TARGETING CELLS WITH ALTERED MICRORNA EXPRESSION
The present invention relates to a method of modulating development of a cell. The method includes the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to modulate development of the cell.
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This application claims priority to International Patent Application No. PCT/AU2006/000750, International Filing Date Jun. 2, 2006, entitled Targeting Cells with Altered MicroRNA Expression; and claims priority to U.S. Provisional Application Ser. No. 60/687,547 filed Jun. 3, 2005, both of which are hereby incorporated by reference.
FIELD OF THE INVENTIONThe present invention relates to a method of modulating the development of a cell with altered microRNA activity, and to a method of preventing and/or treating a disease, condition or state associated with altered microRNA activity.
The present invention also relates to nucleic acids having a binding site for a microRNA, cells and animals including such nucleic acids, and compositions including the nucleic acids.
BACKGROUND OF THE INVENTIONTargeted gene expression is one of the most difficult and important goals in the development of effective therapies for a variety of disorders, including cell proliferative disorders such as cancer. The ultimate aim of such therapies is the provision of controlled, sustained, and site-specific expression of a therapeutic agent such that surrounding healthy tissue remains relatively unaffected by the effects of the therapeutic agent.
However, one major limitation of current gene therapy protocols has been the inability to control expression of the therapeutic gene, and in particular, the inability to restrict expression of the delivered gene to the desired tissue or type of cell. In this regard, although tissue specific promoters may be adequate to achieve specific expression of an agent in some target tissues, for diseases such as cancer they have proved to be of less value as non-diseased cells may also express from the promoter. Accordingly, there is a need to identify new methods of targeting expression of therapeutic nucleic acids.
Recently, short 20-22 nucleotide RNA molecules known as microRNAs (miRNAs) have been identified as regulating gene expression in variety of eukaryotic systems. In Caenorhabditis elegans, miRNAs coordinate the transitions between stages of larval development by regulating the translation of heterochromic genes. A specific miRNA in Arabidopsis has been shown to direct the cleavage of transcripts encoding several putative transcription factors. The Drosophila bantam gene encodes a miRNA that regulates cell proliferation and the proapoptotic gene hid. More recently, human miR143 has been shown to regulate adipocyte differentiation.
miRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the Dicer family of RNase III enzymes. They share this process with an experimental system, RNA interference (RNAi), which may be used to silence the expression of endogenous genes in eukaryotic cells. The products of Dicer cleavage are short dsRNA molecules, one strand of which is retained in a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). The retained RNA acts as a guide to target this complex to a complementary mRNA sequence which is inactivated either by cleavage or translational interference, depending on the degree of complementarity between the miRNA and its target.
The present invention relates to a method of modulating the development of cells with altered microRNA levels, and arises from the recognition that some diseased cells have altered expression levels of endogenous microRNAs and that expression of therapeutic nucleic acids may be effectively targeted to such cells by exploiting the altered expression of the microRNAs in these cells.
A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.
SUMMARY OF THE INVENTIONThe present invention provides a method of modulating development of a cell, the method including the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to modulate development of the cell.
The present invention also provides a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a binding site for a microRNA.
The present invention also provides a nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed and/or has differential activity.
The present invention also provides a cancerous cell including an exogenous nucleic acid including a binding site for a microRNA, wherein the cancerous cell has a reduced activity and/or concentration of the microRNA as compared to a similar non-cancerous cell.
The present invention also provides an animal including cancerous cells, the cancerous cells including an exogenous nucleic acid including a target site for binding of a microRNA.
The present invention also provides a method of preventing and/or treating a disease, condition or state associated with target cells in a subject, the method including the step of introducing into cells in the subject a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a target site for binding of the microRNA, wherein the activity of the microRNA in the target cells results in a level of activity of the nucleic acid sufficient to modulate development of the target cells in the subject.
The present invention also provides a method of detecting altered microRNA activity and/or concentration in a cancerous or pre-cancerous cell, the method including the steps of:
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- determining the level of expression of a reporter nucleic acid in the cancerous or pre-cancerous cells and determining the level of expression of a reporter nucleic acid in non-cancerous cells; and
- detecting a reduced activity of the microRNA in the cancerous cells by an increase in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.
The present invention also provides a method of modulating the concentration of a nucleic acid expressed in a cancerous cell, the cancerous cell having an altered activity and/or concentration of a microRNA as compared to a similar non-cancerous cell, the method including the step of introducing a target site for binding of the microRNA into the nucleic acid to be expressed in the cell.
The present invention arises from the recognition that many cells, including cancerous cells, have altered expression levels of endogenous microRNAs, and that expression of therapeutic nucleic acids may be more effectively targeted to such cells by exploiting the altered expression of the microRNAs in these cells.
In addition, it has also been recognised that the levels of some microRNAs are modulated during differentiation of cells or during their normal developmental programme, and accordingly, expression of nucleic acids may also be targeted to these cells at specific times in their developmental programme and/or at times when the level of the microRNAs changes.
Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined.
The term “development” as used throughout the specification in relation to the development of a cell is to be understood to mean the continuance of a cell in its current state or the continuance of a cell in its normal developmental and/or metabolic program.
In this regard, modulation of the development of a cell may, for example, result in an inhibition or cessation of cell growth, cell death, or an alteration in the normal developmental pathway that a cell undergoes. Alternatively, modulation of the development of a cell may result, for example, in increased cell survival or rescue, or increased cell proliferation.
The term “modulate” or variants thereof as used throughout the specification is to be understood to mean any alteration (increase or decrease) in the activity of a process. For example, alteration may result in activation of a process, inhibition of a process, a change in the timing of a process or a change in probability that a process may occur.
The term “nucleic acid” as used throughout the specification is to be understood to mean to any oligonucleotide or polynucleotide. The nucleic acid may be DNA or RNA and may be single stranded or double stranded. The nucleic acid may be any type of nucleic acid, including a nucleic acid of genomic origin, cDNA origin (ie derived from a mRNA), derived from a virus, or of synthetic origin. It will be appreciated that a nucleic acid with the capacity to modulate development of a cell is a nucleic acid that in a particular cell either inhibits or promotes development of the cell.
In this regard, an oligonucleotide or polynucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups to facilitate the function of the nucleic acid. The oligonucleotide or polynucleotide may be modified at any position on its structure with constituents generally known in the art. For example, an oligonucleotide may include at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyliydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.
The oligonucleotide or polynucleotide may also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. In addition, the oligonucleotide or polynucleotide may include at least one modified phosphate backbone, such as a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or any analogue thereof.
The term “subject” as used throughout the specification is to be understood to mean any multicellular organism, including an animal or human subject. For example, the subject may be a mammal such as a primate, a livestock animal (eg. A horse, a cow, a sheep, a pig, a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
The term “variant” as used throughout the specification is to be understood to mean an amino acid sequence of a polypeptide or protein that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid (e.g., replacement of leucine with isoleucine). A variant may also have “non-conservative” changes (e.g., replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids. The term also includes within its scope any insertions/deletions of amino acids for a particular polypeptide or protein. A “functional variant” will be understood to mean a variant that retains the functional capacity of a reference protein or polypeptide.
As mentioned above, in one form the present invention provides a method of modulating development of a cell, the method including the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to modulate development of the cell.
The present invention arises from the recognition that some diseased cells have altered expression levels of endogenous microRNAs, and that expression of nucleic acids may be effectively targeted to these cells by exploiting the altered expression of the microRNAs in the cells.
It will be appreciated that in the case of a reduced miRNA expression, the expression of the nucleic acids is in fact effectively de-targeted.
Thus, the present invention has application in fields such as genetic engineering and therapeutic gene transfer.
The present invention is suitable for example for targeting expression of a cytotoxic nucleic acid to a diseased cell so as to ablate the cell, or alternatively, for targeting expression of a therapeutic nucleic acid to a diseased cell to improve one or more characteristics of the cell (eg for gene therapy purposes).
For example, previous studies of microRNAs differentially expressed between colonic adenocarcinoma cells and matched normal mucosa have identified two microRNAs, miR-143 and miR-145, that show significantly reduced levels of the fully processed miRNA in tumors compared to normal tissues. These miRNAs are produced from hairpin precursor (pre-miRNAs) that are cleaved to the shorter mature miRNA, as shown in
The alteration in the level and/or activity of the one or more microRNAs in the cell may be a constitutive alteration, or alternatively, may be an alteration that occurs at a specific point(s) in the developmental programme of the cell. For example, the present invention is suitable for modulating development of a cancer cell that shows a reduced constitutive expression and/or activity of a particular miRNA, for modulating development of a cell infected with a virus which causes altered expression and/or activity of a miRNA, or for modulating development of an embryonic or adult stem cell that modulates the activity of a specific miRNA at specific stages of its developmental programme.
Confirmation that a cell has an altered activity and/or expression of a specific miRNA may be achieved by a suitable method known in the art, such as Northern analysis, reverse transcription PCR (RT-PCR) or RNase protection.
Alternatively confirmation that a cell has an altered activity and/or expression of a specific miRNA may be achieved by way of expression of a reporter gene linked to a target site for the particular miRNA. In this case, the level of expression of the reporter gene will inversely reflect the activity and/or expression level of the miRNA in the cell.
Methods for the construction of reporter genes including a target site for a miRNA are known in the art. For example, a target site may be cloned into the 3′UTR of GFP, and the construct introduced into the cell. Methods for the cloning of nucleic acid sequences are as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratroy Press, New York. (1989).
