WO2002034908A2

WO2002034908A2 - Method for temporally controlling antisense-mediated gene inactivation

Info

Publication number: WO2002034908A2
Application number: PCT/EP2001/012248
Authority: WO
Inventors: Alexander D. Crawford; Camilla V. Esguerra
Original assignee: Mermaid Pharmaceuticals Gmbh
Priority date: 2000-10-23
Filing date: 2001-10-23
Publication date: 2002-05-02
Also published as: AU2002224787A1; WO2002034908A3

Abstract

The present invention relates to a novel method for inactivating genes in model organisms in a temporally specific manner. In particular, the invention relates to the delay of antisensemediated inhibition of gene expression by temporarily blocking the transcript-binding activity of antisense oligomers, which facilitates the stage-specific inactivation of individual genes and enables the functional analysis of genes with multiple functional roles in vertebrate development.

Description

Method for temporally controlling antisense-mediated gene inactivation

The present invention relates to a novel method for inactivating genes in model organisms in a temporally specific manner In particular, the invention relates to the delay of antisensemediated inhibition of gene expression by temporarily blocking the transcript-binding activity of antisense oligomers, which facilitates the stage-specific inactivation of individual genes and enables the functional analysis of genes with multiple functional roles in vertebrate development

Background of the invention

The targeted inactivation or inhibition of gene expression in cells and model organisms is an accepted strategy for the analysis of gene function, and thereby an important tool for the identification and validation of disease-relevant drug targets for drug discovery applications Established approaches for the inactivation of gene expression include gene targeting via homologous recombination, RNA interference (RNAi), and antisense-mediated inhibition of gene expression both at the translational and transcπptional level using classical and nbozyme-based approaches

Although the above-mentioned methods have proven to be powerful tools for the analysis of gene function in vivo, the full elucidation of gene function is difficult or impossible if gene inactivation results in embryonic lethality This often occurs in cases where a gene performs multiple functions in an animal including an important role in early embryonic development Thus, the function of such a gene in later stages of development or in the adult can no longer be determined because the embryo does not survive beyond a certain developmental stage Methods capable of temporally controlling gene inactivation are therefore of significant interest in functional genomics Gene targeting has been established for several species using a number of different methods such as homologous recombination in embryonic stem cells in mice (Thompson et a/ , 1989, Mansour, et al , 1990), meiotic recombination in fruit flies (Rong and Golic 2000) and nuclear transfer from cultured somatic cells in sheep (McCreath et al , 2000) In gene targeting approaches, the problem of temporal control has been solved through the use of the bactenophage P1 -derived Cre-loxP recombination system to achieve temporal and spatial regulation of gene expression through somatic mutagenesis in mice (Kuhn et al , 1995, Rajewsky et a/ , 1996, Tsien et al , 1996, Betz et a/ . 1996, Rickert ef a/ , 1997, Alimzhanov et al , 1997, Schwenk et al , 1998) This method requires the generation of two stable lines of transgenic mice, one line containing loxP sites flanking the gene of interest introduced into the target gene locus through homologous recombination in mouse embryonic stem cells, and the second line containing Cre recombinase under the control of a tissue-specific and/or inducible promoter Alteration of the target gene is accomplished through Cre-mediated deletion of loxP-flanked gene segments Mosaic mice are produced, which contain a certain proportion of mutant cells in specific tissues or organs, thus allowing gene function to be analyzed in a temporal and tissue-specific manner The production of stable transgenic mice expressing the appropriate level of Cre recombinase is nevertheless tricky and cumbersome, therefore rendering this method unsuitable for the systematic analysis of gene function on a large scale

Temporally and tissue-specific gene inactivation has also been achieved through the creation of transgenic organisms expressing antisense RNA (Galvm-Parton, et al , 1997, Kaya, ef al , 1997, Schinke, et al , 1999, Eπckson, 1999, Terada et al , 2000, Weber ef al , 2000) The successful use of antisense transgenics has been reported for several cases in plants, rats and mice

