HK1118577B - Rnai expression constructs - Google Patents
Rnai expression constructs Download PDFInfo
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- HK1118577B HK1118577B HK08112495.7A HK08112495A HK1118577B HK 1118577 B HK1118577 B HK 1118577B HK 08112495 A HK08112495 A HK 08112495A HK 1118577 B HK1118577 B HK 1118577B
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Abstract
The present invention provides compositions and methods suitable for expressing 1-x RNAi agents against a gene or genes in cells, tissues or organs of interest in vitro and in vivo so as to treat diseases or disorders.
Description
Background
The use of double-stranded RNA to inhibit gene expression in a sequence-specific manner has revolutionized the drug discovery industry. In mammals, RNA interference, or RNAi, is mediated by double-stranded RNA molecules of 15 to 49 nucleotides in length, known as small interfering RNAs (RNAi agents). RNAi factors can be chemically or enzymatically synthesized extracellularly and subsequently delivered to cells (see, e.g., Fi)re, and the like,Nature(Nature), 391: 806-11 (1998); the general structure of Tuschl, et al,Genes and Dev(gene and development), 13: 3191-97 (1999); and the list of Elbashir, et al,Nature(natural), 411: 494-498 (2001)); or may be expressed in vivo by an appropriate vector within the cell (see, e.g., U.S. patent 6,573,099).
The in vivo delivery of unmodified RNAi agents as effective therapeutics for use in humans faces a number of technical challenges. First, due to cellular and serum nucleases, RNA injected into the body has a half-life of only about 70 seconds (see, e.g., Kurreck,Eur.J.Bioch(European journal of biochemistry) 270: 1628-44(2003)). Efforts to increase the stability of injected RNA by using chemical modifications have also been attempted; however, in many cases, chemical changes result in an increase in cytotoxic effects. In a specific example, the cells are intolerant to dosing of RNAi duplexes that replace every second phosphate with a phosphorothioate (Harborth, et al,Antisence Nucleic Acid Rev.(review of antisense nucleic acid drugs) 13 (2): 83-105(2003)). Other difficulties include providing tissue-specific delivery, and the ability to deliver doses of RNAi agents sufficient to elicit a therapeutic response without toxicity.
Several options for delivery of RNAi have been developed, including the use of viral-based and non-viral-based vector systems that can infect or otherwise transfect target cells, and deliver and express RNAi molecules in situ. Typically, small RNAs are transcribed from viral or non-viral vector backbones as short hairpin rna (shrna) precursors. Once transcribed, the shRNA is postulated to be processed by ribonucleic acid degrading enzymes (enzyme Dicer) into the appropriate active RNAi agents. Viral-based approaches to delivery attempt to exploit the targeting properties of viruses to produce tissue specificity, once properly targeted, rely on endogenous cellular mechanisms to produce sufficient levels of RNAi agents to achieve a therapeutically effective dose.
One useful use of RNAi therapeutics is as antiviral agents. In general, RNA viruses rely on RNA-dependent RNAThe polymerase performs replication. This RNA polymerase replicates the viral genome with relatively low fidelity, the functional consequence of which is the generation of a genome with an abnormally large number of mutations. These quickly led to the generation of progeny of evolved virosomes that evaded conventional immunological or chemical antiviral factors. Thus, similar to the effects observed with small molecule therapeutics, the corresponding efficacy and effect of RNAi therapeutics is reduced as a result of viral evolution during long-term therapy. In one study, an HIV escape variant containing a single nucleotide change appeared 35 days after delivery of the expressed shRNA (Boden, et al,J. Virol.(journal of virology) 77 (21): 11531-11535(2003)). In another study, poliovirus (poliovirus) escape variants were detected in cells transfected with pre-synthesized RNAi within as little as 54 hours of cell infection (Gitlin et al)J. Virol.(journal of virology) 2005 Jan; 79(2): 1027-35). Likewise, other possible RNAi targets, such as genes involved in tumors, have sequence variability. Delivery of two or more RNAi's simultaneously against multiple sequences can provide a better therapeutic effect against any disease that utilizes genetic variation to resist inhibition. There is a need in the art to develop stable, efficient, expressed RNAi agents that can deliver multiple RNAi agents.
Disclosure of Invention
The present invention relates to genetic constructs for delivering RNAi agents to tissues, organs, or cells for the treatment of various diseases or disorders. In one aspect, the invention provides novel nucleic acid molecules comprising two or more RNAi agents for modulating expression of a target gene. In another aspect, the present invention provides an expression cassette comprising a promoter and two or more stem-loop structures separated from each other by a spacer structure (hereinafter referred to as 1-xRNA expression cassette). In another aspect, the invention provides a genetic construct capable of regulating expression of one or more genes, wherein the genetic construct comprises two or more RNAi agents transcribed from a single promoter. Also, other aspects of the invention include methods of treating a disease or disorder in a tissue, cell or organ by expressing two or more RNAi factors from a single promoter to modulate gene expression in the tissue or organ.
Drawings
So that the manner in which the above recited features, advantages and objects of the present invention are attained and can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to the embodiments thereof which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG. 1 is a simplified block diagram of one embodiment of the delivery of a 1-x RNAi expression cassette to a cell, tissue or organ of interest according to the present invention.
FIGS. 2A, 2B, and 2C show three embodiments of 1-x RNAi expression cassettes according to the present invention.
FIGS. 3A and 3B show alternative methods for generating viral particles for delivery of constructs containing 1-x RNAi expression cassettes to relevant cells, tissues or organs.
FIG. 4 shows a schematic of the 1-3RNAi expression cassettes used in the examples described herein. The expected results of siRNA activity are listed below each construct according to the role of the RNA interference mechanism of the pre-established artificial control (art boosting).
FIG. 5 shows the results of the inhibition of the Luc-HCV fusion reporter construct containing the HCV 5' -8 target sequence by the 1-3RNAi expression cassette constructs of the present invention.
FIG. 6 shows the results of the suppression of the Luc-HCV fusion reporter construct containing the HCV encoding-12 target sequence by the 1-3RNAi expression cassette constructs of the present invention.
FIG. 7 shows a schematic representation of the positions of selected RNAi target sequences in the HCV genome. Ten conserved target sequences were identified as located in the HCV IRES region, twelve in the ORF (open reading frame), and eight in the 3 'non-coding region (3' UTR). Also shown are the Luc-HCV fusion reporter constructs used to evaluate 1-x RNAi inhibition.
FIG. 8 shows Luc-HCV fusion reporter constructs used to evaluate 1-x RNAi inhibition.
FIG. 9 shows the results of inhibition of a series of Luc-HCV fusion reporter constructs containing the target sequence by transfected RNA species generated in vitro. RNA was obtained from a run-off transcription reaction using a DNA template containing the 1-3RNAi expression cassette constructs of the invention by T7 RNA polymerase.
Detailed Description
Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular methodology, products, apparatus, and elements described. As such methods, apparatus and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a factor" refers to a factor or a mixture of factors. And references to "methods of manufacture" include references to equivalent steps and methods, etc., known to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference, without limitation, for the purpose of describing and disclosing the devices, formulations and methodologies that are described in the publications, which might be used in connection with the invention described.
In the following description, numerous specific details are provided to provide a more thorough understanding of the invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and steps that are well known to those skilled in the art have not been described in order to avoid obscuring the present invention.
The present invention relates to novel, potent genetic compositions and methods of treating diseases or disorders using novel RNAi cassettes.
Generally, the present invention applies methods in molecular biology, microbiology, recombinant DNA technology, cell biology, and virology within the skill of the art. These techniques are explained fully in the literature, see, for example, Maniatis, Fritsch and Sambrook,Molecular Cloning：A Laboratory Manual(molecular cloning: A laboratory Manual) (1982);DNA Cloning：A Practical Approach(DNA cloning: practical methods), volumes I and II (D.N. Glover, eds. 1985);Oligonucleotide Synthesis(oligonucleotide synthesis) (m.j.gait, editions. 1984);Nucleic Acid Hybridization(nucleic acid hybridization) (b.d.hames and s.j.higgins, editors (1984));Animal Cell Culture(animal cell culture) (r.i. freshney, editors 1986); andRNA Viruses：A practical Approach(RNA Virus: methods of practice), (Alan, J.Cann, eds., Oxford University Press, 2000).
A "vector" is a replicon, such as a plasmid, phage, viral construct or cosmid (cosmid), to which another DNA segment may be attached. The vector is used to transduce and express the DNA fragment in the cell. The terms "construct" and "1-x RNAi expression construct" generally refer to a vector associated with a 1-x RNAi expression cassette.
A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase within a cell and initiating transcription of a polynucleotide or polypeptide coding sequence, such as messenger RNA, ribosomal RNA, small or nucleolar RNA or any type of RNA transcribed by any kind of RNA polymerase.
