US20050222061A1 - Means and methods for the specific inhibition of genes in cells and tissue of the cns and/or eye - Google Patents

Means and methods for the specific inhibition of genes in cells and tissue of the cns and/or eye Download PDF

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US20050222061A1
US20050222061A1 US10/511,656 US51165605A US2005222061A1 US 20050222061 A1 US20050222061 A1 US 20050222061A1 US 51165605 A US51165605 A US 51165605A US 2005222061 A1 US2005222061 A1 US 2005222061A1
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dsrna
cells
eye
organism
tissue
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Ralf Schulte
Frank Gohring
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LYNKEUS BIOTECH GmbH
Phio Pharmaceuticals Corp
Acuity Pharmaceuticals Inc
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LYNKEUS BIOTECH GmbH
Acuity Pharmaceuticals Inc
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Assigned to ACUITY PHARMACEUTICALS, INC. reassignment ACUITY PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LYNKEUS BIOTECH GMBH
Assigned to LYNKEUS BIOTECH GMBH reassignment LYNKEUS BIOTECH GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHULTE, RALF W., GOHRING, FRANK
Priority to US12/828,624 priority patent/US20110021605A1/en
Assigned to RXI PHARMACEUTICALS CORPORATION reassignment RXI PHARMACEUTICALS CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: OPKO PHARMACEUTICALS, LLC
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Definitions

  • the present invention relates to the specific modulation of the expression of genes in cells and tissues of the CNS and/or the eye.
  • the present invention relates to the use of one or more double-stranded oligoribonucleotides (dsRNA) for the preparation of a composition for the specific modulation of the expression of one or more target genes in cells and/or tissues of the CNS and/or eye of a subject, wherein said composition is designed to be applied outside the blood-brain or blood-retina barriers.
  • dsRNA double-stranded oligoribonucleotides
  • the instant invention further relates to methods for the identification and validation, respectively, of the function of a gene comprising the mentioned use of dsRNA for providing a test cell, test tissue or test organism and comparing the resulting phenotype with that of a suitable control, thus allowing information on the function of the gene to be gained.
  • the present invention relates to cells, tissue and non-human organisms obtainable by the method of the invention, wherein said organisms preferably display of phenotype of a CNS or eye disease.
  • the present invention relates to the use of RNA interference technique for the diagnosis and/or therapy of disorders related to CNS and/or eye and to method of identification and isolation of drugs capable of specific modulation of the expression of a target gene in cells and/or tissues of the eye maling use of the afore-mentioned methods.
  • tissues at the back of the eye such as the multi-layer retina, in which functionally highly specialized types of cells mediate the transformation of light energy to electrical impulses, and also the retinal pigment epithelium (RPE) are characterized by an extremely high metabolic activity.
  • RPE retinal pigment epithelium
  • Bruch's membrane The exchange of substances via the RPE and Bruch's membrane is controlled and specific, and on the basis of this functional analogy to the blood-brain barrier one refers to a blood-retina barrier in this case.
  • the human eye is an organ characterized by high complexity and the coordinated functioning of numerous specific structures and tissues. Both are protected by barriers (tear secretion, enzymes, transport mechanisms, blood-retina and blood-CNS barrier) against harmful environmental influences. Like the blood-brain barrier, the blood-retina barrier also represents a physiological barrier for the uptake of medication by the inner part of the eye, and makes pharmacological therapy of ocular diseases very difficult indeed—if at all possible—at the present state of technology.
  • Medication currently available on the market for the treatment of disorders of the CNS including ophthalmological diseases is therefore almost exclusively available for treatment of clinical symptoms often associated with side effects due to the high doses necessary.
  • a causal therapy of the CNS, and particularly of the back sections of the eye, was not possible apart from the injections.
  • the current state of information on the complex molecular metabolic interrelationship underlying the etiology of retinal diseases of multi-factorial origin is only limited. Consequently, medicaments available on the market are suitable to treat the symptoms of such diseases only.
  • the technical problem of the present invention is to provide means and methods for the identification and modulation of genes involved in disorders of the CNS and/or the eye.
  • the technical problem of present invention is to provide non-invasive methods for the controlled modulation of target genes and gene products in the mammalian CNS and/or eye while overcoming the blood-brain and/or blood retina barrier without injuring it.
  • the present invention relates to the use of one or more double-stranded oligoribonucleotides (dsRNA) for the preparation of a composition for the specific modulation of the expression of one or more target genes in cells and/or tissues of the CNS and/or eye of a subject, wherein said composition is designed to be applied outside the blood-brain or blood-retina barriers.
  • dsRNA double-stranded oligoribonucleotides
  • the present invention relates to a method for the specific modulation of the expression of target genes in cells and/or tissues of the CNS and/or eye, wherein a composition comprising one or more double-stranded oligoribonucleotides (dsRNA) is introduced into a cell, tissue or organism outside the blood-brain or blood-retina barriers; wherein preferably said method results in the provision of a non-human organism comprising cells and/or tissue containing said dsRNA, and wherein said organism or the corresponding test cells or tissue are maintained under conditions allowing the degradation of the corresponding mRNA of one or more target genes by RNA interference.
  • dsRNA double-stranded oligoribonucleotides
  • the present invention is based on the surprising finding that in contrast to wide-spread professional opinion active substances of the invention are able to cross the blood-retina barrier as a physiological barrier, enabling a systemic or local application on the outer side of the eye for specific treatment of a disease in the back of the eye. Due to the high similarity in function as well as cellular and molecular architecture of the blood-retina and blood-brain barrier the methods and pharmaceutical compositions provided by the present invention are also be expected to cross the blood-brain barrier and thereby applicable to the treatment of diseases of the CNS.