A related method uses nbozymes instead of antisense RNA Ribozymes are catalytic RNA molecules ith enzyme-like cleavage properties, which can be designed to target specific RNA sequences (Haseloff and Gerlach, 1988) Successful target gene inactivation, including temporally and tissue-specific gene inactivation, using ribozymes has been reported in mouse, zebrafish and fi uitflies (Zhao and Pick, 1993, Larsson et a/ , 1994, Lieber and Kay, 1996 Xie et a/ , 1997, Andang et al , 1999) However, this approach has certain limitations Prior to the identification of a nbozyme targeted to a specific RNA that can effectively inactivate gene function, numerous nbozymes have to be designed and tested RNA interference (RNAi) is a form of post-transcriptional gene silencing. The phenomenon of RNA interference was first observed and described in Caenorhabditis elegans here exogenous double-stranded RNA (dsRNA) was shown to specifically and potently disrupt the activity of genes containing homologous sequences through a mechanism that induces rapid degradation of the target RNA (Montgomery et al., 1999; Timmons and Fire, 1998; Montgomery and Fire, 1998; Fire, 1999), Although this phenomenon is still not completely understood, subsequent reports described the same phenomenon in other organisms, including experiments demonstrating spacial and/or temporal control of gene inactivation, including plant (Arabiclopsis thaliana), protozoan (Trypanosoma bruceii), invertebrate {Drosophila melanogaster), and vertebrate species (Danio rerio and Xenopus laevis) (Wargelius et al., 1999; Nakano et al, 2000; Kennerdell and Carthew, 2000; Chuang and Meyerowitz, 2000). However, unlike the success reported in roundworms or fruit flies, reports as to the reliability or reproducibility of this method when applied to zebrafish have been contradictory. Furthermore, information on the entire target gene sequence is required, which necessitates cloning and sequencing the full-length versions of genes to be analyzed.

A much more rapid method for the inhibition of gene expression is based on the use of shorter antisense oligomers consisting of DNA, or other synthetic structural types such as phosphorothiates, 2'-0-alkylrιbonucleotide chimeras, locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholinos (Toulme and Helene, 1988; Larsen et al., 1999; Soomets et al., 1999; Maier et a/., 2000; Fraser et al, 2000; Wahlestedt et al., 2000; Petersen et al , 2000). With the exception of RNA oligomers, PNAs and morpholinos, all other antisense oligomers act in eukaryotic cells through the mechanism of RNase H-mediated target cleavage. PNAs and morpholinos bind complementary DNA and RNA targets with high affinity and specificity, and thus act through a simple steric blockade of the RNA translationai machinery, and appear to be completely resistant to nuclease attack.

More recently it has been shown that morpholino antisense oligomers in zebrafish and frogs overcome the limitations of RNase H-competent antisense oligomers, which include numerous non-specific effects due to the non target-specific cleavage of other mRNA molecules caused by the low stringency requirements of RNase H (Nasevicius and Ekker, 2000; Heasman et al, 2000).

However, it has not yet been possible to temporally control gene inactivation mediated by antisense oligomers. Therefore, a method achieving this goal would be of significant interest, as it would significantly increase the percentage of genes that could be effectively analysed in model organisms amenable to antisense analysis, such as zebrafish

Definitions

Oligomer

The term"olιgomer/s" refers to a polymer comprising DNA or RNA nucleotides, peptide nucleic acid (PNA) subunits, morpholino subunits, or related structural compounds

Antisense oligomer

'Antisense oligomer" refers to an antisense molecule or anti-gene agent that comprises an oligomer of at least about 5 bases (or any derivatives thereof) in length In embodiments an antisense oligomer comprises at least 6, 7, 8, 9, 10, 12, 15, 18 or 20 bases preferably between 10 and 30 bases

Detailed description of the invention

Although the invention is described with respect to particular materials and methods or equipment, it is not limited thereto as these may 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

It must be noted that as Used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art Although any materials and methods, or equipment similar or equivalent to those described herein can be used to practice or test the present invention, the preferred equipment, materials and methods are now described All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention This invention describes methods for temporally controlling the inhibition of gene expression mediated by antisense oligomers

In particular, the invention is directed to a method for the temporal control of antisensemediated inhibition of gene expression, comprising the preparation of an antisense oligomer and a blocking oligomer, annealing of the antisense oligomer to the blocking oligomer to generate a double-stranded complex, and delivering said complex to cells or embryos

In one embodiment of this invention, said antisense oligomer is a DNA or RNA molecule In a preferred embodiment, said antisense oligomer is an unmodified DNA or RNA molecule Particularly preferred antisense oligomers are modified DNA or RNA molecules, or chimeπc molecules consisting of DNA, RNA, and/or modified DNA or RNA molecules In a further preferred embodiment, an antisense oligomer is at least 20 bases long