When an exogenous or heterologous nucleic acid or vector is introduced into a cell, for example, in admixture with a transfection reagent or packaged into a viral particle, the cell is "transformed", "transduced" or "transfected" with such nucleic acid. The transforming DNA may or may not be integrated (covalently bound) into the genome of the cell. For eukaryotic cells, a stably transformed cell is one in which the transforming DNA has integrated into the host cell chromosome or is maintained extrachromosomally, such that the transforming DNA is passed on to daughter cells during cellular replication, or a non-replicating, differentiated cell in which a stable episome is present.
The term "RNA interference" or "RNAi" generally refers to a process in which a double-stranded RNA molecule alters the expression of a nucleic acid sequence that shares substantial or complete homology with a double-stranded or short hairpin RNA molecule. The term "RNAi agent" refers to an RNA sequence that triggers RNAi; the term "ddRNAi agent" refers to an RNAi agent transcribed from a vector. The term "short hairpin RNA" or "shRNA" refers to an RNA structure having a double-stranded region and a loop region. In some embodiments of the invention, the ddRNAi agent is initially expressed as a shRNA. The term "1-x RNAi expression cassette" refers to a cassette according to an embodiment of the present invention having one promoter and x RNAi constructs, wherein x is two or three or four or five or more, thus 1-2, 1-3, 1-4, 1-5, etc. RNAi expression cassettes. RNAi agents are initially expressed as shrnas and comprise two or more stem-loop structures separated by one or more spacer regions. The term "1-x RNAi expression construct" or "1-x RNAi expression vector" refers to a vector containing a 1-x RNAi expression cassette.
By "derivative" of a gene or nucleotide sequence is meant any individual nucleic acid molecule that contains significant sequence similarity to the gene or nucleotide sequence or a portion thereof. In addition, "derivatives" include such individual nucleic acids containing modified nucleotides or analogs of naturally occurring nucleotides.
FIG. 1 is a simple flow diagram showing the steps of a method according to one embodiment of the invention in which a 1-x RNAi expression construct can be used. The method includes step 200 in which a 1-x RNAi expression cassette targeted to a disease or disorder is constructed. Then, in step 300, the 1-x RNAi expression cassette is ligated into an appropriate viral delivery construct. The viral 1-x RNAi expression delivery construct is then packaged into a virion in step 400, and the virion is delivered to a relevant cell, tissue, or organ in step 500. Details of each of these steps and the components involved will be provided below.
The virus-based 1-x RNAi expression constructs according to the invention can be produced synthetically or enzymatically by a number of different methods well known to the person skilled in the art and can be used, for example, in the field of the invention in the field of the DNA technology of Sambrook et al,Molecular Cloning：A Laboratory Manual(molecular cloning: A laboratory Manual) second edition, Cold Spring Harbor Press (Cold Spring Harbor Press), Cold Spring Harbor, New York (1989), and purification using standard recombinant DNA techniques as specified in, for example, the U.S. department of health and safety services (HHS), National Institutes of Health (NIH) for the guidelines for recombinant DNA research.
FIGS. 2A and 2B are simplified schematic diagrams of 1-3 and 1-5 RNAi expression cassettes containing three and five different RNAi stem-loop structures, respectively, according to embodiments of the present invention. It will be appreciated by those skilled in the art that the 1-x RNAi expression cassettes of the invention can include two, four, six, or more stem-loop structures, and are exemplary in the embodiments shown in this figure. These figures show embodiments of 1-3 and 1-5 RNAi expression cassettes containing three and five stem-loop structures separated by spacer regions. Stem regions 1-5 contain about 17-21 base pairs, preferably 19 base pairs. The loop regions 1-5 contain about 3-20 nucleotides, preferably 5 to 9 nucleotides,more preferably 6 nucleotides. Spacer region between RNAi stems (N)1，N2...) between about 4-10 nucleotides, preferably 6 nucleotides. FIG. 2C shows a specific embodiment of a 1-x RNAi cassette of the present invention comprising three stem-loop structures having 17-21 base pairs, and a fourth stem-loop structure having a shorter stem region with between 2-17 base pairs.
The following is an example of a sequence of multiple hairpin boxes of the invention:
ggatccGTGCACGGTCTACGAGACCTCgaagcttgGAGGTCTCGTAGACCGTGCAtgtacaGCGAAAGGCCTTGTGGTACTgaagcttgAGTACCACAAGGCCTTTCGCccatggATTGGAGTGAGTTTAAGCTgaagcttgAGCTTAAACTCACTCCAATtttttctaga (serial number 57).
When a 1-x RNAi expression cassette is used, the two or more RNAi agents comprising the cassette will all have different sequences; these are RNAi1, RNAi2, RNAi3, RNAi4 and RNAi5, for example, all of which are different from each other. In addition, in a preferred embodiment, the promoter and termination elements used in the 1-x RNAi expression cassette are matched to each other; that is, the promoter and terminator elements are derived from the same gene in which they naturally occur. Promoters may or may not be modified to attenuate transcription levels using molecular techniques, or other techniques, such as through regulatory elements.
Promoters used in some embodiments of the invention may be tissue-specific or cell-specific. The term "tissue-specific" when used in reference to a promoter means capable of mediating selective expression of a nucleotide sequence of interest directed against a specific type of tissue, said nucleotide sequence being relatively devoid of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., brain). The term "cell-specific" when used in reference to a promoter refers to a promoter capable of mediating the selective expression of a nucleotide sequence of interest in a specific type of cell, with relative lack of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue (see, e.g., Higashibata, et al,J.Bone Miner.Res(journal of bone mineral research) Jan 19 (1): 78-88 (2004); hoggatt, et al，Circ.Res(cycling study), dec.91 (12): 1151-59 (2002); the results of the experiments in Sohal, et al,Circ.Resjul 89 (1): 20-25 (2001); and Zhang, et al,Genome Res(genome study.) Jan 14 (1): 79-89(2004)). The term "cell-specific" when used in reference to a promoter also means a promoter capable of promoting the selective expression of a nucleotide sequence of interest in a region within a single tissue. Alternatively, the promoter may be constitutive or regulatable. In addition, promoters may be modified to have different specificities.
The term "constitutive" when used in reference to a promoter means capable of mediating transcription of an operably linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.). In general, a constitutive promoter is capable of mediating expression of a coding sequence in essentially any cell and in any tissue. The promoter for transcription of the RNAi factor is preferably a constitutive promoter, for example the promoter of the ubiquitin, CMV, beta-actin, histone H4, EF-1alfa or pgk gene under the control of RNA polymerase II, or a promoter element under the control of RNA polymerase I. In other embodiments, PolII promoters are used, such as CMV, SV40, U1, β -actin or hybrid PolII promoters. In other embodiments, promoter elements controlled by RNA polymerase III are used, such as the U6 promoter (e.g., U6-1, U6-8, U6-9), H1 promoter, 7SL promoter, human Y promoter (hY1, hY3, hY4 (see Maraia, et al,Nucleic Acids Res(nucleic acid research) 22 (15): 3045-52(1994)) and hY5 (see Maraia, et al,Nucleic Acids Res(nucleic acid research) 24 (18): 3552-59(1994), the human MRP-7-2 promoter, the adenovirus VA1 promoter, the human tRNA promoter, the 5s ribosomal RNA promoter, and hybrids and combinations of these promoter functionalities.
Alternatively, in some embodiments, it is preferred to select a promoter that can induce expression of the plurality of RNAi agents contained in the 1-xRNAi expression cassette. A number of systems for inducible expression using such promoters are known in the art, including but not limited to: tetracycline response systems and lac operator-repressor systems (see publication WO 03/022052A 1; and U.S. patent publication 2002/0162126A 1), ecdysone regulatory systems, or promoters regulated by glucocorticoids, progesterone, estrogens, RU-486, steroids, thyroid hormones, cyclic AMP, cytokines, calciferol family regulators, or metallothionein promoters (regulated by inorganic metals).
One or more enhancers may also be present in the 1-x RNAi expression construct to increase expression of the relevant gene. Enhancers suitable for use in embodiments of the invention include the recently described Apo E HCR enhancer, CMV enhancer (see, Xia et al,Nucleic Acids Res(nucleic acids research) 31-17(2003)), as well as other enhancers known to those skilled in the art.
The 1-x RNAi expression cassette for delivering RNAi agents used in the present invention has two or more stem-loop structures in which the ends of the double-stranded RNA of each stem are linked by single-stranded, circular RNAs. The RNAi sequences encoded by the 1-x RNAi expression cassettes of the invention result in the expression of small interfering RNAs, which are short double-stranded RNAs that are not toxic in normal mammalian cells. The length of the 1-x RNAi expression cassette of the present invention is not particularly limited as long as it does not exhibit cytotoxicity. The length of the RNAi can be 17 to 21 bp (base pairs), and more preferably 19 bp. The RNAi duplex or stem portion may be fully homologous, or may contain unpaired portions due to sequence mismatches (corresponding nucleotides on each strand are not complementary), bulges (lack of corresponding complementary nucleotides on one strand), and the like. Such unpaired portions can be tolerated to the extent that they do not significantly interfere with the formation or efficacy of RNAi duplexes. The length of the single-stranded loop portion of the shRNA may be 3 to 20 nucleotides, and preferably 5 to 9 nucleotides.