  • the present invention provides improved methods as well as components for the treatment of CNS and/or eye diseases.
  • the fundamental idea of the present invention concerns a method for the specific inhibition of the expression of target genes in cells and tissues of the CNS and/or eye, by
  • compositions comprising a dsRNA capable of modulating a target gene or gene product in the CNS or the eye are preferably designed to be administered without any substantial, i.e. substantially effective amount of delivery-enhancing agents facilitating passage of compounds through the blood-brain barrier and/or without the necessity of applying invasive methods and devices; see, e.g., those compounds, methods and devices described in US2002183683 and WO03/000018.
  • dsRNA double-stranded oligoribonucleotides
  • RNA interference double-stranded oligoribonucleotides
  • RNAi could also be demonstrated in plants, protozoa, insects (Kasschau and Carrington 1998) and recently also in mammalian cells (Caplen et al., 2001; Elbashir et al., 2001).
  • the mechanism by which RNAi suppresses gene expression is not yet fully understood.
  • dsRNA molecules are transformed into small interfering RNA molecules (siRNA molecules) by endogenous riboriucleases (Bernstein et al., 2001; Grishok et al., 2001; Hamilton and Baulcombe, 1999; Knight and Bass, 2001; Zamore et al., 2000).
  • the 21 to 23 bp long siRNA molecules are thus the actual mediators of the degradation of the mRNA of the target gene.
  • a double-stranded oligoribonucleotide exhibits a sequence of 21 to 23 nucleotides (base pairs) in length identical to the target gene; see, e.g., Elbashir et al., Methods 26 (2002), 199-213 and Martinez et al., Cell 110 (2002), 563-574
  • Medication for the treatment of ophthalmological diseases currently on the market is almost exclusively available for treatment of clinical symptoms of the front of the eye, since the relatively easy application of eye drops is possible in this case.
  • a causal therapy, particularly of the back sections of the eye, is not possible with conventional pharmaceutical compositions apart from the injections associated with side effects described earlier.
  • CNS related disorders are currently treated mainly by systemic application.
  • surgery or stimulation by implanted probes as for example in Parkinson disease, can relieve the symptoms but do not treat the cause of the disease.
  • the present invention provides a method for the specific intervention in diseases of the CNS or the back of the eye on a molecular level, without requiring direct application to the target tissue.
  • the present invention opens up comprehensively the broad not yet or only unsatisfactorily addressable therapeutic field of diseases of the CNS, the inner eye and the back of the eye, in particular the inner segment of the eye ball.
  • the intervention is based on an inhibition of genes expressed specifically or predominantly in the target tissues of CNS or the eye, respectively, characterized by the ability of the required active substances to cross the blood-brain and/or the blood-retina barrier, allowing systemic or local application outside the respective barrier.
  • CNS disorders are, for example, Alzheimer's disease, Parkinson disease, depression, bipolar disorder, schizophrenia, amnesia, migraine-headache, stroke, insomnia, alcohol abuse, anxiety, obsessive compulsive disorder, cerebral acquired human immuno-deficiency syndrome, chronic pain and many others.
  • compositions of the invention may be administered locally or systemically e.g., intravenously.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
  • the pharmaceutical compositions are administered to a subject in an effective dose of between about 0,1 ⁇ g to about 10 mg units/day and/or units/kg body weigth, preferably between about 0,1 and 2.0 mg.
  • the appropiate dose can also be determined as descibed further below and in the examples.
  • the use according to the present invention is directed to the treatment of a discorder such as of the CNS described above.
  • the disorder to be treated is related to eye.
  • Such disorders include chorioretinitis and herpes retinitis, which may be considered as acquired forms of retinal disease, the majority of retinal disease disorders are reduced to a genetic predisposition.
  • retinal detachment ablatio retinae
  • retinal blastoma retinal blastoma
  • retinal astrocytoma retinal astrocytoma
  • Hippel-Lindau angiomatosis retinae
  • Coat's disease exudative retinitis
  • Eale's disease central serous retinopathy, ocular albinism, retinitis pigmentosa, retinitis punctata albescens, Usher syndrome, Leber's congenital amaurosis, cone dystrophy, vitelliform macular degeneration (Best's disease), juvenile retinoschisis, North Carolina macular dystrophy, Sorsby's fundus dystrophy, Doyne's honey comb retinal dystrophy (Malattia Leventinese), Stargardt's disease, Wagner vitreoretinal degeneration or Age-related macular degeneration (AMD) as well as single-gene retinopathies like Mor
  • the disorder to be treated is wet age-related macular degeneration (AMD) or diabetic retinopathy.
  • AMD wet age-related macular degeneration
  • the following description deals with AMD as example for a complex eye disease with a genetic component.
  • the example shall illustrate the associated technical problems with reference to the study of molecular causes and the development of diagnostic and pharmacological intervention strategies.
  • AMD which can be thought as a sub-type of retinal degeneration, is the most common cause of visual morbidity in the developed world with a prevalence increasing from 9% in persons over 52 years to more than 25% in persons over the age of 75 (Paetkau et al. 1978, Leibowitz et al. 1980, Banks and Hutton 1981, Ghafour et al. 1983, Hyman 1987, Hyman et al. 1983, Grey et al. 1989, Yap and Weatherill 1989, Heiba et al. 1994).
  • AMD is a complex disease caused by exogenous as well as endogenous factors (Meyers and Zachary 1988; Seddon et al. 1997).
