) with 10OnM siRNA, e.g. in ImI medium for application to a 6 well plate.
- concentration of amine group containing dendrimer used can be modified to achieve the charge ratio and/or N/P ratio that is achieved using the conditions set out in Example 12.
- a suitable concentration is O.35 ⁇ g/ml or 0.7 ⁇ g/ml (e.g. 0.1-20, 0.2-15, 0.3-1 O ⁇ g/ml) with 10OnM siRNA, e.g. in ImI medium for application to a 6 well plate.
- these concentrations were used successfully with polyarginine carrier of molecular weight 15000-70000 and >70000.
- the molecular weight and amino acid composition of the peptide will influence the charge of the molecule and as such different concentrations may be appropriate.
- the concentration of cationic peptide used can be modified to achieve a charge ratio and/or N/P ratio as is achieved using the conditions set out in Example 13.
- the ratio of the two components may be expressed as the ratio of charges, and this also needs to be taken into account.
- the charge ratio between the carrier and the siRNA is at least 1+/- (i.e. 1 positive charge per negative charge), 5+/-, 10+/, 20+/-, 30+/-, 40+/-, 50+/-, 60+/-, 70+/-, 80+/-, 90+/-, 100+/-; 200+/-, 300+/-, 400+/- or 500+/-.
- the charge ratio depends on the charge of each component (i.e. the siRNA and the carrier) and on the amount of each component that is present.
- the ratio of the two components may be expressed as the N/P ratio, i.e. the ratio of nitrogen residues to oligonucleotide phosphate. Since not every nitrogen atom of a carrier is always a cation, the N/P ratio is not the same as the charge ratio.
- the N/P ratio depends on the chemical composition of each compound and on the amount of each compound that is present. Suitable values for the N/P ratio include 1-500, e.g. 2-450, 3-400, 4-350, 5-300, 6-250, 7-200, 8-150, 9-100, 10-80, 15-60, 20-50, 30-40, 1-25, 1-20, 1-15, 1-10, 1-5.
- the carrier is such that at the concentration that is chosen for use, there is no release from intracellular compartments without the irradiation step of PCI, and release from intracellular compartments is seen following the irradiation step of PCI.
- concentrations and ratios of siRNA and carrier are mentioned above.
- the carrier is in general formulated for transfection purposes in an aqueous solution (e.g. in water).
- Stock solution e.g. 2OmM in Ethanol
- can then be diluted e.g. in water
- US5,476,962 describes the formulation of lipopolyamines.
- the siRNA carrier may also be used as a carrier for the photosensitising agent.
- the photosensitiser may be used without a carrier or with an alternative carrier riot used according to the invention for siRNA.
- Such alternative carriers are referred to herein as photosensitizer carriers and include polycations such as polylysine (e.g.
- poly-L-lysine or poly-D-lysine polyethyleneimine or dendrimers (e.g. cationic dendrimers such as SuperFect7); cationic lipids such as DOTAP or Lipofectin and peptides.
- dendrimers e.g. cationic dendrimers such as SuperFect7
- cationic lipids such as DOTAP or Lipofectin and peptides.
- the siRNA molecule and/or the photosensitizing agent may be associated or conjugated to specific targeting molecules that will promote the specific cellular uptake of the siRNA molecule into desired cells or tissues.
- the targeting molecule may be associated, bound or conjugated to the siRNA molecule, to the carrier, to the photosensitising agent or to two (e.g. the siRNA and the carrier or the siRNA and the photosensitising agent or the carrier and the photosensiting agent) or all three of these moieties, and the same or different targeting molecules may be used. As mentioned above, more than one targeting molecule may be used simultaneously.
- the method of the invention may be put into practice as described below.
- the siRNA molecule, together with its carrier and a photosensitising compound are applied simultaneously or in sequence to the cells, whereupon the photosensitizing compound, carrier and the siRNA molecule are endocytosed or in other ways translocated into endosomes, lysosomes or other intracellular membrane restricted compartments.
- the siRNA, carrier and the photosensitising compound may be applied to the cells together or sequentially.
- the siRNA is mixed with the carrier as described above so as to form a complex, which is then administered to the cell simultaneously with the photosensitising compound.
- the siRNAxarrier complex and the photosensitising compound can be administered sequentially.
- the siRNA- carrier complex and the photosensitising compound may be taken up by the cell into the same or different intracellular compartments (e.g. they may be co-translocated).
- the siRNA is then released by exposure of the cells to light of suitable wavelengths to activate the photosensitising compound which in turn leads to the disruption of the intracellular compartment membranes and the subsequent release of the siRNA, which may be located in the same compartment as the photosensitizing agent, into the cytosol.
- the final step of exposing the cells to light results in the siRNA being released from the same intracellular compartment as the photosensitizing agent and becoming present in the cytosol.
- WO 02/44396 (which is incorporated herein by reference) describes a method in which the order of the steps could be changed such that for example the photosensitising agent is contacted with the cells and activated by irradiation before the molecule to be internalised (and the carrier) are brought into contact with the cells.
- This adapted method takes advantage of the fact that it is not necessary for the molecule to be internalised to be present in the same cellular subcompartment as the photosensitising agent at the time of irradiation.
- said photosensitising agent, said carrier and said siRNA molecule are applied to the cell together, or said photosensitising agent is applied separately relative to said carrier and said siRNA molecule. As a consequence they may be taken up by the cell into the same intracellular compartment and said irradiation may then be performed.
- the photosensitising agent, carrier and siRNA molecule can be separate, or they can be formulated as a dendrimeric molecule (see e.g. Nishiyama N et ah, (2005) Nat Mater. 4(12):934-41)
- said method can be performed by contacting said cell with a photosensitising agent, contacting said cell with the carrier and the siRNA molecule to be introduced and irradiating said cell with light of a wavelength effective to activate the photosensitising agent, wherein said irradiation is performed prior to the cellular uptake of said siRNA molecule and said carrier into an intracellular compartment containing said photosensitising agent, preferably prior to cellular uptake of said molecule and said carrier into any intracellular compartment.
- Said irradiation can be performed after the cellular uptake of the molecule and the carrier molecule into an intracellular compartment, whether or not said siRNA molecule and the photosensitising agent are localised in the same intracellular compartments at the time of light exposure. In one preferred embodiment however irradiation is performed prior to cellular uptake of the molecule to be internalised.