The sequences of the stem structures of two or more RNAi agents in an expression cassette of the invention can be the same or different, but the sequences of the RNAi agents in each 1-x expression cassette often differ from each other. Likewise, the stem and loop lengths of the different RNAi agents in a 1-x RNAi expression cassette can be the same or different from the other stem and/or loop lengths in the 1-x RNAi cassette. Two or more stem-loop structures of the invention are separated by a spacer region. The spacer region is composed of nucleotides, which may be naturally occurring or synthetic. The spacer region between the stem-loop structures may be about 4 to 10 nucleotides in length, and preferably about 6 nucleotides in length. The spacer regions between the three or more RNAi agents in an expression cassette of the invention can have the same sequence or different sequences, and can be of the same or different lengths.
Nucleic acid sequences targeted by the 1-x RNAi expression cassettes of the invention include viral genes, oncogenes, bacterial genes, developmental genes, and are selected based on the genetic sequence of the gene sequence; and preferably on the basis of conserved regions of gene sequences. Methods of sequence alignment (alignment) and RNAi sequence selection for alignment are well known in the art. Mathematical algorithms can be used to determine the percent identity between two or more sequences. Preferably, non-limiting examples of such mathematical algorithms are the algorithms of Myers and Miller (1988); pearson and Lipman's similarity search method (search-for-similarity-method) (1988); and the method of Karlin and Altschul (1993). Preferably, the mathematical algorithms are performed using a computer. These operations include, but are not limited to: CLUSTAL (available from Intelligenetics, Mountain View, Calif.) in the PC/Gene program; ALIGN program (version 2.0), GAP, BESTFIT, BLAST, FASTA, Megalign (using the Jotun Hein, Martinez, Needleman-Wunsch algorithm), DNAStar Lasergene (see www.dnastar.com) and TFASTA in the Wisconsin Genetics software Package (Wisconsin Genetics software Package), version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Comparison using these programs may be performed using default parameters or parameters selected by the operator. Higgins describes the CLUSTAL program in detail. The ALIGN program is based on the algorithms of Myers and Miller; the BLAST programs are based on the algorithm of Karlin and Altschul. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov /).
For sequence comparison, a reference sequence is typically compared to a sequence that serves as a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence equivalents are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.
Generally, inhibition of a target sequence by RNAi requires a high degree of sequence homology between the target sequence and the sense strand of the RNAi molecule. In some embodiments, this homology is greater than about 70%, and may be greater than about 75%. Preferably, the homology is higher than about 80%, and higher than 85% or even 90%. More preferably, the sequence homology between the target sequence and the sense strand of the RNAi is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In embodiments where the 1-x RNAi expression construct is used to target viral infection, sequence homology of 15 to 30 contiguous nucleotides may be less than 90% or even 80% greater between the genomes of various subspecies of the virus, even between conserved regions. In such cases, the sequence homology between the target sequence of some subspecies and the sense strand of the RNAi can be 80% or less. On the other hand, when the targeted gene of an organism does not show high sequence homology between species, subspecies or variants, embodiments of the 1-x RNAi expression constructs of the invention are particularly beneficial because each ddRNAi agent in the 1-x RNAi expression cassette can be used to localize at a different site in the allele of a subtype, subspecies or variant of the target gene or variant.
A major problem with current antiviral therapies is the emergence of resistant variants, commonly known as escape variants (Gitlin et al)J.of Virol(journal of virology) 79; 1027-1035, 2005). The present invention, in one aspect, neutralizes emerging escape variants. In some embodiments of the inventionIn the protocol, multiple RNAi sequences are selected for treatment of viral infection based on the emergence of escape variants due to treatment of a single sequence of RNAi from infected cells. After infection of the cells with the virus, the escape variants that appeared were determined by treatment with expression constructs containing the RNAi unique sequence. Cells containing the emerging resistant virus were harvested and the viral genome sequenced. Sequencing showed important mutations that caused resistance to viral inhibition. The 1-x RNAi expression constructs of the invention are generated containing RNAi sequences based on the genetic sequence of the target gene and additional sequences that cause point mutations for anti-RNAi therapy.
In addition to selecting RNAi sequences based on conserved regions of a gene, RNAi sequences can be selected based on other factors. Although numerous attempts have been made to design selection criteria for recognition sequences that are effective in RNAi based on the characteristics of the envisaged target sequence (e.g. percentage GC content, position from translation initiation codon, or sequence similarity based on search of electronic sequence databases for analogs of the proposed RNAi, thermodynamic pairing criteria), it is currently not possible to make a good prediction of which of the ever-changing sequences is the candidate RNAi sequence that actually elicits the best resting response to RNA relative to the gene (despite the progress of this approach: Dharmacon currently claims 70% success rate). Instead, individual specific candidate RNAi polynucleotide sequences are typically synthesized and tested to determine whether interference with the expression of the desired target can be elicited.
In some embodiments of the invention, the ddRNAi factor coding region of a 1-x RNAi expression cassette is operably linked to a terminator element. In one embodiment, using the pol III promoter, the terminator includes a stretch of four or more thymine residues. Other terminators include SV40 poly A, Ad VA1 gene, 5S ribosomal RNA gene, and human t-RNA. In addition, promoters and terminators may be mixed and matched, for example, RNA pol II promoters and terminators are typically mixed and matched.
In addition, the 1-x RNAi expression cassette can be configured overall with multiple cloning sites and/or specific restriction endonuclease sites so that promoter, ddRNAi factor, and terminator elements can be easily removed or replaced. 1-x RNAi expression cassettes can be assembled from small oligonucleotide components using generally distributed restriction enzyme sites and/or complementary cohesive ends. The basic vector of a method according to an embodiment of the invention consists of a plasmid with multiple linkers, on which all sites are unique (although this is not absolutely necessary). Next, insertion of the promoter between each unique site that is set generates a basic cassette with promoters that can be redirected. Next, the annealing primer pair is inserted between unique sites downstream of the monomers that produce the single, two or more 1-x RNAi expression cassette constructs. The insert can be moved into, for example, a vector backbone using two unique enzyme cleavage sites flanked by a single, two, or more 1-x RNAi expression cassette inserts.
In step 300 of FIG. 1, a 1-x RNAi expression cassette is ligated into a delivery vector. The vector inserted with the 1-x RNAi expression cassette and used for efficient transduction and expression of the 1-x RNAi expression cassette in different cell types can be derived from a virus and suitable for viral delivery; alternatively, non-viral vectors may be used. Preparation of the resulting construct comprising the vector and 1-x RNA expression cassette may be accomplished using any suitable genetic engineering techniques known in the art, including, but not limited to, PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and standard techniques of DNA sequencing. If the construct is based on a viral construct, the vector preferably includes, for example, the necessary sequences for packaging the 1-x RNAi expression construct into a viral particle and/or sequences for integrating the 1-x RNAi expression construct into the genome of the target cell. The viral construct may also include genes that allow viral replication and propagation, although in other embodiments such genes may be provided in trans (in trans). In addition, the viral construct may comprise genes or genetic sequences derived from the genome of any known organism, either in native form or modified to be integrated. For example, preferred viral constructs may include sequences for replicating the construct in bacteria.
The construct may also contain additional genetic elements. The type of elements that may be included in a construct is not limited in any way and may be selected by one of ordinary skill in the art. For example, additional genetic elements may include reporter genes, e.g., one or more genes for fluorescently labeled proteins such as GFP or RFP; readily detectable enzymes such as beta-galactosidase, luciferase, beta-glucuronidase, chloramphenicol acetyltransferase or secreted embryonic alkaline phosphatase; or proteins, such as hormones or cytokines, can be readily obtained for use in immunoassays. Other genetic elements that may be used in embodiments of the invention include those encoding genetic elements that confer a selective growth advantage on the cell, such as adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin-B-phosphotransferase, drug resistance, or a gene encoding a protein that provides the biosynthetic capability lost to auxotrophy. If a reporter gene is included with the 1-x RNAi expression cassette, an Internal Ribosome Entry Site (IRES) sequence can be included. Preferably, the additional genetic element is operably linked to and controlled by a separate promoter/enhancer. In addition, suitable origins of replication for the propagation of the constructs in bacteria may be used. The replication initiation sequence is typically separate from the 1-x expression cassette and other sequences to be expressed in the relevant cell, tissue, or organ. Such origins of replication are well known in the art and include pUC, CoIEI, 2-micron (2-micron) or SV40 origins of replication.
Any suitable virus-based viral delivery system can be used to deliver the 1-xRNAi expression constructs of the invention. Alternatively, hybrid virus systems may be used. The viral delivery system is selected according to various parameters, such as the efficiency of delivery into the relevant cell, tissue, or organ, the transduction efficiency of the system, pathogenicity, associated immunity and toxicity, and the like. No single viral system is known to be suitable for all applications. When selecting a viral delivery system for use in the present invention, the selection system is important, wherein the virion containing the 1-x RNAi expression construct is preferably: 1) reproducibility and stable proliferation; 2) can be purified to high titer; and 3) can mediate targeted delivery (delivery of 1-x RNAi expression constructs to relevant cells, tissues, or organs without widespread dissemination).