The term antisense oligomer denotes a nucleic acid oligomer of any available structural type, preferably modified DNA or RNA molecules with significantly increased intracellular stability through the use of structural modifications resulting in enhanced resistance to nucleases and other enzymes In a preferred embodiment, the antisense oligomer is a phosphorothioate oligomer or a 2'-0-methyl oligomer Particularly preferred antisense oligomers for the purposes of this invention are peptide nucleic adds (PNAs) and/or morpholino oligomers (Summerton, 1999) Antisense oligomers, both modified or unmodified are designed, synthesized and purified according to methods known to a person skilled in the art (Uhlmann & Peyman 1990) Most antisense structural types can be obtained commercially

As described above, available experimental protocols for the use of antisense oligomers in cells or embryos result in an immediate effect on the function of the targeted genes In order to elucidate the later functions of genes with multiple roles in embryonic development, however, it is often necessary to maintain the early function of said genes requiring an approach allowing antisense oligomers to exert their inhibitory activity only in later developmental stages As most typical procedures for the delivery of antisense oligomers to embryos are dependent upon the use of early-stage embryos (e g microinjection at the single-cell stage), such an approach would require the temporary inactivation of said antisense oligomer According to the method described herein, the temporary inactivation of said antisense oligomers is achieved by the use of a blocking oligomer

The base sequence of the blocking oligomer is partially or completely complementary to the base sequence of the antisense oligomer, whereby the ability of said antisense oligomer to inhibit gene expression by annealing to target transcripts is temporarily prevented Preferably, the blocking oligomer is complementary to the antisense oligomer over at least 20 bases

In one embodiment of this invention, said blocking oligomer is a DNA or RNA molecule In a preferred embodiment, the blocking oligomer is an unmodified DNA or RNA molecule Particularly preferred blocking oligomers are modified DNA or RNA molecules, or chimenc molecules consisting of DNA, RNA, and/or modified DNA or RNA molecules Examples of modified blocking oligomers include phosphorothioate oligomers, 2'-0-methyl oligomers morpholino oligomers and peptide nucleic acids (PNA) In a further preferred embodiment, a blocking oligomer comprises at least 20 bases

Blocking oligomers, either unmodified or modified, are designed, synthesized and purified according to methods known to a person skilled in the art (Uhlmann & Peyman, 1990) and can be obtained commercially

Preferred embryos for the purposes of this invention are embryos of the zebrafish (Barut and Zon, 2000, Dodd, 2000, Long et al , 2000), although embryos of the medaka and of the African clawed frog are also suitable Particularly preferred embryos are transgenic zebi afish embryos (Lin, 2000) With cell type-specific, tissue-specific, and/or stage-specific expression of reporter proteins, whereby preferred reporter proteins include but are not limited to, green fluorescent protein (Chalfie et al , 1994, Amsterdam et al , 1995, ibid, 1996, Long et al , 1997), beta-lactamase (Raz et al , 1998), and beta-galactosidase (Lin et al , 1994)

According to the method of this invention, the antisense oligomer is annealed to the blocking oligomer to create a double-stranded complex which is then delivered to embryos

For annealing the antisense oligomer to the blocking oligomer, appropriate ratio amounts of the antisense oligomer and of the blocking oligomer aie used The way of calculating the annealing temperature is known to the person skilled in the art and described for example in

Uhlmann and Peyman, 1990 The antisense oligomer is thereby temporarily blocked from annealing to its target transcript and inhibiting the translation of that transcript Because the blocking oligomer is an unmodified DNA or RNA molecule, it is more rapidly degraded by cellular enzymes than the antisense oligomer, which is resistant to the activity of such enzymes With this approach it is possible to mask the antisense activity of antisense oligomeroligomers for at least several hours, thereby bypassing many of the crucial stages of early development, such as gastrulation, where many genes with important functions in later developmental stages, such as organ differentiation, also play an essential role in patterning the early embryo

Delivery of the antisense oligomer/blocking oligomer complex to cells or embryos is achieved by introduction of the complex into early embryos or cells by microinjection or any other method known to a person skilled in the art (Westerfield, 1993)