- Internalisation refers to the cytosolic delivery of molecules.
- internalisation thus includes the step of release of molecules from intracellular/membrane bound compartments into the cytosol of the cells.
- cellular uptake or “translocation” refers to one of the steps of internalisation in which molecules external to the cell membrane are taken into the cell such that they are found interior to the outer lying cell membrane, e.g. by endocytosis or other appropriate uptake mechanisms, for example into or associated with intracellular membrane-restricted compartments, for example the endoplasmic reticulum, Golgi body, lysosomes, endosomes etc.
- the step of contacting the cells with a photosensitising agent and with the siRNA molecule and carrier may be carried out in any convenient or desired way.
- the cells may conveniently be maintained in an aqueous medium such as for example appropriate cell culture medium and at the appropriate time point the photosensitising agent and/or siRNA molecule and carrier can. simply be added to the medium under appropriate conditions, for example at. an appropriate concentration and for an appropriate length of time.
- the cells may be contacted with the siRNA molecule and carrier in the presence of serum-free medium.
- the photosensitizing agent is brought into contact with the cells at an appropriate concentration and for an appropriate length of time which can easily be determined by a skilled person using routine techniques and will depend on such factors as the particular photosensitizing agent used and the target cell type and location.
- concentration of the photosensitizing agent must be such that once taken up into the cell, e.g. into, or associated with, one or more of its intracellular compartments and activated by irradiation, one or more cell structures are disrupted e.g. one or more intracellular compartments are lysed or disrupted.
- photosensitising agents as described herein may be used at a concentration of for example 10 to 50 ⁇ g/ml.
- the range can be much broader, e.g. 0.05-500 ⁇ g/ml.
- the photosensitizing agent may be used in the range 0.05-20 mg/kg body weight when administered systemically or 0.1-20% in a solvent for topical application. In smaller animals the concentration range may be different and can be adjusted accordingly.
- the time of incubation of the cells with the photosensitizing agent can vary from a few minutes to several hours, e.g. even up to 48 hours or . longer, e.g. 12 to 20 or 24 hours.
- the time of incubation should be such that the photosensitizing agent is taken up by the appropriate cells, e.g. into intracellular compartments in said cells.
- the incubation of the cells with the photosensitizing agent may optionally be followed by a period of incubation with photosensitiser-free medium before the cells are exposed to light or the siRNA molecule and carrier are added, e.g. for 10 minutes to 8 hours, especially 1 to 4 hours.
- the siRNA molecule and carrier e.g. as a preformed siRNAxarrier complex
- siRNA molecules may be administered at levels of less than 50OnM, e.g. less than 30OnM, especially preferably less than 10OnM or 5OnM, for example from 1 to 10OnM, or 5 to 5OnM, where the concentration indicated reflects the levels in contact with the cell.
- the contact may be initiated even several hours after the photosensitising agent has been added and irradiation taken place.
- transfection time or “cellular uptake time” i.e. the time for which the molecules are in contact with the cells can be a few minutes or up to a few hours, for example a transfection time of from 10 minutes until up to 24 hours, for example 30 minutes up to 10 hours or for example 30 minutes until up to 2 hours or 6 hours can be used. Longer incubation times may also be used, e.g. 24 to 96 hours or longer, e.g. 5-10 days.
- transfection time usually results in increased uptake of the molecule in question.
- shorter incubation times for example 30 minutes to 1 hour, can also result in an improved specificity of the uptake of the molecule.
- an appropriate balance must be struck between obtaining a sufficient uptake of the molecule while maintaining sufficient specificity of Iiie PCI treatment.
- an appropriate method and time of incubation by which the siRNA molecule, carrier and photosensitizing agents are brought into contact with the target cells will be dependent on factors such as the mode of administration and the type of siRNA molecule, carrier and photosensitizing agents.
- the siRNA molecule and carrier are injected into a tumour, tissue or organ which is to be treated, the cells near the injection point will come into contact with and hence tend to take up the siRNA molecule more rapidly than the cells located at a greater distance from the injection point, which are likely to come into contact with the siRNA molecule at a later time point and lower concentration.
- an siRNA molecule administered by intravenous injection may take some time to arrive at the target cells and it may thus take longer post-administration e.g. several days, in order for a sufficient or optimal amount of the siRNA molecule to accumulate in a target cell or tissue.
- the same considerations of course apply to the time of administration required for the uptake of the photosensitizing agent into cells. The time of administration required for individual cells in vivo is thus likely to vary depending on these and other parameters.
- the time at which the molecules come into contact with the target cells must be such that before irradiation occurs an appropriate amount of the photosensitizing agent has been taken up by the target cells and either: (i) before or during irradiation the siRNA molecule has either been taken up, or will be taken up after sufficient contact with the target cells, into the same or different intracellular compartments or (ii) after irradiation the siRNA molecule is in contact with the cells for a period of time sufficient to allow its uptake into the cells.
- the siRNA molecule is taken up into intracellular compartments affected by activation of the photosensitizing agent (e.g. compartments in which the agent is present), the siRNA molecule can be taken up before or after irradiation.
- the light irradiation step- to activate the photosensitising agent may take place according to techniques and procedures well known in the art. For example, the wavelength and intensity of the light maybe selected according to the photosensitising agent used. Suitable light sources are well known in the art.
- the time for which the cells are exposed to light in the methods of the present invention may vary.
- the efficiency of the internalisation of the siRNA molecule into the cytosol increases with increased exposure to light to a maximum beyond which cell damage and hence cell death increases.
- a preferred length of time for the irradiation step depends on factors such as the target, the photosensitizer, the amount of the photosensitizer accumulated in the target cells or tissue and the overlap between the absorption spectrum of the photosensitizer and the emission spectrum of the light source.
- the length of time for the irradiation step is in the order of minutes to several hours, e.g. preferably up to 60 minutes e.g. from 0.5 or 1 to 30 minutes, e.g. from 0.5 to 3 minutes or from 1 to 5 minutes or from 1 to 10 minutes e.g. from 3 to 7 minutes, and preferably approximately 3 minutes, e.g. 2.5 to 3.5 minutes.