In general, the five most commonly used types of viral systems in gene therapy can be divided into two categories depending on whether their genomes are integrated into the host cell chromatin (oncoretroviruses and lentiviruses) or persist in the nucleus primarily as extrachromosomal episomes (adeno-associated viruses, adenoviruses and herpesviruses). If maintenance of stable genetic changes is desired in actively dividing cells, an integrating vector will be selected as a tool.
For example, in one embodiment of the invention, viruses from the parvoviridae family are used. Parvoviridae are a family of small single-stranded, non-enveloped DNA viruses whose genomes are approximately 5000 nucleotides in length. Included among the members of this family are adeno-associated viruses (AAV), dependent parvoviruses, which by definition require co-infection with another virus (usually an adenovirus or a herpes virus) to initiate and maintain a proliferative infection cycle. In the absence of such helper viruses, AAV can still infect or transduce target cells, penetrating into the nucleus in dividing or non-dividing cells, through receptor-mediated binding and internalization.
Once in the nucleus, the virus is decapsulated and the transgene is expressed in a different form-the most stable form being a cyclic monomer. AAV is integrated into the genome of 1-5% of stably transduced cells (Nakai, et al,J.Virol(journal of virology) 76: 11343-349(2002). Expression of transgene expression can be abnormally stable and in a study with AAV to deliver factor IX, the dog model continued to express therapeutic levels of protein for more than 5.0 years after a single direct fusion with the virus. Because AAV infection does not produce progeny virus in the absence of helper virus, the scope of transduction is limited to infecting only the original cells of the virus. It is this feature that makes AAV the preferred choice for the present inventionA gene therapy vector. In addition, unlike retroviruses, adenoviruses, and herpes simplex viruses, AAV has been shown to lack pathogenicity and toxicity to humans (Kay, et al,Nature(Nature) 424: 251(2003) and Thomas, et al,Nature Reviews，Genetics(natural review, genetics) 4: 346-58(2003)).
Typically, the genome of AAV includes only two genes. The "rep" gene encodes at least four independent proteins used in DNA replication. The "cap" gene product is differentially cleaved to produce 3 proteins containing the viral capsid. When packaging the genome into a nascent virus, only Inverted Terminal Repeats (ITRs) are required sequences; rep and cap can be deleted from the genome and replaced by selected heterogeneous sequences. However, in order to generate the proteins required for replication and packaging of AAV-based heterogeneous constructs into nascent virions, rep and cap proteins must be provided in trans. Helper functions are typically provided by co-infection with a helper virus, for example the adenovirus or herpes virus referred to above may be provided in trans in the form of one or more DNA expression plasmids. Since the genome typically encodes only two genes, it is not surprising that AAV packaging capacity as a delivery vector is limited to single strand 4.5 kilobases (kb). However, although this size limitation may limit the genes delivered for alternative gene therapy, it has no side effects on the packaging and expression of short sequences such as RNAi.
The use of AAV in RNAi applications was demonstrated in experiments in which AAV was used to deliver shRNA in vitro to inhibit the expression of p53 and cysteine protease (Caspase)8 (Tomar et al,Oncogene(oncogene) 22: 5712-15(2003)). After cloning of the appropriate sequences into restriction endonuclease digested AAV-2 vectors, AAV virions were produced in HEK293 cells and used to infect HeLa S3 cells. The results indicated a dose-dependent reduction in endogenous levels of Caspase 8 and p 53. Boden et al also use AAV for in vitro delivery of shRNA to inhibit HIV replication in tissue culture systems (Boden, et al,J.Virol(journal of virology) 77 (21): 115231-35(2003)) by culturing in spent stateP24 production in the base was evaluated.
However, technical hurdles must be overcome when using AAV as a 1-x RNAi expression construct. For example, various percentages of the human population may have neutralizing antibodies against a particular AAV serotype. However, since there are several AAV serotypes, the percentage of individuals with neutralizing antibodies is greatly reduced for some of them, other serotypes can be used, or a pseudo-packaging (pseudo-typing) can be used. There are at least ten different serotypes that have been identified (see De et al Mol Ther, 2006 Jan; 13 (1): 67-76), as well as many others that have been isolated but lack detailed descriptions. Another limitation is that AAV-based therapies may be performed only once as a result of a possible immune response to AAV; however, as an alternative, serotypes of non-human origin may be used repeatedly. The route of administration, the serotype, and the components of the genome being delivered all affect tissue specificity (see, e.g., Zhu et al,Circulation(cycle). 2005 Oct 25; 112(17): 2650-9).
Another limitation in using unmodified AAV systems with 1-x RNAi expression constructs is that transduction can be inefficient. Stable transduction in vivo is likely to be within 5-10% of the cell limit. However, different methods of elevating stable transduction levels are known in the art. One approach is to use pseudotyped packaging, in which the AAV-2 genome is packaged with cap proteins derived from other serotypes. For example, Mingozzi et al increased stable transduction to about 15% of hepatocytes by replacing their AAV-2 counterpart with the AAV-5 cap gene (Mingozzi, et al,J.Virol(journal of virology) 76 (20): 10497-502(2002)). Thomas et al, transduced mouse hepatocytes with AAV8 capsid gene for over 30% (Thomas, et al,J Virol(journal of virology) 2004 Mar; 78(6): 3110-22). Grimm et al (Blood(blood) 2003-02-0495) use AAV-1, AAV-3B, AAV-4, AAV-5, and AAV-6 pseudotyped for tissue culture studies to pack AAV-2. The highest level of transgene expression was induced by pseudotyped virions with AAV-6; production of almost 2 over AAV-2000% of transgenes were expressed. Thus, the present invention contemplates the use of pseudotyped AAV viruses to achieve higher levels of transformation with corresponding increases in expression of the 1-x RNAi expression construct.
Self-complementary AAV vectors may also be used according to embodiments of the invention. The most significant difference between standard AAV vectors and self-complementing vectors is the form of their genome and the size of the packaging. Standard AAV vectors have 4.6Kb of single-stranded DNA, while self-complementary AAV vectors have 2.3Kb of double-stranded DNA. AAV vectors can be transformed into self-complementary vectors by introducing mutations/deletions in one of the Inverted Terminal Repeats (ITRs). Each AAV genome has two such repeats located at the 5 'and 3' ends. Replication is usually initiated at one of the ITRs and through the entire genome and is dissociated at the other ITR. For this reason, AAV vectors contain either a plus-or minus-strand genome. The sequences most effective in controlling transcriptional dissociation are the D-sequence and the termination dissociation site (trs). These sequence sites are located between nucleotides 122-144 of the AAV2 genome (Wang et al (2003)Gene Therapy(Gene therapy) 10: 2106-2111), and deletion of them does not allow the dissociation of transcription at the ITR. It should be noted that since the ITRs of the AAV vector are nearly identical, both the D-sequence and the trs can be deleted in either ITR. As a result of deleting the ITR D-sequence and trs, the extended replicative complex is no longer dissociated and the complex is extended in a direction opposite to the original direction, i.e., if the replicative complex first produces a positive strand, it cannot dissociate at the deleted ITR and then produces a negative strand complementary to the positive strand. This in turn results in a self-complementary double stranded DNA molecule to be packaged into an AAV vector, provided that it is no more than 2.3kb in length, and preferably shorter. It should be noted that since the ITRs of AAV vectors recombine during replication, a revertant phenotype, i.e., two ITRs reverting to the wild-type sequence, can be generated. To alleviate this problem, it is necessary to use ITRs for different types of AAV vectors. For example, AAV2 left ITR and AAV4 deleted right ITR, etc. The only criterion controlling the selection of ITRs to be bound is sequence identity between ITR serotypes. ITRs of serotypes 2 and 5 are nearly identical, and I of serotypes 2 and 4TR has a similarity of 81.6%. After deletion of the D sequence and trs, the sequence identity between the ITRs of AAV2 and AAV4 dropped to just over 50%. The binding of these two ITRs thus results in good binding of the differentiated ITRs and will result in self-complementing AAV vectors that no longer reproduce progeny with wild-type ITRs.
Self-complementing vectors have a great advantage over single-stranded vectors by virtue of their ability to transduce cells efficiently. AAV vectors have been shown to transduce over 95% of targeted hepatocytes when pseudotyped with the envelope protein of AAV8 (see Nakai et al)J Virol(journal of virology) 2005 Jan; 79(1): 214-24 and Grimm et al,J Virol.(journal of virology) 2006 Jan; 80(1): 426-39).
Another viral delivery system that can be used with the 1-x RNAi expression constructs of the invention is a retroviral-based system. Retroviruses include single-stranded RNA animal viruses, which have two unique characteristics. First, the retroviral genome is diploid, consisting of two copies of RNA. Second, the RNA is transcribed into double-stranded DNA by the virion-associated enzyme reverse transcriptase. The double stranded DNA or provirus is then integrated into the host genome and passed from parent cells to progeny cells as a component of stable integration of the host genome.