  • endogenous factors such as hypermetropia, light skin and iris colour, elevated serum cholesterol levels, hypertension or cigarette smoking have been suggested (Hyman et al. 1983, Klein et al. 1993, Sperduto and Hiller 1986, The Eye Disease Case-Control Study Group 1992, Bressler and Bressler 1995).
  • a genetic component for AMD has been documented by several groups (Gass 1973, Piguet et al. 1993, Silvestri et al. 1994) and has lead to the hypothesis that the disease may be triggered by environmental/individual factors in those persons who are genetically predisposed.
  • the number of genes which, when mutated, can confer susceptibility to AMD is not known but may be numerous.
  • compositions of the present invention represent an important improvement and alternative therapeutic intervention for the treatment of this particular disease as well as of others.
  • the pharmaceutical compositions are preferably designed to be effective in (and applied to) the posterior segment of the eye, preferably in a form designed to be applied outside the retinal region of the blood-retina barrier.
  • the compound used in accordance with the present invention is dsRNA, which usually substantially consists of ribonucleotides which preferbly contain a portion of double-stranded oligoribonucleotides (dsRNA).
  • dsRNA double-stranded oligoribonucleotides
  • the region of the double stranded RNA that is present in a double stranded conformation includes at least 5, 10, 20, 30, 50, 75, 100 or 200.
  • the double stranded region includes between 15 and 30 nucleotides, most preferably 20 to 25 and particularly preferred 21 to 23 nucleotides. Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites is described for example in Amarzguioui, Nucleic Acids Res.
  • the dsRNA molecule can also contain a terminal 3′-hydroxyl group and may represent an analogue of naturally occurring RNA, differing from the nucleotide sequence of said gene or gene product by addition, deletion, substitution or modification of one or more nucleotides.
  • dsRNA or RNAi are known to the person skilled in the art and are described, for in WO99/32619, WO01/68836, WO01/77350, WO00/44895, WO02/055692 and WO02/055693, the disclosure content of which is hereby incorporated by reference.
  • the target gene subject of the RNA interference is predominantly or more preferably specifically expressed in said cell and/or tissue of the CNS and/or eye.
  • Another technical problem consists in the identification of genes, which cause CNS and retinal diseases as well as in the validation of these genes as targets for diagnosis and for pharmacological intervention.
  • Conventional experimental strategies are often difficult to apply.
  • Positional cloning is often not possible since linkage studies lead to conflicting results or are altogether impossible due to the small number of patients and afflicted families.
  • linkage studies might be inconclusive when the group of patients to be studied includes individuals who suffer from a disorder different to the disease whose etiology is to be elucidated.
  • Schizophrenia e.g. is thought to be caused by several different etiologies with a large number of genes involved.
  • the present invention relates to a method for the specific modulation of the expression of target genes in cells and/or tissues of the CNS and/or eye, wherein a composition comprising one or more double-stranded oligoribonucleotides (dsRNA) is introduced into a cell, tissue or organism outside the blood-brain or blood-retina barriers.
  • dsRNA double-stranded oligoribonucleotides
  • test cell or tissue provided is derived from the animal to which the composition mentioned above has been applied to.
  • Assaying the test cell or tissue can thus be done in vivo or in vitro, for example after the subject cells or tissue have been isolated from the animal, in particular, mammalian animals are preferred.
  • the use or method described earlier said specific modulation of the expression being an inhibition of target gene expression.
  • Said one or more of said target genes preferably encode a cellular mRNA.
  • the target cells and/or tissues are cells and/or tissues of the eye.
  • said cells or tissues are cells or tissues of the inner segment of the eye ball, preferably retinal cells, and particularly preferred cells of the retinal pigment epithelium (RPE) or neurosensory retina cells.
  • RPE retinal pigment epithelium
  • the present invention relates to a drug or pharmaceutical composition, which contains one or more double-stranded oligoribonucleotides (dsRNA), that by means of RNA interference, inhibits the expression of the corresponding mRNA of one or more target genes, whose restricted functions cause an eye disease, and which are applied outside the blood-retina barrier, particularly outside the eye.
  • dsRNA double-stranded oligoribonucleotides
  • Double-stranded oligoribonucleotides are used in the present invention, which cross the blood-retina barrier after application, in order to elicit inhibition of the target genes in the target cells by RNA interference of the corresponding mRNA molecules.
  • the present invention includes further a drug of dsRNA molecules for the specific treatment of genetically caused eye diseases.
  • the present invention opens up the broad so far not or only unsatisfactorily addressable therapeutic field of diseases of the inner eye and the back of the eye.
  • the present invention includes embodiments of the uses or methods described above, wherein one or more of said target genes are predominantly expressed in said cell and/or tissue, or wherein the expression of one or more of said target genes is specific for said cell and/or tissue.
  • the solution of the technical problem underlying the present invention consists of the provision of a method for the specific inhibition of genes, whose aberrant functions are causally associated with CNS or eye diseases of monogenic or multifactorial origin.
  • AMD for example may be taken as one form of degenerative retinal disease.
  • a double-stranded stranded oligoribonucleotide comprises a sequence of 21 to 23 nucleotides (base pairs) in length identical or substantial identical to the target gene.
  • Said dsRNA molecules can contain a terminal 3′-hydroxyl hydroxyl group, have been chemically synthesized and/or represent an analogue of naturally occurring RNA.
  • Said dsRNA analogues can also differ from the corresponding naturally occurring RNA by addition, deletion, substitution or modification of one or more nucleotides.