As discussed previously, a reduced activity and/or expression of a miRNA may be correlated with a disease, condition or state. For example, cancerous cells often have a reduced level of one or more specific miRNAs. Indeed, for many cancers a reduced level of miRNA activity and/or expression is associated with the progression of a normal cell to a cancerous cell. Thus, the cell in this form of the present invention may be a cancerous cell or a pre-cancerous cell.
Examples of cancerous cells that show a reduced activity and/or expression of a miRNA include colorectal cancer cells, lung cancer cells, thymus cancer cells, bladder cancer cells, breast cancer cells and prostate cancer cells.
Examples of cancerous cells generally include cells associated with the cancers such as bladder cancer, bone cancer, brain tumours, breast cancer, cervical cancer, colorectal cancer including cancer of the colon, rectum, anus, and appendix, cancer of the esophagus, Hodgkin's disease, kidney cancer, cancer of the larynx, leukemia, liver cancer, lung cancer, lymphoma, melanoma, moles and dysplastic nevi, multiple myeloma, muscular cancer, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, cancer of the pancreas, prostate cancer, skin cancer, stomach cancer, testicular cancer, teratoma, thyroid cancer, and cancer of the uterus. The present invention also includes pre-cancerous cells, including for example, pre-cancerous cells associated with the aforementioned cancers.
In one form, the cell in the various forms of the present invention is cancerous cell from a colorectal cancer or colorectal polyp, including a cancerous cell from a colorectal adenocarcinoma or an adenomatous polyp.
However, as discussed above, the cell in the various forms of the present invention may also be for example an embryonic stem (ES) cell or an adult stem cell. In this regard, microRNAs have been identified in the mouse for which expression is repressed as the ES cells differentiate into embryoid bodies and is undetectable in adult organs, indicating that these miRNAs may have a role in the maintenance of the pluripotent cell state and in the regulation of early mammalian development.
Examples of other cells suitable for the various forms of the present invention include haemopoietic cells including haemopoietic precursor cells, adipocytes, chronic lymphocytic B cells, neuronal cells, sperm cells or sperm producing cells, pancreatic endocrine cells including pancreatic islet cells, and virally infected cells including EBV, HIV, Hepatitis and Herpes infected cells.
It will be appreciated that the cell for which development is modulated in the various forms of the present invention may be an isolated cell in vitro, or a cell present in a biological system such as a cell in an organ or tissue, or a cell present in an entire organism (eg animal or human subject). In this regard, the term “biological system” is to be understood to mean any multi-cellular system, and includes isolated groups of cells to whole organisms.
Thus, the present invention may be used to modulate the development of a target cell in a biological system.
Accordingly, in another form the present invention provides a method of modulating development of a target cell in a biological system, the method including the step of introducing into a target cell in the biological system a nucleic acid with the capacity to modulate development of the cells in the biological system, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the target cell results in a level of activity and/or concentration of the nucleic acid in the target cell sufficient to modulate the development of the target cell.
In one form, the biological system is an animal or human subject, including an animal or human that is susceptible to, or suffering from, a disease, condition or state associated with altered microRNA activity and/or expression.
The present invention is therefore suitable for preventing and/or treating a disease, condition or state associated with target cells having an altered activity of a microRNA in a subject.
Accordingly, in another form the present invention provides a method of preventing and/or treating a disease, condition or state associated with target cells in a subject, the method including the step of introducing into cells in the subject a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a target site for binding of the microRNA, wherein the altered activity of the microRNA in the target cells results in a level of activity of the nucleic acid sufficient to modulate development of the target cells in the subject.
Examples of diseases, conditions or states associated with altered microRNA activity and/or expression in the various forms of the present invention are as previously discussed, including cancers such as colorectal cancer, lung cancer, thymus cancer, bladder cancer, breast cancer and prostate cancer; human B cell chronic lymphocytic leukemia; B cell (Burkitt) Lymphoma; disorders of pancreatic endocrine cells such as diabetes; diseases and conditions associated with virally infection of cells such as EBV, HIV, Hepatitis and Herpes infected cells; 5q-myelodysplastic syndrome (macrocytic anaemia); diseases and conditions associated with haemopoietic dysfunction; autoimmune and inflammatory diseases (eg Crohn's); fragile X mental retardation; Di George syndrome; Wilms tumour; a disease or condition associated with adipocyte dysfunction; a disease that can be treated with embryonic or adult stem cells; and a disease or condition associated with sperm producing cells.
It will be appreciated that an amount of the nucleic acid effective to provide a therapeutic or desired effect will be introduced/administered to the cell, biological system or subject in the various relevant forms of the present invention.
For example, methods for introducing exogenous DNAs into cells are as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989).
The present invention may be used to either inhibit the development of target cells by use of a nucleic acid that inhibits the development of target cells, or alternatively, to promote the development of target cells by the use of a nucleic acid that promotes the development of target cells. For example, the present invention may be used to selectively ablate or rescue cells. Methods for determining whether the development of a cell have been inhibited or promoted are known in the art.
Thus, in one form of the present invention the development of a cell may be inhibited.
In this case, the nucleic acid introduced into the cell will have the capacity to inhibit development of the cell. As the nucleic acid will include one or more target sites for binding of one or more miRNAs, a reduction in activity and/or expression of the one or more miRNAs in a cell will result in an increased expression of the nucleic acid (as compared to a similar cell which does not have a reduced activity of the one or more miRNAs) and consequently result in a level of expression of the nucleic acid sufficient to inhibit development of the cell.
Accordingly, in another form the present invention provides a method of inhibiting development of a cell, the cell having a reduced activity and/or concentration of a microRNA, the method including the step of introducing into the cell a nucleic acid with the capacity to inhibit development of the cell, the nucleic acid including a target site for binding of the microRNA, wherein the reduced activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to inhibit development of the cell.
In one form, the nucleic acid with the capacity to inhibit development of a cell is a nucleic acid with cytotoxic or cytostatic activity, or a nucleic acid encoding a suicide gene. In this case, the introduction of such a nucleic acid including one or more target sites for binding of one or more miRNAs into cells will result in an inhibition of development in those cells that have a reduced activity and/or expression of the one or more miRNAs. In cells that do not have a reduced activity and/or expression of the one or more miRNAs, the miRNAs will act to reduce the expression of the nucleic acid sufficiently that cell development is not inhibited, or at least less inhibited than the cells with the reduced activity and/or expression of the miRNA.
Thus, the present invention allows the selective inhibition of cells with a reduced activity and/or expression of a specific miRNA. In one form, the present invention may be used to selectively ablate target cells with a reduced activity and/or expression of a specific miRNA.
Examples of cytotoxic or suicide genes are generally as described in Greco and Dachs (2001) J Cell Physiol. 187(1):22-36 and include herpes simplex thymidine kinase [NC—001798:c48000-46870 (HSV2 genome)] [gi| 9629267], E. coli cytosine deaminase [NC—000913: 355395-356678 (E. coli genome)] [gi| 49175990], bacterial E. coli nitroreductase [NC—000913: c603994-604647] [gi| 49175990, P. aeruginosa carboxypeptidase G2 [AE004706.1: 3474-4712], horseradish peroxidase [X57564] [gi|16095], and E. coli purine nucleoside phosphorylase [U00096.2: 4618906-4619625 (E. coli genome)] [gi| 48994873]. It will also be appreciated that active fragments of these genes may be used, or a functional variant may be used.
In one form, the nucleic acid with the capacity to inhibit development of the cell is selected from one of the group consisting of herpes simplex thymidine kinase, a purine nucleoside phosphorylase and a cytosine deaminase.
The nucleotide sequence of the HSV thymidine kinase gene is designated SEQ ID NO. 151.
The nucleotide sequence of E. coli cytosine deaminase is designated SEQ ID NO. 152. In this case, it should be noted that the initiation codon is modified from GTG to ATG for mammalian expression constructs.
An example of a plasmid encoding the HSV thymidine kinase gene and the miRNA target sequence for miR-145 is plasmid pMM-TK/miR-Targ, which is shown in
An example of a plasmid encoding the E. coli cytosine deaminase and the miRNA target sequence for miR-145 is plasmid pMM-CD/miR-Targ, which is shown in
In another form, the present invention allows the development of a cell to be promoted.
In this case, the nucleic acid introduced into the cell will have the capacity to promote development of the cell. As the nucleic acid will include one or more target sites for binding of one or more miRNAs, a reduction in activity and/or expression of the one or more miRNAs will result in an increased expression of the nucleic acid (as compared to a similar cell which does not have a reduced activity of the one or more miRNAs) and consequently result in a level of expression of the nucleic acid sufficient to promote development of the cell.
Accordingly, in another form the present invention provides a method of promoting development of a cell, the cell having a reduced activity and/or concentration of a microRNA, the method including the step of introducing into the cell a nucleic acid with the capacity to promote development of the cell, the nucleic acid including a target site for binding of the microRNA, wherein the reduced activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to promote development of the cell.
Examples of nucleic acids that have the capacity to promote development in a cell include therapeutic genes or a cytokine gene such as granulocyte macrophage-colony stimulating factor (GM-CSF; human: NM—000758), G-CSF (human: NM—000759; NM—172219; NM—172220), Interleukin 11 (human: NM—000641), and Tumour Necrosis Factor alpha (human: NM—000594). Thus, the nucleic acid may encode a cytokine, a therapeutic protein or an active fragment of a cytokine or a therapeutic protein.
In the case of a cytokine gene, the introduction of a nucleic acid encoding a cytokine into cells will result in a promotion of development in cells that have a reduced activity and/or expression of the one or more miRNAs. In this case, it will be appreciated that the cells will be susceptible to the effects of the cytokine. In cells that do not have a reduced activity and/or expression of the one or more miRNAs, the miRNAs will act to reduce the expression of the nucleic acid sufficiently that cell development is not promoted, or at least less promoted to an extent that is less than the cells with the reduced activity and/or expression of the miRNA.