In some embodiments, lentivirus is a preferred member of the transcriptiviridae family for use in the present invention. Lentiviral vectors are often pseudopackaged by vesicular stomatitis virus glycoprotein (VSV-G) and are derived from Human Immunodeficiency Virus (HIV), the causative agent of acquired human immunodeficiency syndrome (AIDS); midie and visna virus (visan-maedi), which causes encephalitis (sheep demyelinating leukomeningitis) or pneumonia in sheep; equine Infectious Anemia Virus (EIAV), which causes autoimmune hemolytic anemia and encephalopathy in horses; feline Immunodeficiency Virus (FIV), which causes immunodeficiency in cats; bovine Immunodeficiency Virus (BIV), which causes lymphadenopathy and lymphoglobus enlargement in cattle; and Simian Immunodeficiency Virus (SIV), which causes immunodeficiency and encephalopathy in non-human primates. HIV-based vectors typically maintain < 5% of the parent genome, and < 25% of the genome is integrated into the packaging construct, which minimizes the possibility of producing HIV capable of replication-back. Biological safety is further increased by the development of self-inactivating vectors with the deletion of regulatory elements downstream of the long terminal repeat, eliminating transcription of packaging signals necessary for vector mobilization.
Reverse transcription of the retroviral RNA genome occurs in the cytoplasm. Unlike C-type retroviruses, lentivirus cDNA complexes with other viral factors-known pre-initiation complexes-can translocate across the nuclear membrane and transduce non-dividing cells. The structural feature of viral cDNA-DNA arms (flaps) -appears to bring about efficient nuclear import. The arms are dependent on the integrity of the central polypurine tract (cPPT) located within the viral polymerase gene, so most lentivirus-derived vectors retain this sequence. Lentiviruses have extensive cell tropism, low inflammatory potential, and produce intact vectors. The main limitation is that in some applications the whole may induce carcinogenesis. The main advantage of using lentiviral vectors is that gene transfer is durable in most tissues or cell types.
The lentivirus-based construct for expression of ddRNAi factors preferably includes sequences from the lentivirus 5 'and 3' LTRs. More preferably, the viral construct comprises an inactivated or self-inactivated lentivirus-derived 3' LTR. Self-inactivation of the 3' LTR may be performed by any method known in the art. In a preferred embodiment, the U3 element of the 3' LTR includes deletion of its enhancer sequences, preferably the TATA box, Sp1 and NF-. kappa.B sites. As a result of self-inactivation of the 3 'LTR, the provirus integrated into the host cell genome will include an inactivated 5' LTR. The LTR sequence may be an LTR sequence from any lentivirus. Lentivirus-based constructs can also incorporate sequences for MMLV or MSCV, RSV or mammalian genes. In addition, the U3 sequence derived from the lentiviral 5' LTR may be replaced by a promoter sequence in the viral construct. This can increase the titer of virus obtained from the packaging cell line. Enhancer sequences may also be included.
Other viral or non-viral systems known to those skilled in the art may also be used to deliver the 1-x RNAi expression cassettes of the invention to relevant cells, tissues or organs, including but not limited to gene-deleted adenovirus-transposon vectors, which stably maintain the virally encoded transgene in vivo by integration into a host cell (see Yant, et al,Nature Biotech(natural biotechnology) 20: 999-; derived from Sindbis (Sindbis) virus or Semliki forest (Semliki forest) virus (see Perri, et al,J.Virol(journal of virology) 74 (20): 9802-07 (2002)); systems derived from newcastle disease virus or Sendai (Sendai) virus; or a small circular DNA (mini-circle DNA) vector lacking bacterial DNA sequences (see Chen, et al,Molecular Therapy(molecular therapy) 8 (3): 495-500(2003)). The small circular DNA as described in U.S. patent publication 2004/0214329 discloses a vector that provides sustained high levels of protein. The circular vector is characterized by the lack of expression of resting bacterial sequences and includes unidirectional site-specific recombination product sequences and expression cassettes.
In addition, hybrid virus systems can be used to combine the features of two or more virus systems. For example, site-specific integration mechanisms of wild-type AAV may be accompanied by efficient internalization and nuclear targeting characteristics of the adenovirus. AAV undergoes a high-yield replication cycle in the presence of adenovirus or herpesvirus; however, in the absence of helper functions, the AAV genome integrates into a specific site on chromosome 19. Integration of the AAV genome requires expression of the AAV rep protein. Since typical rAAV vectors have deleted all genes of the virus, including rep, they cannot be specifically integrated into chromosome 19. However, this feature can be exploited in a suitable hybrid system. In addition, non-viral genetic elements can be used to achieve desirable properties in viral delivery systems, such as genetic elements that can allow site-specific recombination.
In FIG. 1, step 400, a 1-x RNAi expression construct is packaged into a viral particle. Any method known in the art can be used to produce infectious virions whose genomes include one copy of the viral 1-x RNAi expression construct. FIGS. 3A and 3B show alternative methods for packaging the 1-x RNAi expression constructs of the invention into virions for delivery. The method in FIG. 3A utilizes packaging cells that stably express viral proteins necessary for the integration of viral 1-x RNAi expression constructs into virions in trans, as well as other sequences necessary or preferred for a particular viral delivery system (e.g., sequences required for replication, structural proteins, and viral assembly), and for entry into tissues or derived from viruses or artificial ligands. In FIG. 3A, the 1-x RNAi expression cassette is ligated to a viral delivery vector (step 300) and the resulting viral 1-x RNAi expression construct is used to transfect a packaging cell (step 410). The packaging cell then replicates the viral sequences, expresses viral proteins, and packages the viral 1-x RNAi expression construct into an infecting virion (step 420). The packaging cell line may be any cell line capable of expressing viral proteins including, but not limited to 293, HeLa, a549, PerC6, D17, MDCK, BHK, bin chery, phoenix, Cf2Th, or any other cell line known or developed by those skilled in the art. One type of packaging cell is described, for example, in U.S. patent 6,218,181.
Alternatively, a cell line that is not stably expressing the necessary viral proteins may be co-transfected with two or more constructs to achieve efficient production of functional particles. One of the constructs comprises a viral 1-x RNAi expression construct and the other plasmid comprises nucleic acid encoding proteins necessary for the cell to produce a functional virus (replication and packaging construct) as well as other helper functions. The method shown in FIG. 3B utilizes cells that do not stably express viral replication and packaging genes for packaging. In this case, the 1-x RNAi expression construct is ligated to a viral delivery vector (step 300), followed by co-transfection with one or more vectors that express sequences necessary for replication and production of infectious virions (step 415). The cells replicate the viral sequences, express viral proteins and package the viral RNAi expression construct into an infecting virion (step 420).
The packaging cell line or replication and packaging construct may not express the envelope gene product. In these embodiments, the gene encoding the envelope gene may be provided on a separate construct that is co-transfected with the viral 1-x RNAi expression construct. The envelope protein is partly responsible for the host range of the virion, and the virus can be pseudotyped. As previously mentioned, a "pseudotyped packaging" virus is a virion whose envelope protein is from a virus other than the virus from which its genome was derived. One skilled in the art can select an appropriate pseudotype package for the viral delivery system used and the cells to be targeted. In addition to conferring a specific host range, the pseudotyped packaging chosen may allow for very high titers of virus to be concentrated. The virus may alternatively be pseudotyped with a monotropic envelope protein that limits infection to a particular species (e.g., the monotropic envelope may only infect, e.g., murine cells, and the amphotropic envelope may infect, e.g., human and murine cells). In addition, the genetically modified ligands can be used for cell specific targeting, for example asialoglycoprotein for hepatocytes, or for receptor mediated binding of transferrin.
Following production in packaging cell lines, viral particles containing the 1-x RNAi expression cassette were purified and quantified (titrated). Purification strategies include density gradient centrifugation, or preferably, column chromatography methods.
In step 500 of FIG. 1, a 1-x RNAi expression construct is delivered to a cell, tissue, or organ of interest. The 1-x RNAi expression constructs of the invention can be introduced into cells in vitro (in vitro) or ex vivo (ex vivo) and then placed into animals for therapeutic purposes, or administered directly to organisms, organs or cells by in vivo administration. Delivery by viral infection is a preferred method of delivery; however, any suitable method can be used to deliver the 1-x RNAi expression constructs. The cassette-containing vector can be administered to a mammalian host using any conventional method, a number of different such methods being known in the art.
1-xRNAi expression constructs equipped with appropriate promoters and terminators, such as those derived from the T7 or T3 phage, or other promoters and terminators known to those of skill in the art, can be used for in vitro transcription of the template. Thus, RNA hairpin molecules of variable length (single hairpins with 2, 3, 4 or more functionalities) can be produced in vitro, which can be purified and administered as follows.
Various techniques are available for delivering nucleic acids into cells and are well known, such as liposome or micelle-mediated transfection or transformation, transformation of cells or bacterial cells with attenuated viral particles, cell conjugation, transformation or transfection procedures or microinjection, which are well known to those skilled in the art.
