A strategy for suppressing specifically or partially specifically an endogenous gene and introducing a replacement gene, said strategy comprising the steps of: 1. providing suppressing nucleic acids or other suppression effectors able to bind to an endogenous gene, gene transcript or gene product to be suppressed and 2. providing genomic DNA or cDNA (complete or partial) encoding a replacement gene wherein the suppressing nucleic acids are unable to bind to equivalent regions in the genomic DNA or cDNA to prevent expression of the replacement gene. The replacement nucleic acids have modifications in one or more third base (wobble) positions such that replacement nucleic acids still code for the wild type or equivalent amino acids.

SUMMARY OF THE INVENTION

To circumvent difficulties associated with specifically targeting a disease mutation and with the genetic heterogeneity present in inherited disorders, a novel strategy for gene suppression and gene replacement exploiting the degeneracy of the genetic code is described. The invention allows flexibility in choice of target sequence for suppression and provides a means of gene suppression that is independent of the disease mutation.

In summary, the invention involves gene suppression of disease and normal alleles targeting coding sequences in a gene and, when necessary, gene replacement such that the replacement gene cannot be suppressed. Replacement genes are modified at third base positions (wobble positions) so that they code for the correct amino acids but are protected completely or partially from suppression. The same suppression and replacement steps can be used for many disease mutations in a given gene. Suppression and replacement can be undertaken in conjunction with each other or separately.

The invention relates to a strategy for suppressing a gene or disease allele using methods that do not target the disease allele specifically but instead can be targeted towards a broad range of sequences in a particular gene. A particular embodiment of the invention is the use of suppression strategies to target either the disease or normal alleles alone or to target both disease and normal alleles. A further embodiment of the invention is the use of the wobble hypothesis to enable continued expression of a replacement normal or beneficial gene (a gene modified from the wild type such that it provides an additional beneficial effect(s)). The replacement gene will have nucleotide changes from the endogenous wild type gene but will code for identical amino acids as the wild type gene. The strategy circumvents the need for a specific therapy for every mutation within a given gene. In addition, the invention allows greater flexibility in choice of target sequence for suppression of a disease allele.

The invention also relates to a medicament or medicaments for use in suppressing a deleterious allele that is present in a genome of one or more individuals or animals.

Generally, the present invention will be useful where the gene, which is naturally present in the genome of a patient, contributes to a disease state. Generally, one allele of the gene in question will be mutated, that is, will possess alterations in its nucleotide sequence that affects the function or level of the gene product. For example, the alteration may result in an altered protein product from the wild type gene or altered control of transcription and processing. Inheritance or somatic acquisition of such a mutation can give rise to a disease phenotype or can predispose an individual to a disease phenotype. However the gene of interest could also be of wild type phenotype, but contribute to a disease state in another way such that the suppression of the gene would alleviate or improve the disease state or improve the effectiveness of an administered therapeutic compound.

Generally, suppression effectors such as nucleic acids--antisense or sense, ribozymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, peptides and/or antibodies directed to sequences in a gene, in transcripts or in protein, can be employed in the invention to achieve gene suppression.

DETAILED DESCRIPTION OF THE INVENTION

The invention addresses shortcomings of the prior art by providing a novel approach to the design of suppression effectors directed to target alleles of a gene carrying a deleterious mutation. Suppression of every mutation giving rise to a disease phenotype may be costly and problematic. Disease mutations are often single nucleotide changes. As a result differentiating between the disease and normal alleles may be difficult. Some suppression effectors require specific sequence targets, for example, hammerhead ribozymes cleave at NUX sites and hence may not be able to target many mutations. Notably, the wide spectrum of mutations observed in many diseases adds additional complexity to the development of therapeutic strategies for such disorders--some mutations may occur only once in a single patient. A further problem associated with suppression is the high level of homology present in coding sequences between members of some gene families. This can limit the range of target sites for suppression that will enable specific suppression of a single member of such a gene family.

The present invention circumvents shortcomings in the prior art by utilising the degeneracy of the genetic code. In the invention suppression effectors are designed specifically to sequences in coding regions of genes or in gene products. Typically, one allele of the gene contains a mutation with a deleterious effect. Suppression targeted to coding sequences provides greater flexibility in choice of target sequence for suppression in contrast to suppression directed towards single disease mutations. Additionally, the invention provides for the introduction of a replacement gene with modified sequences such that the replacement gene is protected from suppression. The replacement gene is modified at third base wobble positions and hence provides the wild type gene product. Notably, the invention has the advantage that the same suppression strategy could be used to suppress, in principle, many mutations in a gene. This is particularly relevant when large numbers of mutations within a single gene cause disease pathology. The replacement gene provides (when necessary) expression of the normal protein product when required to ameliorate pathology associated with reduced levels of wild type protein. The same replacement gene could in principle be used in conjunction with the suppression of many different disease mutations within a given gene. Target sequences may be found in any part of the coding sequence. Suppression in coding sequence holds the advantage that such sequences are present in both precursor and mature RNAs, thereby enabling suppressors to target all forms of RNA.