In one embodiment of this invention, the antisense oligomer is targeted against a gene that encodes a protein or untranslated RNA molecule of known or Unknown function Gene products with unknown functions of particular interest include secreted proteins, receptors, ion channels, enzymes, proteases, kinases, and phosphatases, with the aim of elucidating the function of potential therapeutic proteins or defined molecular targets for the identification of pharmacological compounds in drug discovery applications Gene products with known functions of particular interest include those capable of generating disease-like phenotypes when inactivated, with the aim of generating fish embryos or larvae with medically relevant index phenotypes for modifier screens to identify genes or gene products functioning as enhancers or suppressors of said index phenotypes through various inactivation methods, including chemical compounds

In a further embodiment, the blocking oligomer is synthesized through transcription from an RNA expression vector, wherein said RNA expression vector contains a promoter for an RNA polymerase in a preferred embodiment, the RNA expression vector is an in vitro expression vector The term in viiio RNA expression vector denotes a vector wherein transcription is driven by a phage RNA promoter such as T7, T3 or SP6 The RNA blocking oligomer synthesized from the in vitro expression vector is annealed to the antisense oligomer and delivered to cells or embryos as described above

In a preferred embodiment the RNA expression vector is an in vivo expression vector If an in vivo expression vectoi is used, the method according to the present invention comprises the steps of (a) the preparation of an antisense oligomer, and (b) co-delivering of an antisense oligomer with an in vivo expression vector driving the in vivo transcription of an RNA blocking oligomer of complementary sequence in cells and embryos to generate a double-stranded complex in said cells or embryos.

This approach allows the selective inhibition of antisense activity in specific tissues and/or at specific time points in development, depending on the activity profile of the promoter used,

The term in ivo RNA expression vector denotes a vector that includes a eukaryotic promoter suitable for expression of the blocking oligomer sequence in vivo.

Previously described experiments have described a related method, based on the use of an in vitro transcribed RNA that is co-injected with the antisense oligomer, which results in the phenotypic rescue of an antisense oligomer-induced phenotype, primarily with the purpose of confirming the specificity of said antisense oligomer (Heasman et a!., 2000; Nasevicius and Ekker, 2000), Said rescue method is different than the blocking method described by this invention because it does not rely on the steric hindrance of the antisense oligomer by annealing said oligomer to a complementary sequence. Rather, it relies on the functional replacement of the gene being inhibited by said antisense oligomer by providing an alternative transcript so modified as to not contain any sequences complementary to said antisense oligomer,

The disadvantages of said rescue technique for the purposes of temporally controlling antisense-mediated inhibition of gene expression include (1) the need for multiple additional experimental procedures to generate a full-length functional transcript for each gene to be targeted by an antisense oligomer in a temporally specific manner, (2) the functional expression of said rescuing transcript throughout the embryo, thereby often resulting in an overexpression phenotype, and (3) the half-life of said rescuing transcript and its protein product are difficult to predict or measure for each gene to be analysed, as compared to the standard half-life of an unmodified DNA oligomer as described in the invention.

The invention described herein is further illustrated by the following example. Example 1

Temporal inhibition of the zebrafish bmp2b transcript

An antisense oligomer of the morpholino structural type was designed to target the region flanking the initiation codon of the zebrafish bmp2b transcript Zebrafish bmp2b is initially expressed in ventral tissues during gastrulation and subsequently develops a dynamic expression pattern, with various levels of transcription in multiple tissues Said bmp2b- specific morpholino oligomer was synthesized with the base sequence 5 CGC GGA CCA CGG CGA CCA TGA TC 3^' Microinjection of 0 5 ni to 3 nl of said morpholino oligomer at 1 10 and 1 30 dilutions of a 4mM stock solution into single-cell stage zebrafish embryos resulted in the complete dorsalization of said embryos at both concentrations By preannealing said morpholino oligomer with suitable amounts of an unmodified DNA blocking oligomer with the complementary base sequence 5^' GAT CAT GGT CGC CGT GGT CCG CG 3^', it was possible to completely suppress the dorsalization effect of said morpholino oligomer upon injection into single-cell stage zebrafish embryos, which developed normally through the gastrulation stage, thereby enabling the phenotypic analysis of bmp2b- knockdown embryos at later developmental stages