The most common transfection reagents are charged lipophilic compounds capable of crossing cell membranes. When they are mixed with nucleic acids, they can carry DNA across cell membranes. A large number of such compounds are commercially available. Polyethyleneimine (PEI) is a new class of transfection reagents with chemical properties that are significantly different from lipophilic compounds that act in a similar manner, but with the advantage that they can cross the nuclear membrane. One example of such a reagent is ExGen 500 (Fermentas). The constructs or synthetic genes according to the invention may be packaged in linear fragments within synthetic liposomes or micelles for delivery into target cells.
Tissue culture cells can be transformed using electroporation. This is to allow for the creation of transient pores in the cell membrane through which DNA or RNA enters the cell. In addition, animal cells can be chemically transformed using reagents such as PEG or calcium phosphate.
Alternatively, ddRNAi expression constructs containing the 1-xRNAi expression cassette can be introduced into the relevant cell, tissue or organ by other routes, including microinjection or vesicle fusion. E.g. Furth et alAnal.Biochem(analytical chemistry) 115 (205): 365-. Nucleic acid can be coated on gold microparticles and passedParticle bombardment devices are delivered intradermally, or according to literature (see, e.g., Tang et al,Nature(Nature) 356: 152- & 154(1992)), in which gold particles are coated with DNA and then bombarded into relevant cells, tissues or organs.
Another delivery method for the method of the present invention includes the use of Cyclosert as described in U.S. Pat. No. 6,509,323 to Davis et alTMProvided is a technique. CyclosertTMThe technical platform is based on a cup-shaped ring-repeat molecule of a sugar called cyclodextrin. The "cup" of the cyclodextrin molecule can form an "inclusion complex" with other molecules, allowing it to bind with other moieties to the cyclodextrin moleculeTMPolymers to improve stability or to add targeting ligands. Additionally, cyclodextrins have been found to be substantially safe in humans (cyclodextrins alone generally promote the solubility of FDA-approved oral and intravenous drugs) and pharmaceutical grade cyclodextrins can be purchased in large quantities at low cost. These polymers are highly water soluble, non-toxic and non-immunogenic at therapeutic doses even when repeatedly administered. The polymers can be easily adapted to carry a wide range of small molecule therapeutics with drug loadings significantly higher than liposomes.
The vector containing the 1-x RNAi expression cassette of the present invention can be formulated into an injectable preparation, or administered by dissolving, suspending or emulsifying in an aqueous or non-aqueous solvent, such as oil, synthetic fatty acid glyceride, ester of higher fatty acid or propylene glycol; and, if desired, with the customary additives, such as solubilizers, isotonizing agents, suspending agents, emulsifiers, stabilizers and preservatives.
Alternatively, vectors containing the RNAi expression cassettes of the invention can be prepared into pharmaceutical compositions by combining with an appropriate, pharmaceutically acceptable carrier or diluent. In pharmaceutical dosage forms, the vector containing the 1-x RNAi cassette can be administered alone or in combination with other pharmaceutically active compounds.
Examples
To select candidate sequences targeted by the 1-x RNAi cassettes of the invention, all published independent full-length or near full-length HCV sequences are aligned; about 100 such sequences representing all genotypes are currently available. Several candidate regions for selection and development of RNAi therapeutics currently exist, and the 5 'and 3' UTR regions are well documented as belonging to the most highly conserved regions in the HCV genome. Although considering that these non-coding sequences may not represent the optimal sequence for targeting due to steric hindrance with cellular transfer complex proteins or regulatory proteins, Yokota et al have identified highly functional RNAi of the 5' UTR in a targeted replicative system (R) ((R))EMBO Rep(European molecular biology society report) 4 (6): 602-608(2003)). Although it is advantageous to identify several regions of absolute identity within individual 21 nucleotide stretches (corresponding in size to the targeting sequences in shRNA species), current analysis shows that such conservation does not occur within different subtypes of a particular genotype, let alone the entire genotype. Thus, fragments are selected that may include genomes in which greater than 80% of the regions remain absolutely conserved. The expression of three independent shrnas compensates for sequence variability and may allow for combination therapies contained in a single delivery vehicle.
Alternatively, if no conserved region was identified that met the selection criteria in all HCV genotyping analyses, the sequence analysis could be limited to genotype 1(1a and 1b), which accounts for nearly 3/4 in the infected population in the united states, which is the dominant genotype worldwide except continent. In addition, the currently most effective anti-HCV therapy, i.e., the combination of pegylated interferon (pegylate interferon) and ribavirin (a guanine nucleoside analog), is quite ineffective against genotype 1, but highly effective against other genotypes. Thus, the greatest need for alternative therapies exists in the largest patient population. Since sequence alignment shows only homology, other selection criteria such as relative GC content and lack of cross-specificity when querying sequence databases are used in selecting the final RNAi agent to be tested.
For example, for one experiment, multiple sequences from HCV subtypes 1a and 1b were compared. Conserved regions were identified from which sufficient length was available to select RNAi agents (> 19 nucleotides) for detection. The 5 'NTR and 3' NTR regions are the most conserved regions. Since the identified regions of homology are rather long, comparisons are also made between different subtypes. Regions that are generally conserved can be selected in conjunction with the two alignments. Some regions are removed from consideration, such as long sections of A or U, or G and C, leaving "qualified" regions for further selection. Only one region of general conservation was identified throughout the coding region (open reading frame) of all genotypes of HCV under consideration; thus, the sequences selected for targeting are in most cases sequences conserved in subtypes 1a and 1 b.
Once a "qualified" region is identified, individual RNAi sequences should be selected using criteria in which the free energy at the 5 'end of the antisense strand of the RNAi agent should be lower than at the 3' end. The "adjacent pairing free energy" rule was used to calculate the free energy of the last five nucleotides at both the 5 'and 3' ends of all potential RNAi agents selected so far. As a result, 56 potential RNAi agents were identified: thirty at 5 'NTR (5' -n), twelve in open reading frame (c-n), and fourteen in 3 'NTR (3' -n) (see Table 1). The positions of the sequences of sequence numbers 1-10 and 31-50 are schematically shown in fig. 7.
Table 1: rnai sequences
| RNAi agents | Sequence of | Serial number | Luc-HCV reporter plasmid |
| 5′-1 | gCTGTGAGGAACTACTGTCT | Sequence 1 | 20 |
| 5′-2 | GTCTAGCCATGGCGTTAGT | Sequence 2 | - |
| 5′-3 | GGAGAGCCATAGTGGTCTG | Sequence 3 | 16，20 |
| 5′-4 | GCGGAACCGGTGAGTACAC | Sequence 4 | 16 |
| 5′-5 | GTCTGCGGAACCGGTGAGTA | Sequence 5 | 16 |
| 5′-6 | GCGAAAGGCCTTGTGGTACT | Sequence 6 | 16，17 |
| 5′-7 | GATAGGGTGCTTGCGAGTG | Sequence 7 | 16 |
| 5′-8 | GAGGTCTCGTAGACCGTGCA | Sequence 8 | 16，17 |
| 5′-9 | gCTTGTGGTACTGCCTGATA | Sequence 9 | - |
| 5′-10 | gCTGCCTGATAGGGTGCTTG | Sequence 10 | 17 |
| 5′-11 | ATCACTCCCCTGTGAGGAA | Sequence 11 | - |
| 5′-12 | ACTCCCCTGTGAGGAACTA | Sequence 12 | - |
| 5′-13 | CGTCTAGCCATGGCGTTAG | Sequence 13 | - |
| 5′-14 | TCTAGCCATGGCGTTAGTA | Sequence 14 | - |
| 5′-15 | CTAGCCATGGCGTTAGTAT | Sequence 15 | - |
| 5′-16 | TGTCGTACAGCCTCCAGGC | Sequence 16 | - |
| 5′-17 | CCGGGAGAGCCATAGTGGT | Sequence 17 | - |