Thus, the present invention allows the selective promotion of development of target cells with a reduced activity and/or expression of a specific miRNA.
It will be appreciated, that the expression of the nucleic acid with the capacity to modulate development of a cell will require various regulatory elements known in the art for the expression of the inserted nucleic acids in particular cell types, such as promoters for driving the expression of an inserted nucleic acid in a particular cell, poly A signals for efficient polyadenylation of mRNA transcribed from inserted nucleic acids, or other regulatory elements to control translation, transcription or mRNA stability.
Depending upon the cell type to be modulated, the promoter driving the expression may be a constitutive promoter, an inducible promoter or a cell or tissue specific promoter.
Constitutive mammalian promoters include hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, phosphoglycerate kinase (which has intrinsic bi-directional activity) and β-actin. Exemplary viral promoters which function constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
Inducible promoters include synthetic promoters regulated by the TetO/TetR system and inducible promoters such as metallothionein promoter, which may be used to induce transcription in the presence of certain metal ions. Other inducible promoters are known in the art.
The tissue-specific promoter will depend upon the particular cell type. For example, promoters that allow expression in colon cancer cells include the regulatory sequences of human carcinoembryonic antigen (CEA) [accession:U17131; gi/967132].
Examples of microRNAs that are correlated with human cancer, or the progression of a normal cell to a cancerous cell in humans, are as follows (5′ to 3′):
An example of a microRNA that is associated with the regulation of insulin secretion is hsa-miR-375:
Based on experiments in mice, hsa-miR-181b-1 is likely to be involved in haemopoiesis (B lineage cell differentiation):
The present invention also provides orthologues of the above human miRNAs, which may identified by method known in the art. A database of miRNAs is found at the miRBase registry (http://microrna.sanger.ac.uk/sequences/).
Thus, the present invention specifically provides in its various forms the following microRNAs: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-155, hsa-miR-181b-1, hsa-miR-124a, hsa-miR-9 and corresponding orthologues from other species.
Methods for identifying a corresponding orthologue are known in the art, such as Weber M J. (2005) New human and mouse microRNA genes found by homology search. FEBS J. 272(1):59-73.
As described previously, miRNAs are small RNA molecules endogenously encoded in the genome of many species that regulate gene expression by binding to specific mRNAs. miRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the Dicer family of RNase III enzymes. The products of Dicer cleavage are short dsRNA molecules, one strand of which is retained in a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). The retained RNA acts as a guide to direct this complex to a target site in the mRNA which is then inactivated either by cleavage or translational interference, depending on the degree of complementarity between the miRNA and its target site.
Accordingly, the modulation of the development of a cell in the various forms of the present invention may be by way of cleavage (or lack of cleavage) of the nucleic acid with the capacity to modulate the development of the cell, and/or by way of translational interference (or lack of translational interference) of the nucleic acid with the capacity to modulate the development of the cell.
The target site in the various forms of the present invention may be a nucleotide sequence that has exact complementarity to the miRNA, or alternatively, be a target site with a reduced degree of complementarity. Methods for determining the extent of complementarity required for binding of a miRNA (so as to cleave the target or translationally interfere with the target) are known in the art, for example Lewis B P, Burge CB, Bartel D P., (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15-20 and Saetrom O, Snove O Jr, Saetrom P., (2005) Weighted sequence motifs as an improved seeding step in microRNA target prediction algorithms. RNA 11(7):995-1003.
The target site may be a natural target site or a non-naturally occurring target site. In the case of a target site occurring in a gene, it will be appreciated that the target site may be introduced into the nucleic acid with one or more addition nucleotides from the gene. For example, the target site may be introduced by way of using the entire 3′UTR of a gene having a suitable target site in that untranslated region of the mRNA.
The ability of a miRNA to bind to a target site and cleave the mRNA and/or interfere with translation may be confirmed experimentally by a suitable method known in the art. For example, the psiCHECK™2 Luciferase assay system (Promega) can be used to determine miRNA activity both in vitro and in vivo. Northern blot and RT-PCR analyses can also be used to determine cleavage of a target transcript, while Western blot analysis will detect reduced translation of encoded proteins.
In the case of the miRNAs discussed previously herein, complementary target sites for the binding of the miRNAs are as follows (5′ to 3′):
In the case of the nucleic acid molecule being a DNA, a person skilled in the art will appreciate that the above target sequences will have the U bases substituted for T bases.
The target site for binding of a microRNA in the various forms of the present invention may be a non-naturally occurring binding site for the particular miRNA, or alternatively, may be a target site present in a naturally occurring gene or mRNA, such as a target site found in the 3′UTR of a gene. In this regard, naturally and non-naturally occurring targets for a microRNA may be identified as described in Krek et al. (2005) Nature Genetics 37(5): 495-500.
For example, in the case of the miR-143 miRNA, Table 1 provides a listing of human mRNAs predicted to contain target sites for hsa-miR-143, using the method as described in Krek et al. (2005) Nature Genetics 37(5): 495-500, using default parameters.
In the case of the miR-145 miRNA, for example, Table 2 provides a listing of human mRNAs predicted to contain target sites for hsa-miR-145, using the method as described in Krek et al. (2005) Nature Genetics 37(5): 495-500, using default parameters.
A target site for the binding of the microRNA may be introduced into the nucleic acid with the capacity to modulate the development of cell at a suitable position in the nucleic acid. For example, in the case of the target site being introduced into a mRNA (by way, for example, of cloning the target site into the appropriate position in a plasmid encoding a gene to be transcribed), the target site(s) may be introduced into one or more of the 3′UTR, coding region and 5′UTR of the mRNA. Methods for the cloning of nucleic acid sequences are essentially as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratroy Press, New York. (1989).
For example, the target site for the miR-143 and/or miR-145 microRNAs may be cloned into the 3′UTR of the HSV thymidine kinase gene. This construct, when expressed in cells with a reduced activity and/or expression of the miR-143 or miR-145 microRNAs, will lead to selective ablation of these cells.
In the case of a non-naturally occurring target site, the target site for introduction into the nucleic acid may be produced by a method known in the art, such as chemical synthesis. For example, phosphorothioate oligonucleotides may be synthesized by the method as described in Stein et al. (1988) Nucl. Acids Res. 16: 3209. The target site may then be introduced into the nucleic acid by a method known in the art. For example, complementary oligonucleotides containing the binding site may be annealed and then introduced into the appropriate restriction site in a plasmid.
The nucleic acid with the capacity to modulate development of a cell may include more than one copy of a target site for binding of the miRNA. For example, the nucleic acid may contain 2, 3, 4 or more copies of a target site. Indeed, it is anticipated that multiple copies of a target site may provide a greater degree of control of the level of expression of the nucleic acid with the capacity to modulate development of the cell.
It will also be appreciated that the copies of the target site may be copies of the same or a similar target sequence, or one or more copies of a target sequence for one or more different microRNA(s).
The present invention also provides a cell including an exogenous nucleic acid including a binding site for a microRNA, or a cell including a nucleic acid with a non-naturally occurring binding site for a microRNA.
In one form, the cell is a cancerous or pre-cancerous cell with a reduced activity of a microRNA. Examples of cancerous and pre-cancerous cells are as previously discussed herein.
Accordingly, in another form the present invention provides a cancerous or pre-cancerous cell including an exogenous nucleic acid including a binding site for a microRNA, wherein the cancerous or pre-cancerous cell has a reduced activity and/or concentration of the microRNA as compared to a similar non-cancerous cell.
The cell, such as a pre-cancerous or cancerous cell, may be present in vitro or in vivo. For example, the cell may be an isolated cell in vitro, or a cell present in a biological system such as an organ, tissue, or an entire organism (eg an animal or human subject).
Thus, the present invention also provides an animal or human including non-cancerous cells and cancerous cells, the non-cancerous and cancerous cells both including and/or expressing an exogenous nucleic acid with a target site for binding of a microRNA.
Accordingly, in another form the present invention provides an animal including cancerous cells, the cancerous cells including an exogenous nucleic acid including a target site for binding of a microRNA.
In one form, the target site for binding of a microRNA is a target site for a microRNA that has reduced expression and/or activity in the cancerous cell, as compared to a similar non-cancerous cell. Examples of such binding sites are as previously herein discussed and include the binding site for the miR-143 and/or miR-145 microRNAs.
In one form, the exogenous nucleic acid has the capacity to modulate the development of a cell in the animal. Examples of such nucleic acids are as previously discussed herein.
The expression of an exogenous nucleic acid in a cell may be by way of introducing the nucleic acid into the cells by a method known in the art. Methods for introducing exogenous DNAs into prokaryotic and eukaryotic cells are essentially as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989).
In the case of an animal, the animal may also be a transgenic animal with the nucleic acid stably integrated into the genome of the cells of the animal. Methods for producing transgenic animals are known in the art.
The present invention also provides a nucleic acid that has the capacity to modulate development of a cell and which includes a binding site for a microRNA.
Nucleic acids in the various forms of the present invention may be produced by a method known in the art, such as cloning, in vitro transcription (for a RNA), chemical synthesis or any combination of such methods. The present invention also provides vectors including the nucleic acids of the present invention, and cells including the vectors and nucleic acids.
Examples of nucleic acids with the capacity to modulate the development of cells are as previously discussed herein. Thus, the nucleic acid may have the capacity to inhibit the development of a cell (eg cytostatic or cytotoxic activity), or alternatively, may have the capacity to promote development of a cell.
Accordingly, in another form the present invention provides a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a binding site for a microRNA. Examples of binding sites for microRNAs are as previously discussed herein.