- Shorter irradiation times may also be used, for example 1 to 60 seconds, e.g. 10-50, 20-40 or 25-35 seconds.
- Appropriate light doses can be selected by a person skilled in the art and again will depend on the photosensitizer and the amount of photosensitizer accumulated in the target cells or tissues.
- the light dose typically used for photodynamic treatment of cancers with the photosensitizer Photofrin and the protoporphyrin precursor 5-aminolevulinic acid is in the range 50-150 J/cm2 at a fluence range of less than 200 mW/cm 2 in order to avoid hyperthermia.
- the light doses are usually lower when photosensitizers with higher extinction coefficients in the red area of the visible spectrum are used.
- the total amount of light needed maybe substantially higher than for treatment of cancers.
- the generation of excessive levels of toxic species is to be avoided and the relevant parameters may be adjusted accordingly.
- the methods of the invention may inevitably give rise to some cell killing by virtue of the photochemical treatment i.e. through the generation of toxic species on activation of the photosensitizing agent. Depending on the proposed use, this cell death may not be of consequence and may indeed be advantageous for some applications (e.g. cancer treatment).
- cell death is avoided and as noted elsewhere herein the method can be carried out so as to cause strong inhibition of expression (i.e.
- the methods of the invention may be modified such that the fraction or proportion of the surviving cells is regulated by selecting the light dose in relation to the concentration of the photosensitizing agent. Again, such techniques are known in the art.
- substantially all of the cells, or a significant majority e.g. at least 50%, more preferably at least 60, 70, 80 or 90% of the cells.
- Cell viability following PCI treatment can be measured by standard techniques known in the art such as the MTS test (see the Examples).
- the siRNA Regardless of the amount of cell death induced by the activation of the photosensitiser, for the siRNA to have an effect in the cells, it is important that the light dose is regulated such that some of the individual cells wherein the PCI effect is manifested are not killed by the photochemical treatment alone (although they may subsequently be killed by molecules introduced into the cells if those molecules have a cytotoxic effect).
- Cytotoxic effects may be achieved by using for example gene therapy in which an siRNA molecule is internalized into a tumour celi by the method of the invention e.g. to down-regulate a gene.
- the methods of the invention may be used in vitro or in vivo, for example either for in situ treatment or for ex vivo treatment followed by the administration of the treated cells to the body, for various purposes including inhibition of expression of specific gene products e.g. in gene therapy methods and the generation of screening assays.
- the present invention provides a method of inhibiting the expression of a target gene by introducing an siRNA molecule into a cell containing said target gene by a method as described hereinbefore, wherein said siRNA molecule specifically inhibits expression of said target gene.
- Specific inhibition refers to sequence-dependent inhibition of the target gene. Expression of genes which contain a sequence that is sufficiently identical at the nucleic acid level to the siRNA molecule used will be affected by the siRNA molecule. As noted above, standard techniques have been developed that allow the skilled person to design siRNA molecules of appropriate sequence to cause sequence-specific inhibition of expression.
- Target gene refers to a gene whose expression is to be down-regulated and which is to be the target of investigation or manipulation.
- the methods of the invention may also be used in treating any disease which benefits from the down-regulation, repair or mutation of one or more genes.
- genes that are overexpressed in cancer may be down-regulated by administering the appropriate siRNA molecule (Lage (2005) Future Oncol l(l):103- 13).
- Alternative diseases that may be treated include neurodegenerative diseases such as Huntington's disease and Alzheimer's disease and viral infections such as hepatitis (e.g. B and C) and HIV.
- a further aspect of the invention provides a composition containing an siRNA molecule, a carrier molecule (preferably as a complex with said siRNA molecule) and optionally separately also a photosensitizing agent as described herein.
- the invention provides said composition for use in therapy.
- the present invention provides a kit comprising an siRNA molecule, a carrier molecule and optionally also a photosensitizing agent as described herein.
- a kit comprising an siRNA molecule, a carrier molecule and optionally also a photosensitizing agent as described herein.
- said kit or product is for simultaneous, separate or sequential use in a medical treatment.
- the present invention provides the use of an siRNA molecule and carrier as described herein in the preparation of a medicament for treating or preventing a disease, disorder or infection by altering expression of one or more target genes in said patient.
- said medicament may contain only one of said siRNA molecule or carrier and may be used in methods in which said siRNA molecule or carrier not present in said medicament is for administration to said patient when treating or preventing said disease, disorder or infection.
- said medicament may contain the photosensitising agent.
- said medicament is for gene therapy, i.e. for treating a disease or disorder which is typified by abnormal gene expression or which would benefit from suppression of one or more genes. Said alteration includes down regulation of said expression.
- the said photosensitizing agent and said siRNA molecule and carrier is contacted with cells or tissues of a patient simultaneously or sequentially and said cells are irradiated with light of a wavelength effective to activate the photosensitizing agent and irradiation is performed prior to, during or after the cellular uptake of said siRNA molecule and the carrier into an intracellular compartment containing said photosensitizing agent, preferably prior to cellular uptake of said transfer molecule into any intracellular compartment.
- the invention provides a method of treating or preventing a disease, disorder or infection in a patient comprising introducing an siRNA molecule and carrier into one or more cells in vitro, in vivo or ex vivo according to the methods as described hereinbefore and where necessary (i.e. when transfection is conducted in vitro or ex vivo) administering said cells to said patient.
- treatment refers to reducing, alleviating or eliminating one or more symptoms of the disease, disorder or infection which is being treated, relative to the symptoms prior to treatment.
- prevention refers to delaying or preventing the onset of the symptoms of the disease, disorder or infection.
- compositions of the present invention may also comprise a cell containing an siRNA molecule which has been internalised into the cytosol of said cell by a method of the invention.
- the invention further extends to such compositions for use in therapy, particularly cancer or gene therapy.
- a yet further aspect of the invention provides a cell or a population of cells containing an siRNA molecule which has been internalised into the cytosol of said cell, which cell is obtainable by a method of the present invention.
- a yet further aspect of the invention provides the use of a such a cell or population of cells for the preparation of a composition or a medicament for use in therapy as described hereinbefore, preferably cancer or gene therapy.