| 5′-18 | AGAGCCATAGTGGTCTGCG | Sequence 18 | - |
| 5′-19 | GCCATAGTGGTCTGCGGAA | Sequence 19 | - |
| 5′-20 | CCGGTGAGTACACCGGAAT | Sequence 20 | - |
| 5′-21 | CGGTGAGTACACCGGAATC | Sequence 21 | - |
| 5′-22 | GACTGGGTCCTTTCTTGGA | Sequence 22 | - |
| 5′-23 | GACCGGGTCCTTTCTTGGA | Sequence 23 | - |
| 5′-24 | ACCGGGTCCTTTCTTGGAA | Sequence 24 | - |
| 5′-25 | TGGGTTGCGAAAGGCCTTG | Sequence 25 | - |
| 5′-26 | TTGCGAAAGGCCTTGTGGT | Sequence 26 | - |
| 5′-27 | AGGCCTTGTGGTACTGCCT | Sequence 27 | - |
| 5′-28 | TAGGGTGCTTGCGAGTGCC | Sequence 28 | - |
| 5′-29 | CGGGAGGTCTCGTAGACCG | Sequence 29 | - |
| 5′-30 | GGTCTCGTAGACCGTGCAT | Sequence 30 | - |
| C-1 | AGATCGTTGGTGGAGTTTA | Sequence 31 | - |
| C-2 | gTTGGGTAAGGTCATCGATA | Sequence 32 | - |
| C-3 | GCCGACCTCATGGGGTACAT | Sequence 33 | 18 |
| C-4 | GGTTGCTCTTTCTCTATCT | Sequence 34 | - |
| C-5 | GGGATATGATGATGAACTG | Sequence 35 | - |
| C-6 | GGATGAACCGGCTAATAGC | Sequence 36 | - |
| C-7 | GGAGATGGGCGGCAACATC | Sequence 37 | - |
| C-8 | GTCTTCACGGAGGCTATGA | Sequence 38 | - |
| C-9 | GTCAACTCCTGGCTAGGCAA | Sequence 39 | - |
| C-10 | gTCCACAGTTACTCTCCAGG | Sequence 40 | - |
| C-11 | gCCTCTTCAACTGGGCAGTA | Sequence 41 | - |
| C-12 | AGCTTAAACTCACTCCAAT | Sequence 42 | C11 and 12, C6-C9-C12-3' 1 |
| 3’-1 | GCTCCATCTTAGCCCTAGT | Sequence 43 | 19 |
| 3’-2 | gTCCATCTTAGCCCTAGTCA | Sequence 44 | 19 |
| 3’-3 | GTCACGGCTAGCTGTGAAA | Sequence 45 | 19 |
| 3’-4 | ACGGCTAGCTGTGAAAGGT | Sequence 46 | 19 |
| 3’-5 | GCTGTGAAAGGTCCGTGAG | Sequence 47 | 19 |
| 3’-6 | GGTCCGTGAGCCGCATGAC | Sequence 48 | - |
| 3’-7 | GCCGCATGACTGCAGAGAGT | Sequence 49 | - |
| 3’-8 | ACTGGCCTCTCTGCAGATCA | Sequence 50 | - |
| 3’-9 | TAGCCCTAGTCACGGCTAG | Sequence 51 | - |
| 3’-10 | AGCTGTGAAAGGTCCGTGA | Sequence 52 | - |
| 3’-11 | TAGCTGTGAAAGGTCCGTG | Sequence 53 | - |
| 3’-12 | CTAGCTGTGAAAGGTCCGT | Sequence 54 | - |
| 3’-13 | CTGTGAAAGGTCCGTGAGC | Sequence 55 | - |
| 3’-14 | GAAAGGTCCGTGAGCCGCA | Sequence 56 | - |
TABLE 2 luciferase-HCV fusion reporter plasmids
| Reporter plasmid | Including a target object |
| #20 | 5 '1 to 5' 5 |
| #16 | 5 '3 to 5' 10 |
| #17 | 5 '6 to 5' 10 |
| #12 | 5 '7 to 5' 10, code-1 |
| #18 | Code-3 |
| #19 | 3 '1 to 3' 8 |
| C2 and 4 | Code-2, code-4 |
| C5 | Code-5 |
| C6 | Code-6 |
| C7 | Code-7 |
| C8 | Code-8 |
| C9 | Code-9 |
| C10 | Code-10 |
| C11 and C12 | Code-11, code-12 |
| C6-C9-C12-3’1 | Code-6, code-9, code-12, 3' 1 |
Example 1:selection and detection of 1-x RNAi expression cassettes against disease or dysfunction
The selection of shrnas as therapeutics against diseases or disorders is not a straightforward subject. In addition to the emergence of escape variants in the treatment of viral infections, the high mutation rates of viral and oncogenes lead to a considerable degree of sequence differentiation in the population of infected individuals. For example, individuals infected with hepatitis virus may carry viruses that differ in genotype by 31-34% in nucleotide sequence, and subtypes (species within a given genotype) may differ by 20-23% based on comparison of full-length genomic sequences. Thus, for HCV, highly conserved regions of the viral genome are identified and selected to ensure the broadest therapeutic application. To select candidate sequences, all published sequences independently full or near full length are aligned. The results of the analysis are summarized to give a list of candidate RNAi sequences. To rank the sequences according to relative strength, the ability of individual presynthesized RNAi agents to inhibit the activity of a target gene was examined. The same method is used when targeting oncogenes, developmental genes and the like.
To test the efficacy of the selected RNAi agents, the presynthesized RNAi agents are transfected into the relevant cells, tissues or organs by standard techniques and reagents. Irrelevant RNAi species were transfected into a parallel set of dishes as negative controls. Transfection efficiency was monitored by small non-specific RNAi containing luciferase or phycoerythrin tagged to the end of the transfection mixture. Relative transfection efficiency was measured by fluorescence microscopy prior to down-regulation efficacy analysis. At various time points after transfection, target gene activity was measured by one of a variety of methods.
Individually, and in the context of the 1-x RNAi cassettes of the invention, highly functional ddRNAi agents are selected and measured. In embodiments using 1-x RNAi expression cassettes, the RNAi factors are validated and the coding sequence for each of the related shRNAs is generated from a long, complementary self-annealing oligonucleotide and cloned into a single site of the 1-x RNAi cassette. The cassette is then inserted into a virus-selecting vector according to the methods described herein, and the construct is then packaged into a virion. Due to the short total length of each shRNAi component of the 1-x RNAi expression cassette (-70 nucleotides); ligating up to five RNAi components together yields a length of less than or equal to about 350 nucleotides, and even including promoters and terminators of the 1-x RNA expression cassette, with a total length well below the upper limit of the payload limit of, for example, self-complementary AAV and other viruses.
The inhibitory activity of the virions was examined on Huh-7 cells. Generation of 1-x RNAi constructs expressing unrelated shRNA species was used for negative controls. The efficacy of the shRNA sequences was monitored by the analytical techniques described above.
Example 2: development of 1-x RNAi expression constructs
Construction of a 1-x RNAi expression construct includes a promoter that drives expression of three or more single shRNA species at similarly sufficient levels. Synthesis of small nuclear RNA and transport rRNA is directed by RNA polymerase III (pol III) under the control of a pol Ill-specific promoter. Due to the relatively high abundance of transcription directed by these regulatory elements, the pol III promoter, including these genes derived from U6 and H1, was used to drive expression of 1-x RNAi. (see, e.g., Domitrovich and Kunkel.Nucl.Acids Res(nucleic acid research) 31 (9): 2344-52 (2003); boden, et alNucl.Acids Res(nucleic acid research) 31 (17): 5033-38(2003 a); and Kawasaki, et alNucl.Acids Res(nucleic acid research) 31 (2): 700-7(2003)).
The detection of the 1-3RNAi expression constructs (#251- #256) using the U6 promoter is shown in FIG. 4. They include three RNAi agents directed against different regions of the HCV genome. The sequences in the cassettes are different in order to test the requirements of the different structures required for the function of the siRNA. In FIG. 4, expected results are presented below each construct according to pre-established techniques for controlling the action of RNA interference mechanisms. The expected successful siRNA functional results are shown as "+", and the expected unsuccessful siRNA functional results are shown as "-". In the case of failure of the expected result, the expected cause is due to sequence deletion or mismatch. Question mark indicates that the question may or may not be significant enough to terminate the activity of the hairpin construct.
To test the efficacy of the selected RNAi sequences, the 1-x RNAi expression constructs of the invention were delivered directly to cultured cells along with the Luc-HCV reporter plasmid. The Luc-HCV plasmid used is the construct shown in fig. 7 and comprises a luciferase sequence that hybridizes to various HCV target sequences of 100, 90, 80, 70, 60, 50, 40, 30 or 20Bp (note: 100Bp is correct for multiple targets in one reporter, while for most single target reporters this sequence corresponds to the targets plus 5 nucleotides at the 5 'and 3' ends), from which the RNAi sequence is derived. RNAi factors directed to sequence fragments within 100bp, if available, will degrade HCV-luciferase transcripts, thus reducing (possibly eliminating) luciferase expression. Table 1 lists RNAi agents, some of which were tested. Table 2 lists some relevant Luc-HCV reporter plasmids and the targeted HCV target regions used. FIG. 7 shows a schematic representation of the positions in the HCV genome of the target targets of the RNAi factors in Table 1.