In one form, the binding site for the microRNA is a binding site for a microRNA that has an altered activity and/or concentration in a cell, as compared to another cell.
Thus, the nucleic acid may include a binding for a microRNA that is differentially expressed and/or differentially active.
The nucleic acid may have the capacity to either inhibit or promote development of a cell. Examples of nucleic acids that have the capacity to modulate development are as discussed previously herein.
It will be appreciated that the nucleic acids of the present invention may be used in a composition for exposure to a cell, so as to introduce the nucleic acid into the cell, or a composition for administration to an animal or human subject, or be part of a vector, such as a viral vector, for introducing the nucleic acids into cells in vitro, or cells in a biological system, such as cells in an animal or human subject.
In this case, the nucleic acid may be used for example to inhibit the development of a cell(s) in an animal or human subject, such as ablating the cell(s) in an animal or human subject.
The nucleic acid in the various forms of the present invention may be an isolated nucleic acid. In this regard, the term “isolated” is to be understood to mean an entity, for example a nucleic acid, a polypeptide, or a cell, which is removed from its natural environment.
The nucleic acid in the various forms of the present invention may also be present and/or expressed in a cell.
Accordingly, in another form the present invention also provides a cell including an exogenous nucleic with the capacity to modulate development of the cell, the nucleic acid including a binding site for a microRNA.
The cell may be a prokaryotic cell or a eukaryotic cell.
An example of a suitable prokaryotic cell is Escherichia coli. Such a cell is useful for maintaining and/or propagating plasmids including the nucleic acid. An example of a suitable eukaryotic cell is a human colon cell, or cell derived therefrom.
In one form, the cell is a eukaryotic cell that has an altered level and/or activity of one or more microRNAs. Examples of such cells are as previously discussed herein.
The cell may be an isolated cell in vitro, or a cell present in a biological system such as an organ, tissue, or an entire organism (eg an animal or human subject).
In one form, the cell is a cancerous or pre-cancerous cell with a reduced activity of a microRNA.
Accordingly, in another form the present invention provides a cancerous or pre-cancerous cell with reduced activity of a microRNA, the cancerous or pre-cancerous cell including an exogenous nucleic with the capacity to modulate development of the cell, the nucleic acid including a binding site for the microRNA. The reduced activity of the microRNA is as compared to a similar non-cancerous cell.
The cancerous or pre-cancerous cell may be an isolated cell in vitro, or a cell present in a biological system such as an organ, tissue, or an entire organism (eg an animal or human subject).
For example, the cells may be present in a whole animal or human that contains non-cancerous cells and cancerous cells, both of which express an exogenous nucleic with the capacity to modulate development of the cell, the nucleic acid including a binding site for a microRNA.
Thus, the present invention also provides an animal including non-cancerous cells and cancerous cells, the non-cancerous and cancerous cells both including and/or expressing an exogenous nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a binding site for a microRNA.
In one form, the binding site for the microRNA is a binding site for a microRNA that has an altered activity and/or concentration in the cancerous cells as compared to the non-cancerous cells.
The present invention also provides a nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed and/or has differential activity.
Accordingly, in another form the present invention provides a nucleic acid including a non-naturally occurring binding site for a microRNA, wherein the microRNA is differentially expressed or differentially active between cells.
The nucleic acid may be an isolated nucleic acid. The nucleic acid may be present and/or expressed in a cell.
The binding site for the differentially expressed or active microRNA is a binding site of a microRNA that has altered expression and/or activity between different types of cells. In one form, the cells are of a similar type.
Examples of differentially expressed microRNAs include the miR-143 and miR-145 microRNAs, which are differentially expressed between colonic tumours and normal colonic tissue.
In one form, the binding site is a non-naturally occurring binding site for a microRNA that has an altered expression and/or activity in cancerous or pre-cancerous cells, as compared to the level of expression and/or activity in normal or non-cancerous cells. For example, the microRNA may have a reduced expression and/or activity in cancerous or pre-cancerous cells, as compared to the level of expression and/or activity in normal or non-cancerous cells.
Accordingly, in another form the present invention provides an isolated nucleic acid including a non-naturally occurring binding site for a microRNA that is down-regulated in a cancerous cell as compared to a similar non-cancerous cell.
In one form, the nucleic acid is a nucleic acid with the capacity to modulate development of a cell. Nucleic acids that have the capacity to modulate the development of a cell are as previously discussed herein. Thus, the nucleic acid may inhibit (eg have cytotoxic or cytostatic activity) or promote the development of a cell.
In one form, the nucleic acid includes two or more binding sites for binding of the same or different microRNAs that are differentially expressed and/or differentially active.
It will be appreciated that the nucleic acid may be used in a composition for exposure to a cell, so as to introduce the nucleic acid into the cell, or for administration to an animal or human subject, or be part of a vector, such as a viral vector, for introducing into cells, including cells in an animal or human subject.
In one form, the nucleic acid is used to inhibit the development of a cell(s) in an animal or human subject, including the ablation of the cell(s) in an animal or human subject.
Methods for the design of target sites for microRNAs are as previously discussed herein.
Methods for determining whether the binding site occurs naturally are known in the art.
For example, the BLAST algorithm can be used for determining the extent of nucleotide homology between a target sequence and sequences in a specific genome. BLAST identifies local alignments between the sequences in the database and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et al. (1990) J. Mol. Biol. 215:403-410.
The nucleic acid may be an exogenous nucleic acid introduced into a cell and then expressed.
Accordingly, the present invention also provides a cell including a nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed and/or differentially active.
The cell may be a prokaryotic cell or a eukaryotic cell.
An example of a suitable prokaryotic cell is Escherichia coli. Such a cell is useful for maintaining and/or propagating plasmids including the nucleic acid.
In one form, the cell is a eukaryotic cell that has an altered level and/or activity of one or more microRNAs. Examples of such cells are as previously described herein.
The cell may be an isolated cell in vitro, or a cell present in a biological system such as cell present in an organ, tissue, or an entire organism (eg an animal or human subject).
In one form, the cell is a cancerous or pre-cancerous cell.
Accordingly, in another form the present invention provides a cancerous or pre-cancerous cell including a nucleic acid including a non-naturally occurring binding site for a microRNA.
In one form, the cell is a cancerous or pre-cancerous cell with a reduced activity of a microRNA.
Accordingly, in another form the present invention provides a cancerous or pre-cancerous cell with reduced activity of a microRNA, the cancerous or pre-cancerous cell including a nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed and/or differentially active in the cancerous or pre-cancerous cell as compared to a similar non-cancerous cell.
For example, the cells may be present in a whole animal or human that contains non-cancerous cells and cancerous cells, either or both of which include and/or express a nucleic acid including a non-naturally occurring binding site for a microRNA.
Accordingly, in another form the present invention provides an animal including cancerous cells, the cancerous cells including a nucleic acid including a non-naturally occurring binding site for a microRNA.
In one form, the microRNA is differentially expressed or differentially active in the cancerous cells as compared to non-cancerous cells.
Accordingly, in another form the present invention provides an animal including non-cancerous cells and cancerous cells, the non-cancerous and cancerous cells both including and/or expressing a nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed or differentially active in the cancerous cell as compared to the non-cancerous cell.
The nucleic acids of the present invention may be introduced into a cell by a suitable method known in art. For example, the nucleic acid may be introduced into a cell by transformation using calcium phosphate, viral infection, electroporation, lipofection, or particle bombardment. In this regard, transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, or cells which transiently express the inserted DNA or RNA for limited periods of time. Methods for introducing exogenous DNAs into prokaryotic and eukaryotic cells are essentially as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989).
In the case of the nucleic acid being a mRNA, the mRNA may be produced in the cell by transcription of the relevant DNA. Alternatively, the mRNA may be an exogenous mRNA introduced into the cell. Methods for producing mRNAs in vitro are known in the art.
For a mRNA expressed in the cell from a DNA template, the target site may be cloned into a suitable expression vector for use in the cell type of interest by methods known in the art. Methods for the isolation of nucleic acid sequences and their cloning into a suitable expression vector are essentially as described in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratroy Press, New York. (1989). The recombinant molecule may then be introduced into the cell and the cloned nucleic acid expressed. The vector may be any nucleic acid capable of having a foreign nucleic acid inserted into the vector. For example, the vector may be a plasmid, all or part of a viral genome, or any other nucleic acid capable of autonomous replication in a prokaryotic or eukaryotic host.
The present invention also provides a vector including the various nucleic acids of the present invention.
In one form, the vector is a viral vector that allows the nucleic acid to be introduced into the target cells by infection. Examples of such viral vectors that can be employed to deliver the nucleic acid to a cell include a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, a recombinant adeno-associated virus (AAV), a recombinant herpesvirus, a recombinant SV-40 virus, an Epstein-Barr virus, or a recombinant pox virus, such as a recombinant vaccinia virus.
Accordingly, in one form the present invention provides a viral vector including a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a binding site for a microRNA.
In another form, the present invention also provides a viral vector including a nucleic acid including a non-naturally occurring binding site for a differentially expressed microRNA.
In this case, it will be appreciated that the viral vector may be formed from all or part of a viral genome. The viral vector may also be a naturally occurring or a recombinant virus and further may be replication deficient or replication proficient.
As will be appreciated, expression of the relevant inserted DNA in plasmid or viral vectors will generally require various regulatory elements known in the art for the expression of inserted nucleic acids, for example promoters for driving the expression of an inserted nucleic acid in a particular cell, poly A signals for efficient polyadenylation of mRNA transcribed from inserted nucleic acids, or other regulatory elements to control translation, transcription or mRNA stability.