- the invention further provides a method of treatment of a patient comprising administering to said patient cells or compositions of the present invention, i.e. a method comprising the steps of introducing an siRNA molecule into a cell as described hereinbefore and administering said cell 1 thus prepared to said patient.
- a method of treatment of a patient comprising administering to said patient cells or compositions of the present invention, i.e. a method comprising the steps of introducing an siRNA molecule into a cell as described hereinbefore and administering said cell 1 thus prepared to said patient.
- said methods are used to treat cancer or in gene therapy.
- any mode of administration common or standard in the art may be used, e.g. injection, infusion, topical administration, both to internal and external body surfaces etc.
- the invention can be used in relation to any tissue -which contains cells to which the photosensitising agent and the siRNA molecule are localized, including body fluid locations, as well as solid tissues. All tissues can be treated as long as the photosensitiser is taken up by the target cells, and the light can be properly delivered.
- compositions of the invention may be formulated in any convenient manner according to techniques and procedures known in the pharmaceutical art, e.g. using one or more pharmaceutically acceptable carrier or excipients.
- “Pharmaceutically acceptable” as referred to herein refers to ingredients that are compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient.
- the nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, purpose of treatment etc. Dosages may likewise be determined in routine manner and may depend upon the nature of the molecule, purpose of treatment, age of patient, mode of administration etc. In connection with the photosensitizing agent the potency/ ability to disrupt membranes on irradiation, should also be taken into account.
- siRNA directed to one or more specific genes may be generated and used in the method of the invention as described above.
- the siRNA may thus be used to reduce the expression of a gene in a population of cells.
- the resulting cell population may then be used as a screening tool to identify downstream effects of gene silencing, with standard techniques.
- the invention provides a method of modifying the gene expression pattern of a cell (e.g. a cell population) to prepare a cell (or cell population) for use as a screening tool (e.g. for high throughput screening), comprising contacting an siRNA molecule capable of inhibiting or reducing the expression of a gene, a carrier and a photosensitizing agent with a cell (e.g.
- modified gene expression pattern it is meant that as a consequence of the presence of said siRNA molecule in the cell nucleus, the transcription or translation of the gene to which it is directed is affected.
- the expression of other genes maybe influenced.
- the normal expression of the gene being studied it is possible to determine the changes in expression pattern of other genes.
- the identification of these genes, and of the influence that the expression of the gene being studied has on them allows the investigator to draw conclusions about the functions of the gene e.g. their downstream functions.
- the genes that are affected by the change in normal expression of the gene being studied may be upregulated or downregulated, but the overall change in the pattern of expression gives an indication of the role of the gene in normal cell function and of the consequences of its misregulation.
- the gene expression profile can be studied directly by analysing mRNA patterns and/or protein expression, again using standard techniques that are well known in the art.
- inhibiting or reducing the expression of a gene it is to be understood that the expression of the gene in question is reduced, when compared to a cell which has not been subjected to the method i.e. a wild-type or normal cell.
- the change in the level of gene expression may be determined by standard techniques known in the art.
- the reduction in expression is to less than or 80% of control levels, e.g. ⁇ 50%, preferably ⁇ 20, 10 or 5% of control levels.
- the cell(s) used will preferably be a cell population, the individual cells of which are genetically identical.
- the cells may be any cells, as discussed above.
- the cell or cell population generated according to methods of the invention may be used to make a library which forms a further aspect of the invention.
- Figure 1 shows the results of a gene silencing experiment using the OHS cell line with various transfection reagents with (black bars) and without (white bars) PCI treatment using siRNA9.
- Figure 2 shows the results of a gene silencing experiment where cells of different types were transfected with siRNA using jetSI-ENDO, with and without PCI treatment.
- results in (A) display S100A4 protein levels in four cell lines when treated with siRNA.
- Grey bars show scrambled control siRNA with PCI, black bars display effector siRNA without PCI and white bars display effector siRNA with PCI.
- the results in (B) show a Western blot showing different cell lines from the top to the bottom, HCT-116, SW620, OHS and RMS.
- the upper panel displays ⁇ -tubulin loading control and the lower panel S100A4 levels.
- lanes 1-3 show protein levels without PCI treatment: untreated control (C), scrambled control (siRNA 11), and effector (siRNA9).
- Lanes 4-6 show protein levels with PCI treatment: untreated control (C), scrambled control (siRNAll), and effector (siRNA9).
- Figure 3 shows results of a gene silencing experiment in OHS cells a) Dose-dependent silencing (1-5 nM siRNA) 96 hrs after irradiation b) Time-dependent silencing (24, 48 and 96 hrs) with siRNA9. Results are represented as percent of untreated control cells. The bars are the mean from three individual experiments. Error bars show standard error of the mean (SEM).
- Figure 4 shows the results of gene silencing with siRNA transfected with jetSI, with and without PCI treatment 96 hrs after irradiation.
- Results show S100A4 protein level (A) and RNA levels (B) after treating the OHS cell line with 100 nM siRNA as indicated below.
- Black bars represent samples without PCI, and white bars represent samples subjected to PCI. Untreated control without PCI treatment was used as control for all samples (not shown).
- Figure 5 shows the distribution of fluorescence labelled siRNA in OHS following transfection (200 nM) with jetSI-ENDO, with and without PCI treatment a) Delivery of siRNA without PCI treatment, from left to right: phase contrast, fluorescence and the corresponding merge, b) Delivery of siRNA with PCI treatment: from left to right: phase contrast, fluorescence and the corresponding merge, c) Fluorescence pictures without PCI treatment displaying from left to right: fluorescence entrapped in endosomes (left hand box) and fluorescence leakage from endosomes (right hand box), enlargement of the image within the left hand box, and enlargement of the image within the right hand box.
- Figure 6 shows the results of gene silencing using jetSI after treating OHS with 100 nM siRNA and jetSI at 50 % of the level recommended compared to the standard protocol. Bars represent; 1) scrambled control siRNA without PCI, 2) effector siRNA without PCI, 3) untreated control with PCI, 4) scrambled control siRNA with PCI, 5) effector control siRNA with PCL The bars represent the mean from three individual experiments.
- Figure 7 shows the structure of preferred lipopolyamines. (See also Ahmed et al, supra and Behr et al (1989) PNAS, 86, 6982-6).