  • said dsRNA molecules inhibit the corresponding target genes by “posttranscriptional silencing”.
  • the central idea of the present invention is surprising in so far as dsRNA molecules of a length of 21 to 23 nucleotides, are able to cross the blood-retina barrier, and specifically inactivate target genes in the tissues of the back of the eye, after systemic application, for example by intravenous injection.
  • This overcoming the blood-retina barrier is all the more remarkable, because no experiment could demonstrate overcoming this barrier by dsRNA so far.
  • Due to the high similarity in function as well as cellular and molecular architecture of the blood-retina and blood-brain barrier the methods and pharmaceutical compositions provided by the present invention will also be able to cross the blood-brain barrier and thereby applicable to the treatment of diseases of the CNS.
  • nucleotides can not only be applied as “naked” dsRNA, preferred are embodiments, wherein said dsRNA molecules are encoded by a vector.
  • RNAi RNA interference
  • Appropriate regulatory sequences with which expression can be selectively controlled both temporarily and specially i.e., at particular times and/or in particular tissue, organs or cells are known to the persons skilled in the art and are also described inter alia in WO 03/006477, that disclosure content or which is hereby incorporated by reference.
  • dsRNA is under control of a cell and/or tissue specific promoter.
  • Vectors that can be used in accordance with the teaching of the present invention are known to the person skilled in the art; see, e.g., heritable and inducible genetic interference by double-stranded RNA encoded by transgenes described in Tavemarakis et al., Nat. Genet. 24 (2000), 180-183. Further vectors and methods for gene transfer and generation of transgenic animals are described in the prior art; see, e.g., adeno-associated virus related vectors described in Qing et al., Virol.
  • HIV-2 human immunodeficiency virus type 2 vector-mediated in vivo gene transfer into adult rabbit retina described in Cheng et al. Curr. Eye Res. 24 (2002), 196-201, long-term transgene expression in the RPE after gene transfer with a high-capacity adenoviral vector described in Kreppel et al., Invest. Ophthalmol. Vis. Sci. 43 (2002), 1965-1970 and non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo described in Borras et al., J. Gene Med. 3 (2001), 437-449.
  • CNS gene transfer has also been described in Leone et al., Curr. Opin. Mol. Ther. 1 (1999), 487-492.
  • the dsRNAs can be introduced into the cells or tissues bound to other molecules and/or combined with one or more suitable carriers.
  • a carrier can be a micellar structure, preferably a liposome, a coat protein, derived from a virus such as the cytomegalovirus (CMV) or produced synthetically, adeno-associated virus (AAV) or adenovirus.
  • CMV cytomegalovirus
  • AAV adeno-associated virus
  • the dsRNA can also be bound to cationic porphyrins, cationic polyamines, polymeric DNA-binding cations or fusogenic peptides. Packaging of the dsRNA into coat proteins or liposomes and/or associating it with carriers will not only improve the targeting but also elongate the half-life.
  • dsRNA molecules preferably of the length specified above
  • the detection is based on the specific inhibition of specified target genes in cells or tissues of the inner eye by RNA interference.
  • the composition is in a form designed to be introduced into the cells or tissue of the CNS or eye by a suitable carrier, characterized by the application occurring outside the blood-CNS and/or blood-retina barriers, for instance as eye drops. It can also be administered systemically, iontophoretically or by retrobulbar injection.
  • Iontophoresis has been defined as the active introduction of ionised molecules into tissues by means of an electric current. The technique has been used to enhance drug delivery into tissues underlying the donor electrode (e.g. skin) as well as to the general blood circulation, thus providing systemic delivery of a drug to the entire body. Iontophoresis devices require at least two electrodes, both being in electrical contact with some portion of a biological membrane surface of the body.
  • One electrode commonly referred to as the “donor” or “active” electrode is the electrode from which the biologically active substance, such as a drug or prodrug, is delivered into the body.
  • Another electrode having an opposite polarity flnctions to complete the electric circuit between the body and the electrical power source.
  • This electrode is commonly referred to as the “receptor” or “passive” electrode.
  • receptor or “passive” electrode.
  • Iontophoresis an electrical potential is applied over the electrodes, in order to create an electrical current to pass through the drug solution and the adjacent tissue. Iontophoresis has been described for the treatment of blood-vessel related disorders (e.g. restenosis), bladder, uterus, urethra and prostate disorders.
  • the subject or organism can be a vertebrate, preferably a mammal or a human.
  • the described methods can be applied to cells and/or tissues of vertebrate origin and particularly of mammalian origin. Human cells and/or tissues are preferred.
  • animal models may easily be generated, which reproduce the symptoms of diseases of the inner eye and/or CNS of predominantly genetic origin. These animal models are suitable to initiate the development of specific pharmaceutical products for ophthalmology and CNS-related diseases and can be used in the validation of products.
  • the method illustrated below by examples of the procedure thus, is suitable for the provision of cell culture as well as animal models with which targets, whose restricted function cause diseases of the eye and/or the CNS, can be identified and validated.
  • the method is moreover suitable for the specific intervention in eye diseases on a molecular level, without necessitating direct application to the back of the eye.
  • the specificity of RNAi for the inhibition of genes expressed specifically in target cells minimizes the risk of unwanted side effects.
  • the present invention relates to a transgenic non-human animal which due to the presence of one or more dsRNA molecules, displays an aberrant expression of one or more target genes, and which obtained by the methods described above, especially when said animal reproduces a disorder of the CNS and/or the eye.