Depending upon the cell type to be modulated, the promoter driving the expression may be a constitutive promoter, an inducible promoter or a cell or tissue specific promoter.
Constitutive mammalian promoters include hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, phosphoglycerate kinase, pyruvate kinase, and β-actin. Exemplary viral promoters which function constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
Inducible promoters include synthetic promoters regulated by the TetO/TetR system and inducible promoters such as metallothionein promoter, which may be used to induced transcription in the presence of certain metal ions. Other inducible promoters are known in the art.
The tissue-specific promoter will depend upon the particular cell type. For example, promoters that allow expression in colon cancer cells include the regulatory sequences of human carcinoembryonic antigen (CEA) [accession:U17131; gi/967132].
The present invention also provides a composition including the various nucleic acids described herein.
Accordingly, in one form the present invention also provides a composition including a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a binding site for a microRNA.
In another form, the present provides a composition including a nucleic acid including a non-naturally occurring binding site for a differentially expressed or differentially active microRNA.
The compositions are suitable for exposing the nucleic acids of the present invention to cells. Methods of introducing nucleic acids into cells are as previously discussed herein.
The compositions of the present invention are also suitable for administration to a subject to prevent and/or treat a disease, condition or state associated with cells that have an altered activity and/or expression of a microRNA.
For example, the nucleic acids may be combined with a pharmaceutically acceptable carrier, stabilizer, buffer or diluent to produce a composition for administration to a subject. Thus, the present invention also provides a pharmaceutical composition including one or more nucleic acids of the invention in an acceptable carrier.
The nucleic acid may be delivered to a cell or a subject by a method known in the art. For example, the nucleic acid molecules can be administered to cells by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins, poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres. The nucleic acid molecules may also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalac-tosamine (PEI-PEG-triGAL) derivatives.
For administration to a subject, the nucleic acids can be administered and introduced by standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, by injection, or delivery by way of infection with a virus.
When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.
Surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) offer a method for increasing the accumulation of drugs in target tissues. Such liposomes have been shown to accumulate selectively in tumors.
Administration routes that lead to systemic absorption include intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular routes. Each of these administration routes exposes the nucleic acids of the present invention to cells or tissue.
The nucleic acids of the present invention will generally be delivered at a pharmaceutically effective dose to prevent, inhibit the occurrence, or treat (so as to alleviate a symptom to some extent) a disease, condition or state. The pharmaceutically effective dose depends on the type of disease or condition being treated, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered.
The nucleic acid molecules of the present invention, and compositions and formulations thereof, can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term “parenteral” includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
As discussed previously, the present invention also provides a pharmaceutical composition including a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use can be prepared according to a suitable method known in the to the art. The composition may contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide a pharmaceutically acceptable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
Formulations for oral use can also be presented as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
Pharmaceutical compositions of the present invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.
Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated as known in the art using suitable dispersing or wetting agents and suspending agents that have been discussed previously. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The nucleic acid molecules of the present invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the nucleic acid with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the nucleic acid. Such materials include cocoa butter and polyethylene glycols.
The nucleic acid molecules of the present invention can also be administered parenterally in a sterile medium. The nucleic acid, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
It is understood that the specific dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
It will be appreciated that the various compositions of the present invention can also be formulated for administering the nucleic acid molecules of the invention to specific cell types. For example, receptors such as the asialoglycoprotein receptor are unique to hepatocytes and bind branched galactose-terminal glycoproteins, such as asialoorosomucoid. Alternatively, some receptors such as the folate receptor are overexpressed in many cancer cells. The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers.
The nucleic acid molecules of the present invention may also be complexed with or covalently attached to nanoparticles, such as Hepatitis B virus or L envelope proteins.
Alternatively, certain the nucleic molecules of the present invention can be expressed within cells from eukaryotic promoters as previously discussed.
Viral vectors expressing the nucleic acids of the present invention may be constructed based upon, for example, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The viral vectors can either be use to introduce the nucleic acid into cells, or alternatively, the viral vector can be packaged and infections used to introduce the nucleic acids of the present invention to cells. The viral vectors can provide for transient expression or stable expression of the nucleic acid molecules.
Therapeutic delivery of biolomolecules is generally as described in Bladon, C. (2002) “Pharmaceutical Chemistry: Therapeutic Aspects of Biomolecules” John Wiley & Sons Ltd.
Viral and gene therapy techniques are as generally described in “Viral Vectors for Gene Therapy Methods and Protocols” Edited by Jules G Constant, Curtis A Machida (2003) Humana Press Inc., “Gene Delivery to Mammalian Cells: Viral Gene Transfer Techniques” Edited by William C Heiser (2004) Humana Press Inc., “Viruses in Human Gene Therapy” Edited by J. H. Vos (1995) Carolina Academic Press, and “Viral Therapy Of Human Cancers” Edited by J. G. Sinkovics and J. C. Horwath (2005) Marcel Dekker.
The present invention is also suitable for modulating the concentration of a nucleic acid in a cancerous cell or pre-cancerous cell.
In this case, an altered activity and/or concentration of a microRNA in a cancerous cell allows the concentration of the nucleic acid in the cell to be modulated by including in the nucleic acid a target site for binding of the microRNA. As will be appreciated, the modulation of the concentration of the nucleic acid is as compared to a similar cell not having an altered activity and/or expression of the microRNA.
For example, a reduced activity and/or concentration of a microRNA in a cancerous cell allows the concentration of the nucleic acid in the cell to be modulated by including in the nucleic acid a target site for binding of the microRNA.
Accordingly, the present invention also provides a method of modulating the concentration of a nucleic acid expressed in a cancerous cell, the cancerous cell having an altered activity and/or concentration of a microRNA as compared to a similar non-cancerous cell, the method including the step of introducing a target site for binding of the microRNA into the nucleic acid to be expressed in the cell.
Methods for cloning and introducing nucleic acids into cells are as previously discussed. Determination that the concentration of a nucleic acid may be accomplished by a suitable method known in the art, such as Northern analysis, RT-PCR or RNase protection.
The present invention is also suitable for detecting an altered microRNA activity and/or concentration in a cancerous or pre-cancerous cell, such as a cancerous cell in vitro, or a cancerous or pre-cancerous cell in an animal or human. In this case, the cancerous cells and non-cancerous cells both express a reporter nucleic acid including a target site for binding of a microRNA, and an altered activity of the microRNA may be detected in the cancerous cells by determining the level of expression of the reporter nucleic acid in the cancerous and non-cancerous cells, and detecting a change in the activity of the microRNA in the cancerous cells by a change in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.
For example, the present invention is suitable for detecting a reduced microRNA activity and/or concentration in a cancerous or pre-cancerous cell. a reduced activity of the microRNA may be detected in the cancerous cells by determining the level of expression of the reporter nucleic acid in the cancerous and non-cancerous cells, and detecting a reduced activity of the microRNA in the cancerous cells by an increase in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.
Accordingly, in another form the present invention provides a method of detecting altered microRNA activity and/or concentration in a cancerous or pre-cancerous cell, the method including the steps of:
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- determining the level of expression of a reporter nucleic acid in the cancerous or pre-cancerous cells and determining the level of expression of a reporter nucleic acid in non-cancerous cells; and
- detecting a reduced activity of the microRNA in the cancerous cells by an increase in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.
In one form, the cancerous cells are present in an animal or human subject.
The non-cancerous cell may be the same or similar cells. In one embodiment, the cancerous (or precancerous cells) and the non-cancerous cells are both present in an animal or human subject.
In one form, the method may be used to detect a reduced activity of the microRNA by detecting an increase in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.
In one form, the reporter nucleic acid in the cancerous (or pre-cancerous cells) is the same or a similar reporter nucleic acid as present in the non-cancerous cells.
Suitable reporter nucleic acids are known in the art. For example, the reporter nucleic acid may produce a detectable product such as LacZ, or GFP. Alternatively, the reporter nucleic acid may be detected by an immunological detection method, with use of antibodies raised to the protein encoded by the reporter nucleic acid. Another method of detecting the reporter nucleic acid is by use of hybridization with a detectably labelled complementary nucleic acid probe.
Finally, standard techniques may be used for recombinant DNA technology, oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
DESCRIPTION OF THE PREFERRED EMBODIMENTSReference will now be made to experiments that embody the above general principles of the present invention. However, it is to be understood that the following description is not to limit the generality of the above description.
Example 1 RNA Isolation From Tissue Samples and Cell LinesCell lines may be maintained in an appropriate medium. Colorectal tumors and the corresponding normal mucosae may be obtained from fresh surgical resections, following informed consent from patients, and then classified according to standard histopathological classification methods.
RNA may isolated from cell lines, using Trizol reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. RNA may be purified from colorectal tissues using the procedure of Chomczynski, P. and Sacchi, N. (1987). Anal. Biochem. 162: 156-159.