- Figure 8 shows the results of gene silencing using PEI as the carrier.
- the Western blot displays a loading control (alpha-tubulin) in the upper panel and S100A4 protein levels in the lower panel.
- Lane 1 untreated control (without PEI)
- 2 1 ⁇ l PEI + effector siRNA
- 3 10 ⁇ l PEI + effector siRNA
- 4 untreated control (without PEI)
- 5 1 ⁇ l PEI + effector siRNA
- 6 10 ⁇ l PEI + effector siRNA.
- Lanes 1-3 are without PCI
- lanes 4-6 are with PCI.
- Figure 9 shows the results of gene silencing using 25kDa PEI as the carrier. The samples used are indicated on the Figure.
- Figure 10 shows the effect of PCI on siRNA activity with different PEI carriers used in different concentrations. S100A4 protein levels were quantified by scanning of Western blots. Black bars represent transfections without PCI, while white bars represent transfection with PCI. The results are the mean of 3 individual experiments.
- Figure 11 shows the results of experiments to determine the toxicity of PEI alone and in combination with PCI and siRNA.
- the amounts of PEI and the light doses used in the PCI experiments are indicated.
- Control - untreated control (with PEI but without PCI)
- Scrambled - scrambled siRNA control (with PEI but without PCI)
- siRNA - S100A4 siRNA (with PEI but without PCI).
- Control +, Scrambled +, and siRNA + are the same as Control -, Scrambled -, and siRNA -, but with PCI (mean of 5 individual experiments).
- Figure 12 shows PCI-induced delivery of siRNA molecules using Beta-Cyclodextrin amine as a carrier.
- Lanes 1 and 4 control without siRNA
- lanes 2 and 5 control scrambled siRNA
- lanes 3 and 6 S100A4 siRNA.
- the tubulin control band and the S100A4 band are as shown.
- Figure 13 shows PCI-induced delivery of siRNA molecules using poly amido amide (PAMAM) dendrimers (G2-7) with ethylenediamine core.
- PAMAM poly amido amide
- G2--7 poly amido amide dendrimers
- FIG. 13 shows PCI-induced delivery of siRNA molecules using poly amido amide (PAMAM) dendrimers (G2-7) with ethylenediamine core.
- A Western blot in which upper bands represent loading control (alpha tubulin), lower bands represent S100A4 levels. The samples in the different lanes are: 1. PAMAM G6 with PCI. 2. PAMAM Gl with PCI. 3. Control with PCI. 4. PAMAM G6 without PCI. 5. PAMAM Gl without PCI. 6. Control without PCI.
- B S100A4 protein level quantified by scanning of Western blots. Black bars represent transfections without PCI, while white bars represent transfection with PCI. The results are the mean of 3 individual experiments. The different forms of PAMAM used
- Figure 14 shows the structural formula of GO, Gl and G2 PAMAM dendrimers.
- Figure 15 shows the effect of PCI on polyarginine-mediated siRNA delivery.
- the level of S 100A4 protein was analysed by Western blotting.
- the upper lane represent a loading control (alpha tubulin), the lower lane represent S100A4 levels.
- HCT-116 colonal adenocarcinoma
- SW620 colonal adenocarcinoma
- the OHS (osteosarcoma) and the RMS cell line was established at the Norwegian Radium Hospital. All cell lines were cultured using RPMI- 1640 medium (Bio Whittaker, Verviers, Belgium or GibcoBRL, Paisley, UK), without antibiotics, but supplemented with 10 % fetal calf serum (FCS; PAA Laboratories, Linz, Austria) and 2 mM L-glutamine (Bio Whittaker, Verviers, Belgium). Cells were grown and incubated at 37°C in a humidified atmosphere containing 5 % CO2. AU cell lines were tested and found negative for Mycoplasma infection prior to the experiments. Light source and photosensitizer.
- a Lumisource ® (PCI Biotech AS, Oslo, Norway) was used as the light source.
- the Lumisource ® is a bank of four fluorescent tubes designed to provide homogeneous illumination of the treatment area, emitting mainly blue light with a peak at 420 nm.
- the photosensitizer, disulfonated tetraphenylporphine (TPPS 28 ) was purchased from Porphyrin Products (Logan, UT, USA).
- TPPS 2a was first dissolved in 0.1 M NaOH, and thereafter diluted in phosphate-buffered saline (PBS), pH 7.5, to a concentration of 5 mg/ml and a final concentration of 0.002 M NaOH.
- PBS phosphate-buffered saline
- the photosensitizer was light protected and stored at -20°C until use.
- transfection reagents for siRNA delivery were evaluated by using: LipofectinTM Reagent from Life Technologies Inc. (Gaithersburg, MD, USA), LipofectamineTM 2000 from Invitrogen (Carlsbad, CA, USA), FuGene 6 from Roche Diagnostics (Mannheim, Germany), siPORTTM Lipid Transfection Agent from Ambion (Austin, TX, USA), jetSITM and jetSITM-ENDO from Polyplus transfection (Illkirch, France). All transfection reagents were handled according to the manufacturers' specifications. All cell lines were cultured as described in "Cell Lines and Culture Conditions", and cultivated for 24 hrs in 6-well plates to 50-70% confluence before transfection for 24, 48 or 96 hrs. Transfection reagent alone was applied to the cells as an untreated control, in addition to scrambled siRNA with transfection reagents.
- Step 1 For each well, dilute 4.2(8.4) ⁇ l of jetSI/jetSI-ENDO solution into 100 ⁇ l of medium. Vortex vigorously (important: do not pipet to mix) and wait for lOmin (important: do not exceed 30 minutes).
- Step 2 For each well, dilute 1.4 ⁇ g (100 nM) of siRNA duplex and into 100 ⁇ l of medium. Vortex gently.
- RNA transfection without PCI Cells were cultured and transfected as in "siRNA transfection without PCI", except for a few modifications.
- the photosensitizer TPPS 23 (0,5 ⁇ g/ml) was added to the medium upon transfection. After 18 hrs of incubation, cells were washed 3 times with fresh medium, and incubated for 4 hrs before light treatment. After 4 hrs, cells were exposed to blue light (7mW/cm 2 ) for different durations (60-90 sec), depending on the cell line, and re-incubated for 24, 48 and 96 hrs before harvesting.