  • a method for the production of a transgenic non-human animal comprises introduction of a polynucleotide or targeting vector encoding said polypeptide into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom.
  • the non-human animal can be used in accordance with a screening method of the invention described herein. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
  • a general method for making transgenic non-human animals is described in the art, see for example WO 94/24274.
  • ES cells embryonal stem cells
  • Murine ES cells such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62: 1073-1085 (1990)) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting.
  • ES lines include, but are not limited to, the E14 line (Hooper et al., Nature 326: 292-295 (1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87: 27-45 (1985)), the CCE line (Robertson et al., Nature 323: 445-448 (1986)), the AK-7 line (Zhuang et al., Cell 77: 875-884 (1994)).
  • E14 line Hooper et al., Nature 326: 292-295 (1987)
  • D3 line Doetschman et al., J. Embryol. Exp. Morph. 87: 27-45 (1985)
  • the CCE line Robot et al., Nature 323: 445-448 (1986)
  • the AK-7 line Zhuang et al., Cell 77: 875-884 (1994)
  • the blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant nonhuman females and are born as chimeric mice.
  • the resultant transgenic mice are chimeric for cells having either the recombinase or reporter loci and are backcrossed and screened for the presence of the correctly targeted transgene (s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozygous for either the recombinase or reporter locus/loci.
  • tissue specific see supra
  • developmental and/or cell regulated and/or inducible promoters which drive the expression of, e.g., an antisense or ribozyme directed against the RNA transcript encoding the target gene mRNA; see also supra.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
  • the expression of a mutant target gene may be controlled by such regulatory elements.
  • the presence of the transgenes in cells of the transgenic animals leads to to various physiological, developmental and/or morphological changes, preferably to conditions related to disorders of the CNS and/or eye such as those described above.
  • mammlian animals are preferred, especially mice and rats.
  • Corresponding animal systems that can be adapted in accordance with the present invention are known to person skilled in the art; see, e.g., molecular biological approaches to neurological disorders including knockout and transgenic mouse models described in Shibata et al., Neuropathology 22 (2002), :337-349.
  • the widely used zebra fish may also be used since this model system has also been shown to provide valuable predicitve results; see, e.g. Gerlai et al., Pharmacol. Biochem. Behav. 67 (2000), 773-782.
  • said transgenic non-human animal is used for a process in the discovery of drugs for the treatment of a disorder of the CNS and/or the eye.
  • Preferred non-human transgenic animals are mammals, for example mice. Particularly preferred are transgenic organisms, especially if the organism displays the phenotype of an eye disease.
  • the phenotype of a disease of the inner segment of the eye ball, a retinal disease and particularly a degenerative retinal disease is preferred.
  • the organism can be for example mouse, rat or zebra fish; se supra.
  • a preferred embodiment of this invention is a pharmaceutical composition useful for the treatment of disease as defined above, comprising a composition disclosed in the description of this invention. Also included in the embodiments of this invention is a diagnostic composition useful for detecting a gene or gene expression involved in diseases of the CNS and/or eye, comprising a composition as defined in the description or a cell, tissue or an organism described above.
  • dsRNA molecules of a length of 21 to 23 nucleotides are capable of, after systemic application, for example by intravenous injection, to cross the blood-retina barrier, and specifically inactivate target genes in the tissues of the back of the eye.
  • This overcoming the blood-retina barrier is all the more remarkable, because no experiment could demonstrate overcoming the blood-brain barrier by dsRNA so far.
  • the methods and uses of the invention explained below by means of examples, are thus suitable for the provision of animal models with which targets, the restricted function of which causing diseases of the eye, can be identified and validated.
  • RNAi RNAi
  • the dosage regimen of the pharmaceutical compositions in all of the above described methods and uses of the present invention will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 ⁇ g to 10 mg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 0.01 ⁇ g to 10 mg units per day.
  • the regimen is a continuous infusion, it should also be in the range of 0.01 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of nucleics acids is from approximately 10 6 to 10 12 copies of the nucleic acid molecule.
  • compositions of the invention are administered to an individual in an effective dose sufficient to treat or diagnose disorders in which modulation of a target gene or gene product is indicated.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as by intracoronary, intraperitoneal, subcutaneous, intravenous, transdermal, intrasynovial, intramuscular or oral routes. In addition, co-administration or sequential administration of other agents may be desirable.
  • a therapeutically effective dose refers to that amount of compounds described in accordance with the present invention needed to ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population.
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the present invention provides a method for the identification and isolation of a drug capable of specific modulation of the expression of a target gene in cells and/or tissues of the eye, comprising the steps:
  • test substances which can be tested and identified according to a method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs, aptamers or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra).
  • the test substances to be tested also can be so called “fast seconds” of known drugs.
  • the invention also relates to further contacting the test cells with a second test substance or mixture of test substances in the presence of the first test substance.
  • the present invention provides convenient in vivo assays for identifying and obtaining drugs capable of modulating the gene activity, thereby being usefil as a therapeutic agent for the treatment of diseases related to CNS disorders including (e.g.) Schizophrenia, Parkinson's Disease, Alzheimer's Disease, and eye diseases such as those described above.
  • the present invention provides also a use for compounds which have been known in the art, properly also known to be able to modulate target gene activity but which hitherto have not been suggested for medical use because of the lack of knowledge of phenotypic responses of an organism evoked by target gene activity or the lack of it.
  • One embodiment of this invention comprises a method for the production of a drug or prodrug identified by such a screening as a modulator or a derivative thereof, particularly if the substance has hitherto not been known as a drug for the treatment of a disorder of the CNS or the eye.