Example 2 Cloning of MicroRNAs miRNAs may be cloned essentially as described by Elbashir et al. (2001) Genes Dev. 15: 188-200, except that nucleic acids may be electroeluted from acrylamide gel slices using the Biotrap system (Schleicher and Schuell GmbH, Dassel, Germany). Briefly, small RNA fractions of between 18 and 26 bases may be size selected on a denaturing polyacrylamide gel. Adapter oligonucleotides, containing EcoRI restriction sites, may then be directionally ligated to the RNA molecules. The adapter-ligated RNA may then be amplified by RT-PCR. Concatamerized fragments, containing multimers of religated, EcoRI-digested PCR products, between 200 and 650 bp, are size selected on an agarose gel and recovered by electroelution. The concatamers may then be end-repaired and dA-tailed with Taq DNA polymerase, then cloned into pGEM T-easy (Promega, Madison, Wis.) or pTOPO (Invitrogen) according to the manufacturers' instructions. Plasmid inserts from the resultant colonies may be analyzed by PCR using primers to vector sequences. The nucleic acid sequence of selected inserts may then be determined following treatment of the PCR products with Exonuclease I and Shrimp Alkaline Phosphatase according to the ExoSAP-IT protocol (USB Corporation, Cleveland, Ohio). Clones created by this procedure will contain concatamers of PCR products, and generally likely to represent between two and five independent small RNAs. Example 3 Northern AnalysisTotal RNA (20 μg) may be separated on a 15% denaturing polyacrylamide gel. Loadings are visualized by ethidium bromide staining. The RNA may then be transferred to Hybond N+ nylon membrane by semi-dry blotting (OWL Separation Systems, Portsmouth, N.H.). Probes may be generated by T4 Polynucleotide Kinase (New England Biolabs, Beverly, Mass.) mediated end-labeling of DNA oligonucleotides with [γ-32P]ATP. To increase the specific activity of the probes, the miRNA sequence may be concatamerized as a trimer of direct repeats, then cloned into pGEM T-easy and the insert amplified using PCR with M13 forward and reverse primers. Antisense probes may then be synthesized using Taq polymerase-generated linear amplification from the Sephadex G-50-purified PCR products to incorporate multiple [γ-32P]dCTP bases.
Filter hybridization may be performed in QuikHyb Solution (Stratagene, La Jolla, Calif.) containing 106 cpm/ml probe for 1 h, with washes, as per the manufacturers' recommendations. Filters may be analyzed using a Fujifilm-BAS 2500 phoshorimager and signal intensity quantitated (as photostimulated luminescence/mm2) using Analytical Imaging Station (version 3.0) software (Imaging Research Inc., Brock University, Ontario, Canada).
miRNA sequences may be identified by BLAST (as described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410) by comparison to the Genbank and EMBL public nucleotide databases. MicroRNAs may also be identified by comparison with the databases of the miRNA registry. The secondary structures of putative pre-miRNA hairpins may be determined using the Mfold 3.1 algorithm (as described in Mathews et al. (1999) J. Mol. Biol. 288: 911-940). Potential mRNA target sequences may be identified by searching the Genbank nonredundant and dbEST databases using BLAST and FASTA algorithms (as described in Pearson, W. R. and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA, 85: 2444-2448) algorithms.
Example 4 Identification of Colorectal MicroRNAsSmall RNA fragments (between 18 and 27 bases) in total RNA, purified from both a colonic adenocarcinoma and its matched normal mucosa, may be size fractionated and cloned. The clones from the cancer-derived sample and clones representing normal mucosa may then be sequenced. Sequence analysis and comparison with public database nucleotide sequences will enable identification of many of the clones or assignment to a possible genomic origin for the transcripts.
Example 5 Accumulation of MicroRNAs in Colorectal Tissues and in Cancer Cell LinesTo confirm that the various sequences accumulate as miRNAs and investigate whether changes in miRNA steady-state levels are associated with neoplastic epithelium, Northern blot analysis may be undertaken against a panel of RNAs from matched colorectal cancer and normal mucosa specimens.
Northern blot analysis may be used to determine the levels of mature miRNAs and precursor hairpin molecules in cell lines derived from a variety of cancerous human tissues.
Example 6 Cloning of the miR145 Target Sequence into GFPA mammalian Enhanced Green Fluorescence Protein (EGFP) expression cassette (pMM043;
(i) Pri-miR145 Expression Constructs:
A fragment of the pri-miR145 transcript was cloned by PCR amplification of the sequence corresponding to positions 184 and 734 of cDNA clone F1136638 fis (Genbank ID:21752921). The oligonucleotides used were #556 (5′-TCCGGTACTTTTCAGGGCAA-3′; SEQ ID NO.4) and #557 (5′-CAAGAAACGCATGCCTGATG-3′; SEQ ID NO.5) in a standard PCR reaction using 50 ng HeLa genomic DNA as template with cycling conditions: 94° C. 3 minutes; 40 amplification cycles 94° C. 30 sec., 55° C. 30 sec., 72° C. 1 min; 72° C. 10 minutes and final extension 72° C. 10 min. The 550 bp product was agarose gel purified and cloned into pGEMT-easy (Promega) to create plasmid pMM105. The EcoRI insert of pMM105 (SEQ ID NO. 154) was then ligated into EcoRI linearised pcDNA3.1(+) (Invitrogen) to create the expression constructs: pMM109 (single sense insert;
(ii) Transfections:
Cotransfections of HeLa cells involved FuGene 6 (Roche)-mediated transfection of 0.1 μg of pMM095 with between 0.1 μg and 1 μg pri-miR145 expression vector (pMM109, pMM106, pMM107) in 24 well culture plates, using standard FuGene 6 (Roche) protocols. EGFP activity was detected as direct fluorescence of live cells, 3 days following transfection, on a Typhoon fluorimager (Amersham Biosciences) and quantified using Imagequant software.
The miRNA, miR145, accumulates to only very low levels in HeLa (cervical carcinoma) cells. It was found that an enhanced Green Fluorescence Protein (EGFP) reporter gene construct containing the miR145 target sequence (from the RIGS transcript) is as active in these cells as an EGFP construct lacking the target sequence, as detected by fluorescence of the transfected cells.
However, as shown in
A series of constructs will be created to contain combinations of the complementary target sequences for miR143 and miR145 in the 3′UTR of reporter genes (EGFP, LacZ, Renilla luciferase) within constitutive expression vectors. These vectors will include plasmids (for liposome mediated transfection and mouse transgenesis) and lentiviral systems.
As the contribution of independent targets sequences (or miRNA response elements; MREs) to the repression of a transcript are known to be cumulative, combinations that include up to four copies of each MRE will also be created.
The EGFP construct described previously will be used, in conjunction with clones that contain several (up to four copies of the miR145 and miR143 complementary sequences), cloned into the NotI site of the 3′UTR. LacZ constructs will be based on the synthetic, codon-optimised β-galactosidase sequence, described by Anson and Limberis (2004) J. Biotechnology 108: 17-30.
The LacZ plasmid vectors will be based on the pcDNA3.1(+) expression backbone and that target sequences will also be inserted into the NotI site of the 3′UTR.
Luciferase constructs will be created using the psiCHECK™-2 vector (Promega) by insertion of target sequences into the multiple cloning region in the 3′UTR of the synthetic Renilla luciferase gene. The psiCHECK™-2 vector also provides firefly luciferase as an internal control to normalise for transfection efficiency. Luciferase activities of transfected and transduced (in the case of lentiviral derivations) cells will be detected using the Dual Luciferase® Assay system (Promega) according to manufacturer's instructions.
Example 9 Effect of miRNA Target Sequences in Cultured CellsThe discovery that diseased (cancer) cells lack the repressive function provided by miR143 and miR145 allows us to address the possibility that we can exploit this phenomenon to control the expression of therapeutic genes in these cells.
Results from experiments using the RIGS MRE in EGFP constructs show that the presence of miR145 will limit expression of foreign genes (that contain complementary 3′UTR sequences) in those cells.
A mammalian cell line that accumulates significant levels of mature miR143 or miR145 has not yet been identified. A lack of miR143 accumulation in cell lines has also been reported by others and is postulated to be a consequence of the control of fundamental processes (such as proliferation) by these miRNAs.
To generate systems in which these miRNAs can be induced, to mimic the status of “normal” cells, dox-inducible miR145 HeLa cell lines will be produced to allow an investigation of the stoichiometry between miR145 levels and target sequences and the ability to silence gene expression. This will enable us to determine, in vitro, whether a threshold level of cytoplasmic miR145 is required to suppress reporter gene (EGFP, LacZ, luciferase) activity and cytotoxic gene (herpes simplex virus thymidine kinase) function.
Doxycycline-inducible pri-miR145 expression constructs were created by incorporating the PmeI insert from pMM106 (containing the pri-miR145 subfragment described earlier) to displace EGFP in the Tet-inducible expression cassette pMM060 between the unique Ascl (blunted) and PmII sites. pMM060 utilises both the Tet-responsive promoter and tTR modified transrepressor described by Rossi et al. (1998) Nat. Genet. 20(4):389-93, with the transrepressor under the control of constitutive murine phosphoglycerate kinase regulatory sequences. The construct with a single sense copy of the 550 bp pri-miR145 subsequence is called pMM110.
HeLa Tet-On cells (Clontech) have been stably-transformed with the pMM110 construct using FuGene 6-mediated transfection and following selection on both genticin and puromycin, clonal lines have been isolated. Different HeLa Tet-On/pMM110 lines display varying levels of pri-miR145 induction and mature miR145 accumulation, following 24 h exposure to 1 μg doxycycline/mL growth medium. The data is shown in
It is necessary to determine whether miR143 and miR145MREs will enable disease specific expression of transgenes in mammalian tissues. To develop a rapid assay for miR143/miR145 retarded gene expression in colonic epithelium and adenocarcinomas, the expression of LacZ (+/−multiple miR145 MREs in 3′UTR) in colonic mucosa and compare that with expression in tumour cells will be examined.
Normal murine intestinal tissues and tumours from an azoxymethane (AOM)-treated p53-knockout mouse (Hu et al. (2005) Int. J. Cancer 115: 561-567), will be maintained in culture to establish this study and also to refine the process and vectors.
Culture conditions will be essentially as described by Whitehead et al. (1999) Gastroenterology 117:858-65.
Normal intestinal mucosa, adenomatous and cancer tissues will also be obtained from the resections of consenting cancer patients and cultured as above.