- effector siRNA, scrambled siRNA and transfection reagent alone were applied in the different wells of the same plate, with or without photosensitizer, and given the same treatment. Cells were light protected by aluminium foil during these experiments.
- Total cellular RNA was isolated with the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Steinheim, GER) and the iScript cDNA synthesis kit (BioRad, Hercules, CA) was used for reverse transcription. Both kits were used according to the manufacturers' manuals. All PCRs were run in parallels, real-time detection was obtained by use of SYBR Green I. For each PCR, 10 ⁇ l cDNA, 30 ⁇ l iQ SYBRGreen Supermix (BioRad), 300 nM of each primer and nuclease free water were added to a final volume of 60 ⁇ l. Samples of 25 ⁇ l each was then applied to the PCR plate.
- Primer design was accomplished using the software Primer Express from Applied Biosystems (Applied Biosystems, Foster City, CA).
- the primer set used forward primer 5'-AAGTTCAAGCTCAACAA GTCAGAAC-3' and reverse primer 5'-CATCTGTCCTTTTCCCCAAGA-S' amplifies a 79-bp segment in exon 2 and 3 of the S100A4 sequence.
- RNA samples The quality of the RNA samples was verified by amplification of two housekeeping genes, TBP (forward primer 5'-GCCCGAAACGCCGAATAT-3 and reverse primer 5'-CGTG GCTCTCTTATCCTCATGA-3') and RPLPO (forward primer 5'-CGCTGCTGAA CATGCTCAAC-3' and reverse primer 5'-TCGAACACCTGCTGGATGAC-S').
- TBP forward primer 5'-GCCCGAAACGCCGAATAT-3 and reverse primer 5'-CGTG GCTCTCTTATCCTCATGA-3'
- RPLPO forward primer 5'-CGCTGCTGAA CATGCTCAAC-3' and reverse primer 5'-TCGAACACCTGCTGGATGAC-S'.
- the Gene Expression Macro version 1.1 (Biorad) was used for the quantitative calculations.
- the program performs calculations based on the ⁇ CT method, which allows comparison of cycle threshold values obtained using different sets of primers on the same set of samples.
- Protein lysates were prepared in 50 mM Tris-HCl (pH 7.5), containing 150 mM NaCl and 0.1 % NP-40 with 2 g/ml pepstatin, aprotinin (Sigma Chemical Company, St Louis, MO) and leupeptin (Roche Diagnostics, Mannheim, Germany). Total protein lysate (30 ⁇ g) from each sample was separated by 12% SDS-polyacrylamide gel electrophoresis, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA) according to the manufacturer's manual. As a loading and transfer control, the membranes were stained with 0.1% amidoblack.
- the membranes were subsequently incubated in 20 mM Tris-HCl (pH 7.5), containing 0.5 M NaCl and 0.25% Tween 20 (TBST) with 10% dry milk (blocking solution) before incubation with rabbit polyclonal anti S100A4 (diluted 1:300, DAKO, Glostrup, Denmark) and mouse monoclonal anti ⁇ -tubulin (diluted 1 :250, Amersham Life Science, Buckinghamshire, England) in TBST containing 5% dry milk.
- immunoreactive proteins were visualized using horseradish peroxidase conjugated secondary antibodies (diluted 1 :5000 DAKO, Glostrup, Denmark), and the enhanced chemiluminescense system (Amersham Pharmacia Biotech, Buckinghamshire, England). S100A4 protein levels were reported as percentages of control sample and ⁇ -tubulin was used as a loading control.
- OHS cells were transfected with designed to target the expression of the S 100 A4 protein using various transfection systems.
- the standard transfection protocol was used in each case, according to the manufacturers' instructions.
- photosensitiser it was TPPS 2a (0.5 ⁇ g in ImI transfection volume, apart from jetSI where a 2ml transfection volume was used).
- the irradiation time in each case was 60 seconds.
- siRNA stock solution 20 ⁇ M siRNA stock solution was mixed with each different transfection reagents and transfected in 6-well plates to a final volume of 1 ml (except for jetSI and jetSI- ENDO, where the final volume was 2 ml).
- siPORT Lipid 2 and 4 ⁇ l were tested, combined with 1.4 ⁇ g siRNA in 1000 ⁇ l
- FuGene 6 4.2 ⁇ l and 8.4 ⁇ l were tested, combined with 1.4 ⁇ g siRNA in 1000 ⁇ l
- Lipofectamine 2000 4.2 and 7 ⁇ l were tested, combined with 1.4 ⁇ g siRNA in
- siRNA molecule used was against the S100A4 mRNA sequence (GenBank accesssion number NM_002961).
- a specific siRNA having the sequence 5 ' -UGAGCAAGUUCAAUAAAGA- 3 ' 3 ' -ACUCGUUCAAGUUAUUUCU- 5 ' was designed according to Elbashir et al, ((2001), Genes Dev. 15, 188-200).
- control siRNA was designed by making a scrambled siRNA (5'-CGCAUAAGUGAAAUAGAAU-S', 3'-GCGUAUUCACUUUAUCUUA-S') and a BLAST search was performed to eliminate false hybridisation.
- siRNA molecules were ordered from Eurogentec (Seraing, Belgium).
- Dried siRNA oligonucleotides were re-suspended to 100 ⁇ M in DEPC-treated water and stored at -20°C. Annealing was performed by separately aliquoting and diluting each RNA oligonucleotide to a concentration of 50 ⁇ M. Then, 30 ⁇ l of each RNA oligonucleotide solution and 15 ⁇ l of 5X annealing buffer were combined, to a final concentration of $0 mM Tris, pH 7.5, 100 mM NaCl in DEPC-treated water. The solution was then incubated for 3 min in a water bath at 95 0 C, followed by gradual cooling for 45 min on the workbench. Successful annealing was confirmed by 4 % NuSieve agarose gel electrophoresis (data not shown).
- PCI dependent gene silencing can be achieved through using the transfection agents jetSI and jetSI-ENDO.