As shown in fig. 5 and 6, the relative strength of each 1-x RNAi construct was evaluated in vitro by a decrease in co-transfected luciferase reporter activity. The test and reporter constructs are transfected into permissive cells using standard techniques. The Luc-fusion reporter plasmid shown in figure 8 was co-transfected into Huh-7 cells with the expression plasmid encoding the three hairpin shRNA species in figure 4. Plasmids for renilla luciferase expression were also included to normalize sample-to-sample transfection efficiency. In each case a total of n-4 independent transfections were used. At 72 hours post-transfection, samples were harvested and analyzed for relative levels of luciferase activity of the firefly. Plasmids containing the promoter driving expression of a single shRNA U6 against the 5' -8 or C-12 target sequences served as positive controls for these experiments, and plasmids containing the U6 promoter but no downstream shRNA sequence served as negative controls, and thus served as criteria by which to evaluate the level of inhibition induced by shRNA expressed from a single or three hairpin plasmids. 1-3RNAi constructs containing RNAi directed to the 5 '8 region of the HCV's 5 'UTR and encoding the 12 region were co-transfected with a luciferase HCV reporter plasmid containing the 5' 8 or c12 target site. The activity of luciferase is effectively inhibited only when the hairpin is directed at the corresponding target site as expected (i.e. there is only one or no mismatch in the expected hairpin).
Example 3 in vivo evaluation of three hairpin constructs
Three hairpin constructs were evaluated in vivo by co-transfecting murine hepatocytes with the appropriate luc-fusion reporter plasmid and 1-3RNAi constructs 251, 252, 253, 254, 255, 256, a positive control plasmid, or a negative control plasmid using the hydraulic tail vein injection method. In addition, mice were injected with plasmids expressing human α 1 antitrypsin (α 1-AT), which was used for normalization of transfection efficiency. At 48 hours post injection, animal sera were collected, sacrificed and livers harvested. The luciferase activity of the firefly from the liver lysate was analyzed using the Promega luciferase kit, and the serum samples were analyzed for α 1-AT using ELISA. The level of inhibition induced by 1-x RNAi expressed from the hairpin construct was evaluated relative to a negative control.
Example 4
The 1-x RNAi expression cassette can also be used as a template to produce RNAi species that can be directed directly to cells or tissues by using one of the aforementioned nucleic acid delivery techniques. To demonstrate the utility of this type of approach, the T7 promoter was introduced upstream of a 1-x RNAi expression cassette containing three shRNA hairpins oriented to separate reporter constructs containing HCV5 '-8, 5' 6 or C-12 target sequences. To use the runaway transcription technique to generate RNA containing RNAi sequences in vitro, the template is prepared using restriction enzyme digestion, resulting in the cleavage of the plasmid just downstream of the 1-x RNAi expression cassette. T7 RNA polymerase was added to the reaction to produce an RNA transcript containing the 1-x expression cassette sequence. In the current example, a lipophilic compound is used as a transfection reagent and the runaway transcript is introduced into the cells at various concentrations along with a set concentration of a series of related luciferase reporter constructs. Separate DNA plasmid expressions encoding the renilla luciferase protein were used for normalization of the transfection efficiency differences between different samples. In each case total N-4 transfections were used and the percent inhibition for each reporter construct was calculated using a set of samples transfected with a mixture containing only the appropriate reporter construct and the renilla plasmid. The results are shown in FIG. 9, showing a dose-dependent decrease in all three reporter constructs by in vitro transcribed 1-x expression constructs. Nonspecific inhibition was monitored by a non-targetable reporter construct (C9 luc fusion) and showed very low inhibition of the triple hairpin in response to in vitro transcription.
While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material or process to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the present invention.
All references cited herein are intended to aid in the understanding of the present invention and are incorporated in their entirety for all purposes without limitation.
Sequence listing
<110> P.W. Roercoke
L. koto
D.A. Suhai
A.a. korea harloff
<120> RNAi expression constructs
<130>BENI 0017
<150>US 60/649,641
<151>2005-02-03
<150>US 60/653,580
<151>2005-02-15
<160>57
<170>PatentIn versi on 3.3
<210>1
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>1
gctgtgagga actactgtct 20
<210>2
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>2
gtctagccat ggcgttagt 19
<210>3
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>3
ggagagccat agtggtctg 19
<210>4
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>4
gcggaaccgg tgagtacac 19
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>5
gtctgcggaa ccggtgagta 20
<210>6
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>6
gcgaaaggcc ttgtggtact 20
<210>7
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>7
gatagggtgc ttgcgagtg 19
<210>8
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>8
gaggtctcgt agaccgtgca 20
<210>9
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>9
gcttgtggta ctgcctgata 20
<210>10
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>10
gctgcctgat agggtgcttg 20
<210>11
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>11
atcactcccc tgtgaggaa 19
<210>12
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>12
actcccctgt gaggaacta 19
<210>13
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>13
cgtctagcca tggcgttag 19
<210>14
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>14
tctagccatg gcgttagta 19
<210>15
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>15
ctagccatgg cgttagtat 19
<210>16
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>16
tgtcgtacag cctccaggc 19
<210>17
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>17
ccgggagagc catagtggt 19
<210>18
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>18
agagccatag tggtctgcg 19
<210>19
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>19
gccatagtgg tctg cggaa 19
<210>20
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>20
ccggtgagta caccggaat 19
<210>21
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>21
cggtgagtac accggaatc 19
<210>22
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>22
gactgggtcc tttcttgga 19
<210>23
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>23
gaccgggtcc tttcttgga 19
<210>24
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>24
accgggtcct ttcttggaa 19
<210>25
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>25
tgggttgcga aaggccttg 19
<210>26
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>26
ttgcgaaagg ccttgtggt 19
<210>27
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>27
aggccttgtg gtactgcct 19
<210>28
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>28
tagggtgctt gcgagtgcc 19
<210>29
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>29
cgggaggtct cgtagaccg 19
<210>30
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>30
ggtctcgtag accgtgcat 19
<210>31
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>31
agatcgttgg tggagttta 19
<210>32
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>32
gttgggtaag gtcatcgata 20
<210>33
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>33
gccgacctca tggggtacat 20
<210>34
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>34
ggttgctctt tctctatct 19
<210>35
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>35
gggatatgat gatgaactg 19
<210>36
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>36
ggatgaaccg gctaatagc 19
<210>37
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>37
ggagatgggc ggcaacatc 19
<210>38
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>38
gtcttcacgg aggctatga 19
<210>39
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>39
gtcaactcct ggctaggcaa 20
<210>40
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>40
gtccacagtt actctccagg 20
<210>41
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>41
gcctcttcaa ctgggcagta 20
<210>42
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>42
agcttaaact cactccaat 19
<210>43
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>43
gctccatctt agccctagt 19
<210>44
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>44
gtccatctta gccctagtca 20
<210>45
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>45
gtcacggcta gctgtgaaa 19
<210>46
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>46
acggctagct gtgaaaggt 19
<210>47
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>47
gctgtgaaag gtccgtgag 19
<210>48
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>48
ggtccgtgag ccgcatgac 19
<210>49
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>49
gccgcatgac tgcagagagt 20
<210>50
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>50
actggcctct ctgcagatca 20
<210>51
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>51
tagccctagt cacggctag 19
<210>52
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>52
agctgtgaaa ggtccgtga 19
<210>53
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>53
tagctgtgaa aggtccgtg 19
<210>54
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>54
ctagctgtga aaggtccgt 19
<210>55
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>55
ctgtgaaagg tccgtgagc 19
<210>56
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>56
gaaaggtccg tgagccgca 19
<210>57
<211>171
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide
<400>57
ggatccgtgc acggtctacg agacctcgaa gcttggaggt ctcgtagacc gtgcatgtac 60
agcgaaaggc cttgtggtac tgaagcttga gtaccacaag gcctttcgcc catggattgg 120
agtgagttta agctgaagct tgagcttaaa ctcactccaa ttttttctag a 171
Claims (8)
1. A 1-x RNAi expression cassette encoding x stem-loop structures comprising a promoter, wherein x is at least 2 and said stem-loop structures are separated from each other by one or more spacer regions, wherein said stem-loop structures are RNAi agents and at least one of said RNAi agents is encoded by sequence No. 42.
2. The 1-x RNAi expression cassette of claim 1, wherein at least one other RNAi agent is encoded by sequence No. 6.
3. The 1-x RNAi expression cassette of claim 1, wherein the spacer region is 6 nucleotides.
4. The 1-x RNAi expression cassette of claim 1, wherein the loop region of the stem-loop structure comprises 5-9 nucleotides.
5. The 1-x RNAi expression cassette of claim 1, further comprising a terminator, and wherein the promoter and terminator are taken from the same gene.
6. A genetic construct capable of regulating the expression of one or more genes, said genetic construct comprising an expression cassette according to any one of claims 1 to 5.
7. Use of a genetic construct according to claim 6 in the manufacture of a medicament for modulating the expression of one or more genes.
8. Use of an RNAi agent that is the product of in vitro transcription of a 1-x RNAi expression cassette according to any one of claims 1-5 in the manufacture of a medicament for modulating expression of one or more genes.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US64964105P | 2005-02-03 | 2005-02-03 | |
| US60/649641 | 2005-02-03 | ||
| US65358005P | 2005-02-15 | 2005-02-15 | |
| US60/653580 | 2005-02-15 | ||
| PCT/US2006/004003 WO2006084209A2 (en) | 2005-02-03 | 2006-02-03 | Rnai expression constructs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1118577A1 HK1118577A1 (en) | 2009-02-13 |
| HK1118577B true HK1118577B (en) | 2013-10-11 |
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