Reporter gene constructs will be inserted into a lentiviral vector system as described in Anson and Fuller (2003) J. Gene Med. 5:829-838, and Fuller and Anson (2001) Human Gene Therapy 12: 2081-2093, and used to infect cultured explants.
All constructs will also incorporate a constitutive EYFP expression cassette to define and normalise the level of lentiviral infection between samples. Initial experiments will use only a constitutive EYFP expression cassette in the lentivirus vector, to establish this system. It will also be determined whether a dual luciferase assay (psiCHECK™ 2; Promega) is more informative, than the lacZ marker, in this system.
If this approach is successful, the LacZ reporter gene will be replaced with the conditional cytotoxic gene, thymidine kinase, to determine whether MRE-derived tissue specificity is sufficient to selectively ablate tumour cells. Naturally, this will lead to the creation of constructs with tissue-specific promoters to enhance disease-specificity.
Example 11 Effect of miRNA Target Sequences on Gene Expression In VivoTransgenic mice will be created that express a LacZ reporter gene, containing multiple miR145 and miR143 complementary sequences in the 3′UTR, under the control of a constitutive promoter (CMV). The transgene construct will also comprise a second reporter gene (EGFP) that does not contain such targeting sequences, but uses the same promoter. Direct fluorescence (or immunohistochemical detection of EGFP) will define the tissue distribution of transgene expression while the subset of cells that also stain for LacZ activity will indicate those which are not affected by microRNA-mediated silencing.
Fluorescence will entail direct observation of fresh or paraformaldehyde-fixed tissues using an inverted fluorescence microscope. Fixed tissue may be pre-treated with 0.1% sodium borohydride, to reduce autofluorescence. GFP specific antibodies (Living Colours® Peptide Antibody; Clontech) will be used for immunohistochemical detection, using standard procedures.
While the entire transgenic mouse will be assessed, particular attention will be paid to colorectal tissues.
Favourable transgenic mouse lines will be crossed with a p53 knockout mouse strain that is currently being used by the Young group to generate a murine model of colorectal cancer following administration of the carcinogen, azoxymethane (Hu et al. (2005) Int. J. Cancer 115: 561-567). The progeny of this cross will be examined for enhanced LacZ expression in tumour cells relative to surrounding epithelium.
To create transgenic mice, pronuclear injection of constructs (linear DNA fragments containing expression cassettes) into embryos isolated from pregnant, superovulated C57BL/6, females will be performed and embryos reimplanted commercially at the GenSA facility (IMVS, Adelaide). Resultant lines will be genotyped (by PCR) for appropriate genomic insertion of the injected sequences, as described in Rulicke T. and Hubscher U. (2000) Exp. Physiol. 85: 589-601.
These founder lines will be assessed for low copy number insertion of the intact introduced expression cassettes and screened for appropriate expression of the transgenes using real-time RT-PCR and immunohistochemistry. Relative copy number determinations will be made using PCR of genomic DNA and/or Southern Blot analysis. Real time RT-PCR will utilize transgene mRNA-specific primers and cDNA templates in a 1×SYBR green PCR Master Mix (Applied Biosystems) reaction. A Corbett Rotorgene 2000 (Corbett Research Pty. Ltd., Australia) will be used for PCR amplification and detection.
The final hybrid line will be inbred to ensure homozygosity for the transgenes, before cross-breeding with the p53 mutant mouse line and other murine models for cancer and polyposis.
Example 12 Effect of miRNA Target Sequences on Gene Expression in Stem Cells and Their DerivativesMicroRNA target sequences in the 3′UTR of reporter and therapeutic genes will be used to assess the potential for exploiting tissue (or cell lineage)-specific microRNAs to limit transgene expression to defined cell lineages and tissues.
For example, target sequences for the neural-specific miRNAs, miR124a and miR9, will be inserted into the 3′UTR of the EGFP or LacZ reporter genes. A lentiviral delivery system will then deliver the synthetic gene into the genomes of murine embryonic stem cells. Transduced embryonic stem cells will then be induced to differentiate into a variety of cells types, including neural progenitors (using the stromal-cell derived induction method of Kawasaki et al. (2000) Neuron 28: 31-40). Reporter gene activity will be correlated with the expression of molecular markers to define which cell lineages allow expression of the introduced gene and which display miR9/124a-mediated silencing. Alternatively, the transduced murine ES cells will be transferred into blastocysts to generate chimeric mice, from which stable transgenic germ lines will be generated (Pfeifer et al. (2001) Proc. Natl. Acad. Sci. USA 99: 2140-2145). The spatial expression of reporter genes will be determined using direct detection (EGFP fluorescence or 3-galactosidase staining) or immunohistochemistry.
Other experiments will study miRNA-mediated transgene regulation in cells that derive from transgenic adult stem cells. These will involve in vitro and in vivo studies of transduced bone marrow-derived stem cells, or haematopoietic progenitor cells, containing reporter genes (or therapeutic genes) with embedded miRNA target sequences. The target sequences will bind with haematopoietic lineage-specific miRNAs, such as miR142-3p. For example, the methodology may be accomplished as described in Brown et al. (2006) Nature Medicine 12: 585-591.
Example 13 The Effect of Increasing miRNA Target Sequences in the 3′UTR of a TransgeneCells of the stable pMM110 transgenic HeLa Tet On cell line, HT0110e, were grown in the presence, or absence, of 2 □g doxycycline/mL medium and FuGene6-transfected, one day after plating, with 80 ng plasmid. The plasmids used for transfection were all derived from pMM043, with varying numbers of miR145 target sequences inserted in the EGFP 3′UTR NotI site. Plasmids were: pMM043 (no targets), pMM095 (1 target), pMM117 (2 targets), pMM119 (8 targets). In this cell line, doxycycline induces the expression of mature miR145 above the low background level present in HeLa cells. Values displayed are the mean fluorescence (n=3) at 46 hours after transfection.
The data is shown in
Finally, it will be appreciated that various modifications and variations of the methods and compositions of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.
Claims
1. A method of modulating development of a cell, the method including the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to modulate development of the cell.
2. A method according to claim 1, wherein the nucleic acid has the capacity to inhibit development of the cell.
3. A method according to claim 2, wherein the nucleic acid has cytotoxic or cytostatic activity.
4. A method according to claim 3, wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase, E. coli cytosine deaminase, E. coli nitroreductase, P. aeruginosa carboxypeptidase, horseradish peroxidase, and E. coli purine nucleoside phosphorylase, or an active fragment or variant of any of these genes.
5. A method according to any one of claims 1 to 4, wherein the cell is selected from the group consisting of a cancerous cell, a pre-cancerous cell, an embryonic stem cell, an adult stem cell, a haemopoietic cell including a haemopoietic precursor cell, an adipocyte, a neuronal cell, a sperm cell or a sperm producing cell, a pancreatic islet cell, and a virally infected cell.
6. A method according to claim 5, wherein the cancerous cell is a colorectal cancer cell, a lung cancer cell, a thymus cancer cell, a bladder cancer cell, a breast cancer cell, a prostate cancer cell or a cancerous B cell.
7. A method according to any one of claims 2 to 7, wherein the method is used to inhibit development of a cell with a reduced activity and/or concentration of the microRNA.
8. A method according to claim 7, wherein the method is used to ablate the cell.
9. A method according to claim 8, wherein the microRNA is selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs.
10. A method according to claim 1, wherein the nucleic acid has the capacity to promote development of the cell.
11. A method according to claim 2, wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned.
12. A method according to claims 10 or 11, wherein the method is used to promote development of a cell with a reduced activity and/or concentration of a microRNA.
13. A method according to any one of claims 1 to 12, wherein the nucleic acid includes two or more target sites for binding of the same or different microRNAs.
14. A method according to any one of claims 1 to 13, wherein the cell is a cell in an animal or human subject.
15. A method according to any one of claims 1 to 14, wherein the method is used to prevent and/or treat a disease, condition or state in an animal or human subject.
16. A nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a binding site for a microRNA.
17. A nucleic acid according to claim 16, wherein the nucleic acid has the capacity to inhibit development of a cell.
18. A nucleic acid according to claim 17, wherein the nucleic acid has cytotoxic or cytostatic activity.
19. A nucleic acid according to claim 18, wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase, E. coli cytosine deaminase, E. coli nitroreductase, P. aeruginosa carboxypeptidase, horseradish peroxidase, and E. coli purine nucleoside phosphorylase, or an active fragment or variant of any of these genes.
20. A nucleic acid according to any one of claims 16 to 19, wherein the binding site is a binding site for a microRNA selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs.
21. A nucleic acid according to any one of claims 16 to 20, wherein the nucleic acid includes two or more binding sites for binding of the same or different microRNAs.
22. A nucleic acid according to claim 16, wherein the nucleic acid has the capacity to promote development of the cell.
23. A nucleic acid according to claim 22, wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned.
24. A nucleic acid according to any one of claims 16 to 23, wherein the binding site is a binding for a microRNA that is differentially expressed and/or differentially active.
25. A vector including the nucleic acid according to any one of claims 16 to 24.
26. A vector according to claim 25, wherein the vector is a viral vector.
27. A composition for administration to an animal or human subject, the composition including a nucleic acid according to any one of claims 16 to 26.
28. A cell including a nucleic acid according to any one of claims 16 to 26.
29. An animal including a cell according to claim 28.
30. A nucleic acid according to any one of claims 16 to 26, wherein the nucleic acid is used to inhibit the development of a cell in an animal or human subject.
31. A nucleic acid according to claim 30, wherein the nucleic acid is used to ablate cells in an animal or human subject.