- these two transfection agents are used in the absence of PCI (i.e. irradiation, but in the absence of photosensitiser)
- gene expression is around 75% of the control based on a > measurement of protein levels using western blotting however when PCI is additionally used, gene expression is reduced to around 15% of the control.
- siRNA molecule designed to silence expression of the S100A4 protein was transfected into OHS cells at concentrations of 1 to 5nM, using 4.2 ⁇ l jetSI-ENDO, and subjected to PCI treatment as described above. Protein levels in cell lysates were measured using western blotting and are shown as a percentage of protein levels in untreated control cells in Figure 3 A.
- the gene silencing effect increased with the concentration of siRNA to which the cells were exposed, although there is little difference between the silencing effects seen with 4nM and 5nM siRNA.
- the scrambled siRNA molecule was also used in all experiments and was shown not to affect gene expression. As such, the gene inhibition effect is specific.
- Figure 2B shows the effect of time on gene silencing by altering the period of time between irradiation of the cells and harvesting of cell lysates for analysis. It can be seen that the longer the cells are left after irradiation and before harvesting, the more inhibition of gene expression is observed.
- OHS cells were transfected with 10OnM siRNA or scrambled siRNA as described above and the effect of PCI treatment on protein and mRNA levels were determined 96h after irradiation by western blotting and RT-PCR and compared to protein and mRNA levels in untreated controls.
- the cells were inspected using phase contrast microscopy and fluorescence microscopy. By comparing the images, it was seen that in the absence of PCI treatment, the siRNA remains in a punctate distribution, which is representative of a distribution in endocytic vesicles ( Figure 5a and c). There is also some leakage seen (right hand box in Figure 5c). In contrast, following PCI treatment the labelled siPvNA is seen to be distributed throughout the cytoplasm and in the nucleus (see Figure 5b).
- OHS cells were transfected with 10OnM siRNA using jetSI.
- jetSI was used at a lower concentration, i.e. at 50% of that recommended in the standard protocol.
- the standard protocol for a 6-well plate is: 2.8 ⁇ g siRNA + 8.4 ⁇ l jetSI in 2000 ⁇ l medium, resulting in 100 nM siRNA in each well.
- siRNA was mixed with 4.2 ⁇ l jetSI in 1000 ⁇ l medium, resulting in 100 nM siRNA in each well. The total amount of complexes was thus reduced by 50%.
- siRNA target was selected against the S100A4 mRNA sequence (Gene Bank accession number NM_0Q2961). siRNA 481-499 was used as effector (for sequence see Example 1).
- PEI Polyethylenimine
- the PEI used was from Sigma and was diluted in sterile water and a stock solution containing 1000 ⁇ l PEI and 9000 ⁇ l sterile water was made. From the stock solution 1 and 10 ⁇ l was used to transfect of cells with 1.4 ⁇ g siRNA (100 nM in each well).
- PEI Polyethylenimine (average Mw ⁇ 800 by LS, average Mn ⁇ 600 by GPC, branched, Low molecular weight, water-free)).
- solution A siRNA was diluted in 100 ⁇ l of serum free (OPTI-MEM I) medium.
- Solution B PEI was diluted in 100 ⁇ l of serum free medium. Solutions A and B were mixed by gently mixing and incubated in room temperature for 30 min. The mixed solution was then added to the cells (1 ml of 100 nM siRNA). PCI treatment was performed as described above for jetSI. Light doses for the PEI experiment was 40 sec. Protein levels were measured by Western blotting 96 hrs after irradiation.
- TPPS 28 ' 0,5 ⁇ g/ml
- siRNA mediated S100A4 gene silencing was measured at the protein level with and without PCI treatment using branched 25 kDa polyethylenimine (PEI) (1 ⁇ g/ml) and an siRNA targeting the S100A4 gene at a 100 nM concentration. The protocol described above was followed, using a light dose of 30 s, with both complexation and transfection in serum containing medium.
- Fig. 9A shows that with the use of PCI S100A4 siRNA typically reduced the S100A4 level down to 5-15 % of the level in untreated controls (treated with PEI but without siRNA). In contrast, S100A4 siRNA without PCI was only able to reduce the S100A4 level down to 100-80 % of the control.
- toxicity increased with increasing molecular weight of the PEI formulation e.g. 25000 PEI vs. 800 PEI
- PEI formulations at 1 ⁇ g/ml (not shown) and 10 ⁇ g/ml did not show significant toxicity.
- a significant biological effect of the siRNA could be achieved with PCI, while the effect without PCI was very modest (see Example 9).
- the toxicity of PEI with siRNA/PCI treatment was also assessed. OHS cells were plated in 6-well plates and allowed to adhere overnight in seruitn containing medium.
- Fig. 1 IB shows the toxicity of the combined treatment with PEI (MW 25 000) at 1 ⁇ g/ml, 100 nM siRNA and PCI at different light-doses.
- the PCI treatment did not induce significant toxicity at the light-dose used (30 s) in all the PEI gene silencing experiments, which is a light-dose at which a strong gene silencing effect was induced by PCI (see Figure 10). Even at the higher light dose of 40 s no cytotoxicity of the PCI treatment could be observed, showing that PCI can induce fully enhance siRNA delivery without giving cytotoxic effects.
- Example 11 PCI-induced delivery of siRNA molecules using ⁇ -Cyclodextrin amine as a carrier.
- Beta-cyclodextrin amine used in this study consists of Beta- cyclodextrin molecules conjugated through an amine bridge responsible for binding to siRNA, and is described in Hwang SJ. et al. (2001, Bioconjugate Chem. 12, 280- 290).
- Example 12 PCI-induced delivery of siRNA molecules using poly amido amide (PAMAM) dendrimers (G2-7) with ethylenediamine core
- PAMAM Poly amido amide dendrimers were evaluated for PCI-induced siRNA delivery.
- PAMAM was diluted in sterile water and transfections were performed in serum containing medium.
- FIG. 13A Western blot
- PCI could strongly enhance the activity of siRNA (lanes 1 and 2) under conditions where siRNA/PAMAM alone was ineffective for gene silencing (lanes 4 and 5).
- Fig. 13B this effect was apparent also for several other types of PAMAM dendrimers, indicating that PCI can generally enhance siRNA delivery by polyamine-based dendrimers.