Example 2 Temporal inhibition of the zebrafish Tfar19 transcript

An antisense oligomer of the morpholino structural type was designed to target the region flanking the initiation codon of the zebrafish orthologue of the human Tfar19 transcript The Tfarl 9 transcript in human is upregulated in tumour cells undergoing apoptosis Said Pdcdδ- specific morpholino oligomer was synthesized with the base sequence 5 TCG AAT TGT

GTG TTC TGC CTG TGT G 3 Microinjection of 0 5 nl to 3 nl of said morpholino oligomer at 1 10 and 1 400 dilutions of a 4 mM stock solution into single-cell stage zebrafish embryos results in an underdevelopment of organs during early organogenesis and leading to premature death By preannealing said morpholino oligomer with suitable amounts of an unmodified DNA blocking oligomer with the complementary base sequence 5 CAC ACA

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Claims

1 A method for the temporal control of antisense-mediated inhibition of gene expression, comprising

(a) the preparation of an antisense oligomer and a blocking oligomer,

(b) annealing of the antisense oligomer to the blocking oligomer to generate a double-stranded complex, and

(c) delivering said complex to cells or embryos

2 A method according to claim 1 , wherein said antisense oligomer is a DNA or RNA molecule

3 A method according to claim 1 , wherein said antisense oligomer is an unmodified DNA or RNA molecule

4 A method according to claim 1 , wherein said antisense oligomer is a modified DNA or RNA molecule, or a chimeric molecule consisting of DNA, RNA, and/or modified DNA or RNA molecules

5 A method according to claim 1 , wherein said antisense oligomer is a morpholino oligomer

6 A method according to claim 1 , wherein said antisense oligomer is a peptide nucleic acid (PNA)

7 A method according to claim 1 , wherein said antisense oligomer is a phosphorothioate oligomer

8 A method according to claim 1 , wherein said antisense oligomer is a 2 -O-methyl oligomer

9 A method according to claim 1 wheiein said antisense oligomer is targeted against a gene that encodes a protein or untranslated RNA molecule of unknown or known function 10 A method according to claim 1 , wherein said blocking oligomer is a DNA or RNA molecule

1 1 A method according to claim 1 , wherein the blocking oligomer is complementary over at least 20 nucleotides to the antisense oligomer

12 A method according to claim 10, wherein said blocking oligomer is an unmodified DNA or RNA molecule

13 A method according to claim 10, herein said blocking oligomer is a modified DNA or RNA molecule, or a chimeric molecule consisting of DNA, RNA, and/or of modified DNA or RNA molecules

14 A method according to claim 10, wherein said blocking oligomer is a morpholino oligomer

15 A method according to claim 10, wherein said blocking oligomer is a peptide nucleic acid (PNA)

16 A method according to claim 10, wherein said blocking oligomer is a phosphorothioate oligomer

17 A method according to claim 10, wherein said blocking oligomer is a 2'-0-methyl oligomer

18 A method according to claim 10, wherein said RNA molecule is synthesized through transcription from an RNA expression vector, wherein said RNA expression vector contains a promoter for an RNA polymerase

19 A method according to claim 18, wherein said RNA expression vector is an //? vitto expression vector

20 A method according to claim 18, wherein said RNA expression vector is an in vivo expression Vector 21 , A method for the temporal control of antisense-mediated inhibition of gene expression, comprising

(a) the preparation of an antisense oligomer, and

(b) the co-delivery of an antisense oligomer with an in vivo expression vector driving the transcription of an RNA blocking oligomer of complementary sequence to cells or embryos to generate a double-stranded complex in said cells or embryos.

22, A method according to any one of claims 1 to 21 , wherein the delivery of the antisense oligomer and the blocking oligomer to cells or embryos is carried out by a method selected from the group consisting of microinjection, electroporation, transfection and ballistic methods (DNA-gun).

23, A method according to any one of claims 1 to 22, wherein said embryos are teleost embryos.

24, A method according to claim 19, wherein said teleost embryos are embryos of the species Da:7/^'o rerio or Oryza latipes,

25, A method according to any one of claims 1 to 22, wherein said embryos are embryos of the species Xenopus laevis or Xenopus tropicalis.

26, A method according to claim 23 to 25, wherein said embryos are transgenic.

27, A method according to any one of claims 23 to 25, wherein said embryos are homozygous or heterozygous for mutations.