32. A nucleic acid including a non-naturally occurring binding site for a microRNA that is differentially expressed and/or has differential activity.
33. A nucleic acid according to claim 32, wherein the binding site is a binding site for a microRNA that is differentially expressed and/or differentially active in a cancerous cell as compared to a similar non-cancerous cell.
34. A nucleic acid according to claim 32 or 33, wherein the binding site is a binding site for a microRNA that is downregulated in the cancerous cell as compared to the non-cancerous cell.
35. A nucleic acid according to any one of claims 32 to 34, wherein the binding site is a binding site for a microRNA selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs.
36. A nucleic acid according to any one of claims 32 to 35, wherein the nucleic acid includes two or more binding sites for binding of the same or different microRNAs that are differentially expressed and/or differentially active.
37. A nucleic acid according to any one of claims 32 to 36, wherein the nucleic acid has the capacity to inhibit development of a cell.
38. A nucleic acid according to claim 37, wherein the nucleic acid has cytotoxic or cytostatic activity.
39. A nucleic acid according to claim 37 or 38, wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase, E. coli cytosine deaminase, E. coli nitroreductase, P. aeruginosa carboxypeptidase, horseradish peroxidase, and E. coli purine nucleoside phosphorylase, or an active fragment or variant of any of these genes.
40. A nucleic acid according to claim 32, wherein the nucleic acid has the capacity to promote development of the cell.
41. A nucleic acid according to claim 40, wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned.
42. A vector including the nucleic acid according to any one of claims 32 to 41.
43. A vector according to claim 42, wherein the vector is a viral vector.
44. A composition for administration to an animal or human subject, the composition including a nucleic acid according to any one of claims 32 to 43.
45. A cell including a nucleic acid according to any one of claims 32 to 43.
46. An animal including a cell according to claim 45.
47. A nucleic acid according to any one of claims 32 to 43, wherein the nucleic acid is used to modulate the development of cells in an animal or human subject.
48. A nucleic acid according to claim 47, wherein the nucleic acid is used to inhibit the development of cells in an animal or human subject.
49. A nucleic acid according to claim 47, wherein the nucleic acid is used to ablate cells in an animal or human subject.
50. A cancerous cell including an exogenous nucleic acid including a binding site for a microRNA, wherein the cancerous cell has a reduced activity and/or concentration of the microRNA as compared to a similar non-cancerous cell.
51. A cancerous cell according to claim 50, wherein the cancerous cell is a colorectal cancer cell, a lung cancer cell, a thymus cancer cell, a bladder cancer cell, a breast cancer cell, a prostate cancer cell or a cancerous B cell.
52. A cancerous cell according to claim 50 or 51, wherein the microRNA is selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs.
53. A cancerous cell according to any one of claims 50 to 52, wherein the exogenous nucleic acid includes two or more binding sites for binding of the same or different microRNAs that are differentially expressed and/or differentially active in the cancerous cell.
54. A cancerous cell according to any one of claims 50 to 53, wherein the exogenous nucleic acid has the capacity to inhibit development of the cell.
55. A cancerous cell according to claim 54, wherein the nucleic acid has cytotoxic or cytostatic activity.
56. A cancerous cell according to claim 55, wherein the exogenous nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase, E. coli cytosine deaminase, E. coli nitroreductase, P. aeruginosa carboxypeptidase, horseradish peroxidase, and E. coli purine nucleoside phosphorylase, or an active fragment or variant of any of these genes.
57. An animal including a cancerous cell according to any one of claims 50 to 56.
58. An animal according to claim 57, wherein the animal is a transgenic animal.
59. An animal according to claim 57 or 58, wherein the animal is used to identify microRNAs that are differentially expressed between cancerous and non-cancerous cells.
60. A method of preventing and/or treating a disease, condition or state associated with target cells in a subject, the method including the step of introducing into cells in the subject a nucleic acid with the capacity to modulate development of a cell, the nucleic acid including a target site for binding of the microRNA, wherein the activity of the microRNA in the target cells results in a level of activity of the nucleic acid sufficient to modulate development of the target cells in the subject.
61. A method according to claim 60, wherein the disease, condition or state is selected from the group consisting of a cancer, including colorectal cancer, lung cancer, thymus cancer, bladder cancer, breast cancer and prostate cancer; human B cell chronic lymphocytic leukemia; B cell (Burkitt) Lymphoma; a disease or disorder of pancreatic endocrine cells including diabetes; a disease or condition associated with viral infection of cells including EBV, HIV, Hepatitis and Herpes infection of cells; 5q-myelodysplastic syndrome (macrocytic anaemia); a disease or conditions associated with haemopoietic dysfunction, an autoimmune and inflammatory diseases including Crohn's disease; fragile X mental retardation; Di George syndrome; Wilms tumour; a disease or condition associated with neuron dysfunction; a disease or condition associated with adipocyte dysfunction; a disease that can be treated with embryonic or adult stem cells; and a disease or condition associated with sperm producing cells.
62. A method according to claim 61, wherein the disease is a cancer.
63. A method according to claim 62, wherein the target cell is a cancerous cell or a pre-cancerous cell.
64. A method according to claim 63, wherein the cancerous cell or pre-cancerous is a colorectal cancer cell, a lung cancer cell, a thymus cancer cell, a bladder cancer cell, a breast cancer cell, a prostate cancer cell or a cancerous B cell.
65. A method according to any one of claims 60 to 64, wherein the nucleic acid has the capacity to inhibit development of the cell.
66. A method according to claim 65, wherein the nucleic acid has cytotoxic or cytostatic activity.
67. A method according to claim 66, wherein the nucleic acid encodes a gene selected from the group consisting of herpes simplex thymidine kinase, E. coli cytosine deaminase, E. coli nitroreductase, P. aeruginosa carboxypeptidase, horseradish peroxidase, and E. coli purine nucleoside phosphorylase, or an active fragment or variant of any of these genes.
68. A method according to any one of claims 60 to 67, wherein the target cells have a reduced activity and/or expression of the microRNA.
69. A method according to any one of claims 60 to 68, wherein the method is used to ablate the target cells in the subject.
70. A method according to any one of claims 60 to 69, wherein the microRNA is selected from the group consisting of hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7f, hsa-miR-10b, hsa-miR-15a, hsa-miR-15b, hsa-miR-16, hsa-miR-19b mature miRNA, hsa-miR-20, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-24, hsa-miR-189, hsa-miR-24, hsa-miR-26, hsa-miR-26b, hsa-miR-26a, hsa-miR-27b, hsa-miR-29a, hsa-miR-30a-3p, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-199b, hsa-miR-200b, hsa-miR-200c, hsa-miR-320, hsa-miR-321, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-29b, hsa-miR-125b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126*, hsa-miR-126, hsa-miR-188, hsa-miR-331, hsa-miR-181b-1, hsa-miR-155, hsa-miR-124a, hsa-miR-9 and the corresponding orthologues of the aforementioned microRNAs.
71. A method according to claim 60, wherein the nucleic acid has the capacity to promote development of the cell.
72. A method according to claim 71, wherein the nucleic acid is a nucleic acid encoding a cytokine, a therapeutic protein, or an active fragment or variant of the aforementioned.
73. A method according to claim 71 or 72, wherein the method is used for the promotion of development a cell with a reduced activity and/or concentration of a microRNA.
74. A method of inhibiting development of a cell, the cell having a reduced activity and/or concentration of a microRNA, the method including the step of introducing into the cell a nucleic acid with the capacity to inhibit development of the cell, the nucleic acid including a target site for binding of the microRNA, wherein the reduced activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to inhibit development of the cell.
75. A method of promoting development of a cell, the cell having a reduced activity and/or concentration of a microRNA, the method including the step of introducing into the cell a nucleic acid with the capacity to promote development of the cell, the nucleic acid including a target site for binding of the microRNA, wherein the reduced activity and/or concentration of the microRNA in the cell results in a level of activity and/or concentration of the nucleic acid in the cell sufficient to promote development of the cell.
76. A method of detecting altered microRNA activity and/or concentration in a cancerous or pre-cancerous cell, the method including the steps of:
- determining the level of expression of a reporter nucleic acid in the cancerous or pre-cancerous cells and determining the level of expression of a reporter nucleic acid in non-cancerous cells; and
- detecting a reduced activity of the microRNA in the cancerous cells by an increase in the expression of the reporter nucleic acid in the cancerous cells as compared to the level of expression of the reporter nucleic acid in the non-cancerous cells.
77. A method of modulating the concentration of a nucleic acid expressed in a cancerous cell, the cancerous cell having an altered activity and/or concentration of a microRNA as compared to a similar non-cancerous cell, the method including the step of introducing a target site for binding of the microRNA into the nucleic acid to be expressed in the cell.
Type: Application
Filed: Jun 2, 2006
Publication Date: Jul 26, 2012
Applicant: VIMAR S.P.A. (MAROSTICA)
Inventor: Michael Zenon Michael (Belair)
Application Number: 11/916,276
International Classification: A61K 31/7088 (20060101); C12N 5/09 (20100101); C12N 5/0735 (20100101); C12N 5/0793 (20100101); C12N 5/076 (20100101); C12N 15/11 (20060101); C12N 15/33 (20060101); C12N 15/38 (20060101); C12N 15/54 (20060101); C12N 15/53 (20060101); C12N 15/57 (20060101); C12N 15/19 (20060101); C12N 15/63 (20060101); A61P 35/00 (20060101); A61P 35/02 (20060101); A61P 31/18 (20060101); A61P 31/14 (20060101); A61P 31/22 (20060101); A61P 29/00 (20060101); A61P 1/00 (20060101); C12N 5/071 (20100101);