  • Substances are metabolized after their in vivo administration in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459).
  • a corresponding formulation as a pro-drug can be used which is converted into its active form in the patient by his/her metabolism.
  • Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329.
  • the present invention relates to the use of a compound identified, isolated and/or produced by any of these methods for the preparation of a composition for the treatment of said CNS and eye disorders.
  • a method for treatment the identified substance or the composition containing it can be administered to a subject suffering from such a disorder.
  • Compounds identified, isolated and/or produced by the method described above can also be used as lead compounds in drug discovery and preparation of drugs or prodrugs.
  • Methods for the lead generation in drug discovery also include using proteins and detection methods such as mass spectrometry (Cheng et al. J. Am. Chem. Soc. 117 (1995), 8859-8860) and some nuclear magnetic resonance (NMR) methods (Fejzo et al., Chem. Biol. 6 (1999), 755-769; Lin et al., J. Org. Chem. 62 (1997), 8930-8931). They may also include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, J. Med. Chem. 41 (1993), 2553-2564, Kubinyi, Pharm.
  • QSAR quantitative structure-action relationship
  • the present invention also relates to the use of a component selected from the group consisting of a composition, nucleic acid, non-human organism, host cell, cell line, tissue, organ, drug, carrier and/or vector for the specific modulation of expression of one or more target genes in cells and/or tissue of the CNS and/or eye, wherein said component comprises one or more dsRNA molecules which are applicable outside the blood-brain barrier or the retinal region of the blood-retina barrier.
  • a component selected from the group consisting of a composition, nucleic acid, non-human organism, host cell, cell line, tissue, organ, drug, carrier and/or vector for the specific modulation of expression of one or more target genes in cells and/or tissue of the CNS and/or eye, wherein said component comprises one or more dsRNA molecules which are applicable outside the blood-brain barrier or the retinal region of the blood-retina barrier.
  • the invention further relates to the use of the any one of these methods, cells or non-human organism in drug discovery or target gene isolation and/or validation as well as the use of RNA interference and the nucleic acid, non-human organism, host cell, cell line, tissue, organ, carrier and/or vector utilized for the diagnosis and/or therapy of disorders related to the CNS and/or eye.
  • kits containing oligonucleotides, dsRNA or vectors may be prepared. Such kits are used to detect for example the function of a target gene. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, diagnostic applications, and epidemiological studies in accordance with the above-described methods of the present invention.
  • the recombinant RNA molecules for example lend themselves to the formulation of kits suitable for the detection and typing of the target gene.
  • a kit would typically comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant protein or antibodies suitable for detecting the expression or activity of the target gene or gene product.
  • the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
  • Literature regarding the materials, methods, applications and components, which can be used in accordance with the invention may be obtained from public libraries and data bases, for example by using electronic devices.
  • the public data base ‘Medline’ may for instance be used, which is supported by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health.
  • Other data bases and Internet addresses such as the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL), are known to the person skilled in the art, and can be found by using Internet search engines.
  • EBI European Bioinformatics Institute
  • EBL European Molecular Biology Laboratory
  • a survey of patent information in biotechnology and a summary of relevant sources for patent information, which are useful for a retrospective search and current awareness are described in Berks, TIBTECH 12 (1994), 352-364.
  • the present invention may be carried out by making use of conventional techniques of cell biology, cell culture, molecular biology, transgenetic biology, microbiology, recombinant DNA and RNA technology, which belong to the skill of the person skilled in the art.
  • conventional techniques of cell biology, cell culture, molecular biology, transgenetic biology, microbiology, recombinant DNA and RNA technology which belong to the skill of the person skilled in the art.
  • DNA Cloning Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No.
  • FIG. 1 eGFP-expression in retina and retinal pigment epithet (RPE) of systemically dsRNA-treated FVB.CG-TG(GFPU)5NAGY mice.
  • the figure shows eGFP-expression in eye paraffin sections of dsRNA-treated FVB.Cg-Tg(GFPU)5Nagy mice.
  • eGFP green fluorescent protein
  • RPE retinal pigment epithelium
  • FVB.Cg-Tg(GFPU)5Nagy The Jackson Laboratory
  • dsRNA of the target gene eGFP in the mouse animal model, during which the optimal dsRNA concentration for post transcriptional gene silencing on systemic application is to be determined (experimental procedure 1, results see table 1 and FIG. 1 ).
  • the procedure involves the in vivo treatment of transgenic mice, which express the enhanced form of green fluorescent protein (eGFP) in their body cells, by systemic application of dsRNA oligoribonucleotide molecules against the target gene eGFP.
  • Control animals are also treated systemically with non-silencing dsRNA molecules.
  • the animals not under analgesic or anesthetic influence receive daily i.v.
  • tail vein injections (1 st day of treatment: day 0, final day of treatment: day 20) of 100 or 200 ⁇ g eGFP-specific dsRNA/kg body weight (BW) and the control group of 200 ⁇ g non-silencing dsRNA/kg BW.
  • a control group of animals treated with buffer (daily i.v. injection of 0.1 ml buffer into the tail vein) is also kept.
  • Each group of experimental animals consists of 8 animals, the maximum injection volume/injection being 0.1 ml. On day 21, the animals are sacrificed by CO 2 inhalation.
  • eGFP fluorescence fluorescence microscopic evaluation
  • eGFP-specific immunofluorescence staining fluorescence microscopic evaluation
  • the procedure involves the in vivo treatment of transgenic mice, which express the enhanced form of green fluorescent protein (eGFP) in their body cells, by systemic application of dsRNA oligoribonucleotide molecules against the target gene eGFP.