- Polyarginine was diluted in sterile water and transfections were performed in serum free medium. Two types of polyarginine carriers (molecular weight of 15000-70000 and >70000) were tested. Polyarginine at 0.35 or 0.7 ⁇ g/ml (ImI was used per well) were complexed with 100 nM (1.4 ⁇ g) siRNA, and the protocol described above ' was followed, assessing S1004A expression by Western blotting after giving a light- dose of 30 s. As can be seen from Fig. 15, there is a significant difference in gene silencing efficacy between PCI-treated and non-treated samples. Thus, while all the PCI-treated samples (R+-samples in Fig.
- PCI is effective in inducing gene silencing with both the different polyarginine carriers tested and at both the concentrations used, showing that PCI can significantly enhance siRNA delivery by a cationic peptide-based carrier.
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Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK07733508.1T DK2037962T3 (en) | 2006-07-11 | 2007-07-11 | PROCEDURE FOR INTRODUCING SIRNA INTO CELLS BY PHOTochemical INTERNALIZATION |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996007432A1 (en) | 1994-09-08 | 1996-03-14 | Radiumhospitalets Forskningsstiftelse | Transfer of molecules into the cytosol of cells |
WO2000054802A2 (en) | 1999-03-15 | 2000-09-21 | Photocure Asa | Method of expressing antigens on the surface of antigen presenting cells by photochemical internalization |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4897355A (en) | 1985-01-07 | 1990-01-30 | Syntex (U.S.A.) Inc. | N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
FR2645866B1 (en) | 1989-04-17 | 1991-07-05 | Centre Nat Rech Scient | NEW LIPOPOLYAMINES, THEIR PREPARATION AND THEIR USE |
FR2714830B1 (en) | 1994-01-10 | 1996-03-22 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
RU2066552C1 (en) | 1996-02-12 | 1996-09-20 | Товарищество с ограниченной ответственностью "Био Прогресс" | Composition for photodynamic target-cell impairment and a method of photodynamic target-cell impairment |
FR2763943B1 (en) * | 1997-05-28 | 1999-07-09 | Rhone Poulenc Rorer Sa | COMPOUNDS, THEIR PREPARATION AND THEIR USE FOR TRANSFERRING NUCLEIC ACIDS INTO CELLS |
KR100980153B1 (en) | 2000-11-29 | 2010-09-03 | 피씨아이 바이오테크 에이에스 | Photochemical Internalization for Delivery of Molecules into the Cytosol |
KR101180548B1 (en) | 2000-11-29 | 2012-09-06 | 피씨아이 바이오테크 에이에스 | Photochemical Internalization for Virus-Mediated Molecule Delivery into the Cytosol |
HU230458B1 (en) * | 2000-12-01 | 2016-07-28 | Europäisches Laboratorium für Molekularbiologie (EMBL) | Rna interference mediating small rna molecules |
GB0121023D0 (en) | 2001-08-30 | 2001-10-24 | Norwegian Radium Hospital Res | Compound |
AU2003279010A1 (en) * | 2002-09-28 | 2004-04-19 | Massachusetts Institute Of Technology | Compositions and methods for delivery of short interfering rna and short hairpin rna |
DE60332277D1 (en) * | 2002-11-26 | 2010-06-02 | Univ Massachusetts | ADMINISTRATION OF SIRNAS |
FR2858628B1 (en) | 2003-08-04 | 2008-01-04 | Polyplus Transfection | NOVEL NUCLEIC ACID COMPLEXES FOR RNA INTERFERENCE AND THEIR USE FOR INHIBITING PROTEIN EXPRESSION |
GB0415263D0 (en) * | 2004-07-07 | 2004-08-11 | Norwegian Radium Hospital Res | Method |
US20060051405A1 (en) | 2004-07-19 | 2006-03-09 | Protiva Biotherapeutics, Inc. | Compositions for the delivery of therapeutic agents and uses thereof |
GB0613753D0 (en) | 2006-07-11 | 2006-08-23 | Norwegian Radium Hospital Res | Method |
-
2006
- 2006-07-11 GB GBGB0613753.3A patent/GB0613753D0/en not_active Ceased
-
2007
- 2007-07-11 WO PCT/GB2007/002569 patent/WO2008007073A2/en active Application Filing
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2008
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-
2012
- 2012-01-12 US US13/349,334 patent/US9700622B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996007432A1 (en) | 1994-09-08 | 1996-03-14 | Radiumhospitalets Forskningsstiftelse | Transfer of molecules into the cytosol of cells |
WO2000054802A2 (en) | 1999-03-15 | 2000-09-21 | Photocure Asa | Method of expressing antigens on the surface of antigen presenting cells by photochemical internalization |
Non-Patent Citations (3)
Title |
---|
ESFAND ET AL., DRUG DISCOVERY TODAY, vol. 6, no. 8, 2001, pages 427 - 36 |
KUKOWSKA-LATALLO ET AL., PNAS, vol. 93, no. L0, 1996, pages 4897 - 902 |
LAGE, FUTURE ONCOL, vol. 1, no. 1, 2005, pages 103 - 13 |
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GB0613753D0 (en) | 2006-08-23 |
CA2657999C (en) | 2015-02-24 |
EP2037962B1 (en) | 2014-09-03 |
CA2657999A1 (en) | 2008-01-17 |
RU2008150847A (en) | 2010-06-27 |
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NO20085259L (en) | 2009-02-10 |
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EP2037962A2 (en) | 2009-03-25 |
JP2010504080A (en) | 2010-02-12 |
NO342202B1 (en) | 2018-04-16 |
AU2007274056B2 (en) | 2013-08-01 |
CN101573140A (en) | 2009-11-04 |
CN101573140B (en) | 2014-04-23 |
PL2037962T3 (en) | 2015-03-31 |
US9700622B2 (en) | 2017-07-11 |
JP5269783B2 (en) | 2013-08-21 |
ES2525222T3 (en) | 2014-12-19 |
DK2037962T3 (en) | 2014-12-15 |
US20090297487A1 (en) | 2009-12-03 |
AU2007274056A1 (en) | 2008-01-17 |
WO2008007073A3 (en) | 2008-04-10 |
US20120264807A1 (en) | 2012-10-18 |
NZ573775A (en) | 2012-11-30 |
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