  • the control animals are also treated systemically with non-silencing dsRNA molecules.
  • the animals not under analgesic or anesthetic influence receive a single i.v. tail vein injection on day 0 of 200 ⁇ g eGFP-specific dsRNA/kg body weight (BW) and the control group of 200 ⁇ g non-silencing dsRNA/kg BW.
  • Each group of experimental animals consists of 8 animals, the maximum injection volume/injection being 0.1 ml.
  • the animals are sacrificed by CO2 inhalation on day 2, 3, 5, and 10 after i.v. injection.
  • eGFP fluorescence fluorescence microscopic evaluation
  • eGFP-specific immunofluorescence staining fluorescence microscopic evaluation
  • Example procedure 3 describes specific post transcriptional gene silencing by dsRNA of the target gene eGFP in the mouse animal model, during which the optimal dsRNA concentration for post transcriptional gene silencing on local (retrobulbar) application is to be determined (Experimental procedure 3).
  • the procedure involves the in vivo treatment of transgenic mice, which express the enhanced form of green fluorescent protein (eGFP) in their body cells, by retrobulbar application of dsRNA oligoribonucleotide molecules against the target gene eGFP. Control animals also receive a retrobulbar injection with non-silencing dsRNA molecules or buffer.
  • eGFP green fluorescent protein
  • the animals under analgesic and anesthetic influence receive a single retrobulbar injection (1 st day of treatment: day 0) of 200 ⁇ g eGFP-specific dsRNA/kg body weight (BW) and the control groups of 200 ⁇ g non-silencing dsRNA/kg BW or buffer.
  • Each group of experimental animals consists of 3 -8 animals, the maximum injection volume/injection being 0.005 ml.
  • the retrobulbar dsRNA injection is carried out both on the left and right eye. On day 3 or day 6, the animals are sacrificed by CO 2 inhalation.
  • eGFP fluorescence fluorescence microscopic evaluation
  • eGFP-specific immunofluorescence staining fluorescence microscopic evaluation
  • the procedure involves the in vivo treatment of transgenic mice, which express the enhanced form of green fluorescent protein (eGFP) in their body cells, by retrobulbar injection of dsRNA oligoribonucleotide molecules against the target gene eGFP. Control animals also receive retrobulbar application of non-silencing dsRNA molecules or buffer.
  • eGFP green fluorescent protein
  • the animals under analgesic and anesthetic influence receive a single retrobulbar injection on day 0 of 200 ⁇ g eGFP-specific dsRNA/kg body weight (BW) and the control groups of 200 ⁇ g non-silencing dsRNA/kg BW or buffer.
  • Each group of experimental animals consists of 8 animals, the maximum injection volume/injection being 0.005 ml.
  • the retrobulbar dsRNA injection is carried out both on the left and right eye. The animals are sacrificed by CO 2 inhalation on day 2, 3, 5, and 10 after retrobulbar injection.
  • eGFP fluorescence fluorescence microscopic evaluation
  • eGFP-specific immunofluorescence staining fluorescence microscopic evaluation
  • Example procedure 5 describes specific post transcriptional gene silencing by dsRNA of the target gene eGFP in the mouse animal model, during which the optimal dsRNA activity for post transcriptional gene silencing on repeated local (retrobulbar) application is to be determined (Experimental procedure 5).
  • the procedure involves the in vivo treatment of transgenic mice, which express the enhanced form of green fluorescent protein (eGFP) in their body cells, by retrobulbar application of dsRNA oligoribonucleotide molecules against the target gene eGFP. Control animals also receive a retrobulbar injection of non-silencing dsRNA molecules or buffer.
  • eGFP green fluorescent protein
  • the animals under analgesic and anesthetic influence receive a retrobulbar injection on days 0, 7 and 14 (1 st day of treatment: day 0, final day of treatment: day 14) of 200 ⁇ g eGFP-specific dsRNA/kg body weight (BW) and the control group of 200 ⁇ g non-silencing dsRNA/kg BW or 0.005 ml buffer.
  • Each group of experimental animals consists of 8 animals, the maximum injection volume/injection being 0.005 ml.
  • the retrobulbar dsRNA injection is carried out both on the left and right eye. On day 15, the animals are sacrificed by CO 2 inhalation.
  • eGFP fluorescence fluorescence microscopic evaluation
  • eGFP-specific immunofluorescence staining fluorescence microscopic evaluation
  • sequences of the type AA(N19)TT were selected from the sequence of the target mRNA, in order to obtain 21 nucleotide (nt) long sense and antisense strands with symmetrical 3′-overhangs of two nucleotides in length.
  • nt nucleotide
  • 2′-deoxy-thymidine was used instead of uridine.
  • the chosen dsRNA sequences are tested against the mouse genome in a BLAST analysis.
  • the 21-nt RNA molecules are synthesized chemically and purified.
  • dsRNA molecules are precipitated from ethanol and resuspended in sterile buffer (100 mM potassium acetate, 30 mM HEPES-KOH, 2 mM magnesium acetate, pH 7.4). The integrity and double strand character of the dsRNA are verified by gelelectrophoresis. Alternatively, the dsRNA molecules are obtained from commercial suppliers. The sequences of the target genes and the corresponding dsRNA molecules are as follows:
  • GFP dsRNA DNA target sequence 5′ G CAA GCT GAC CCT GAA GTT CA (SEQ ID NO 1) Coding region, 121-141 relative to the first nucleotide of the start codon (Acc. No. U55761) dsRNA (sense) 5′ r(GCA AGC UGA CCC UGA AGU U) (SEQ ID NO 2) dsRNA (antisense) 5′ r(AA CUU CAG GGU CAG CUU GC) (SEQ ID NO 3)
  • Non-Silencing dsRNA, Control DNA target sequence 5′ AATTCTCCGAACGTGTCACGT (SEQ ID NO 4) dsRNA (sense) 5′ r(UUCUCCGAACGUGUCACGU)d(TT) (SEQ ID NO 5) dsRNA (antisense) 5′ r(ACGUGACACGUUCGGAGAA)d(TT) (SEQ ID NO 6)
  • Analgesia and Anesthesia of the Mice 5′ AATTCTCCGAACGTGTCACGT (SEQ ID NO 4) dsRNA (sense) 5′ r(UUCUCCGAACGUGUCACGU)d(TT) (SEQ ID NO 5) dsRNA (antisense) 5′ r(ACGUGACACGUUCGGAGAA)d(TT) (SEQ ID NO 6)
  • Analgesia and Anesthesia of the Mice 5′ AATTCTCCGAACGTGTCACGT (SEQ ID NO 4) dsRNA (sense) 5′
  • the animals are immobilized and the dsRNAs are injected i.v. in the tail vein (maximal injection volume: 0.1 ml), where analgesia or anesthesia are refrained from, since this would put more stress on the animals than the i.v. injection itself.
  • maximal injection volume: 0.1 ml the animals are however subjected to short-term isofluorane inhalation anaesthesia and provided with Metamizole sodium for analgesic purposes.
  • the animals are then kept in their accustomed animal cage surroundings. After completion of in vivo diagnosis (the end of each animal experiment is stated respectively in example 1 -5) the animals are killed by CO 2 inhalation, enucleated and the eyes are studied histologically (immunohistology).
  • the eyes are fixed in 4% formalin/PBS solution for 24 hours.
  • the fixed samples are subsequently dehydrated in a series of increasing alcohol and embedded in paraffin.
  • standard 5 to 12 ⁇ m serial slices are produced, stretched in a heated water bath and transferred to a polylysin-coated cover slip.
  • the sections are then dried in an incubator for 2 hours at a temperature of 52° C.
  • the dried sections are deparaffinated in xylol, transferred to a decreasing series of alcohol followed by Tris/HCl pH 7.4.
  • the sections are incubated for 2 hours with primary anti-eGFP antiserum (polyclonal goat anti-eGFP antiserum, Santa Cruz No.
  • Detection occurs by means of immunofluorescence staining by using a Cy2-conjugated rabbit anti-goat IgG (Dianova, No. 305-225-045). The samples are embedded and then mounted for microscopy with an Eclipse TE-2000-S microscope (Nikon), equipped with a 20 ⁇ and 40 ⁇ /1.3 objective. The spontaneous, eGFP-specific fluorescence in deparaffinated, untreated sections is analyzed using a fluorescence microscope.
  • Experimental procedure 1 Systemic siRN application. Determination of optimal dsRNA concentration for post transcriptional gene silencing. Group Substance Number of animals Control animals Buffer 8 Negative control non-silencing dsRNA 8 200 ⁇ g dsRNA/kg BW 200 ⁇ g dsRNA/kg eGFP-specific dsRNA 8 BW 100 ⁇ g dsRNA/kg eGFP-specific dsRNA 8 BW Animals per 32 experiment
  • Experiment Number of Group Substance ended after animals Negative control (8 non-silencing 2, 3, 5, 10 days 32 animals per point in dsRNA time) 200 ⁇ g dsRNA/kg eGFP-specific 2 days 8 BW dsRNA 200 ⁇ g dsRNA/kg eGFP-specific 3 days 8 BW dsRNA 200 ⁇ g dsRNA/kg eGFP-specific 5 days 8 BW dsRNA 200 ⁇ g dsRNA/kg eGFP-specific 10 days 8 BW dsRNA Animals per 64 experiment
  • Negative control (8 non- non-silencing 2, 3, 32 animals per point in si- dsRNA 5, 10 days time) lencing dsRNA 200 ⁇ g dsRNA/kg eGFP- eGFP-specific 2 days 8 BW specific dsRNA dsRNA 200 ⁇ g dsRNA/kg eGFP- eGFP-specific 3 days 8 BW specific dsRNA dsRNA 200 ⁇ g dsRNA/kg eGFP- eGFP-specific 5 days 8 BW specific dsRNA dsRNA 200 ⁇ g dsRNA/kg eGFP- eGFP-specific 10 days 8 BW specific dsRNA dsRNA Animals per 64 experiment
  • Experimental procedure 5 Repeated retrobulbar dsRNA injection for the determination of post transcriptional gene silencing.
  • Cited literature the disclosure content of which is hereby incorporated by reference in the present description:
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WO2003087367A3 (fr) 2004-04-29
US8202845B2 (en) 2012-06-19
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AU2003224087B2 (en) 2009-03-05
US20060003915A1 (en) 2006-01-05
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EP1495120A2 (fr) 2005-01-12
AU2003222820A1 (en) 2003-10-27
AU2009200699A1 (en) 2009-03-12
CA2482903A1 (fr) 2003-10-23
CA2482904A1 (fr) 2003-10-23
WO2003087368A2 (fr) 2003-10-23
WO2003087368A3 (fr) 2004-04-29
AU2003224087A1 (en) 2003-10-27
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US20120277288A1 (en) 2012-11-01
US20110021605A1 (en) 2011-01-27

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