There is now an armament with which to obtain gene suppression. This, in conjunction with a better understanding of the molecular aetiology of disease, results in an ever increasing number of disease targets for therapies based on suppression. In many cases, complete suppression of gene expression has been difficult to achieve. Possibly a combined approach using a number of suppression effectors may aid in this. For some disorders it may be necessary to block expression of a disease allele completely to prevent disease symptoms whereas for others low levels of mutant protein may be tolerated. In parallel with an increased knowledge of the molecular defects causing disease has been the realisation that many disorders are genetically heterogeneous. Examples in which multiple genes and/or multiple mutations within a gene can give rise to a similar disease phenotype include osteogenesis imperfecta, familial hypercholesterolemia, retinitis pigmentosa, and many others. The utility of the degeneracy of the genetic code (wobble hypothesis) to enable suppression of one or both alleles of a gene and the introduction of a replacement gene such that it escapes suppression has been exploited in the invention. According to the present invention there is provided a strategy for suppressing expression of an endogenous gene with a deleterious mutation, wherein said strategy comprises providing suppression effectors such as antisense nucleic acids able to bind to sequences of a gene to be suppressed, to prevent the functional expression thereof.

Generally the term suppression effectors means nucleic acids, peptide nucleic acids (PNAs), peptides, antibodies or modified forms of these used to silence or reduce gene expression in a sequence specific manner.

Suppression effectors, such as antisense nucleic acids can be DNA or RNA, can typically be directed to coding sequence; however suppression effectors can be targeted to coding sequence and can also be targeted to 5' and/or 3' untranslated regions and/or introns and/or control regions and/or sequences adjacent to a gene or to any combination of such regions of a gene. Antisense nucleic acids including both coding and non-coding sequence can be used if required to help to optimise suppression. Binding of the suppression effector(s) prevents or lowers functional expression of the endogenous gene.

Generally the term `functional expression` means the expression of a gene product able to function in a manner equivalent to, or better than, a wild type product. In the case of a mutant gene or predisposing gene `functional expression` means the expression of a gene product whose presence gives rise to a deleterious effect or predisposes to a deleterious effect. By deleterious effect is meant giving rise to or predisposing to disease pathology or altering the effect(s) and/or efficiency of an administered compound.

In a particular embodiment of the invention the strategy further employs ribozymes that can be designed to elicit cleavage of target RNAs. The strategy further employs nucleotides that form triple helix DNA. The strategy can employ peptide nucleic acids for suppression. Nucleic acids for antisense, ribozymes, triple helix forming DNA and peptide nucleic acids may be modified to increase stability, binding efficiencies and uptake. Nucleic acids can be incorporated into a vector. Vectors include naked DNA, DNA plasmid vectors, RNA or DNA virus vectors, lipids, polymers or other derivatives and compounds to aid gene delivery and expression.

The invention further provides the use of antisense nucleotides, ribozymes, PNAs, triple helix nucleotides, or other suppression effectors alone or in a vector or vectors, wherein the nucleic acids are able to bind specifically or partially specifically to coding sequences of a gene to prevent or reduce the functional expression thereof, in the preparation of a medicament for the treatment of an autosomal dominant or polygenic disease or to increase the utility and/or action of an administered compound.

In a further embodiment of the invention, target sequences for suppression can include non-coding sequences of the gene.

According to the present invention there is provided a strategy for suppressing specifically or partially specifically an endogenous gene and (if required) introducing a replacement gene, said strategy comprising the steps of: 1. providing nucleic acids or other suppression effectors able to bind to an endogenous gene, gene transcript or gene product to be suppressed and 2. providing genomic DNA or cDNA (complete or partial) encoding a replacement gene wherein the nucleic acids are unable to bind to equivalent regions in the genomic DNA or cDNA to prevent expression of the replacement gene. The replacement nucleic acids will not be recognised by suppression nucleic acids or will be recognised less effectively than the endogenous gene. The coding sequence of replacement nucleic acids can be altered to prevent or reduce efficiency of suppression. Replacement nucleic acids have modifications in one or more third base (wobble) positions such that replacement nucleic acids still code for the wild type or equivalent amino acids.

In a particular embodiment of the invention there is provided a strategy for gene suppression targeted to coding sequences of the gene to be suppressed.

Suppression will be specific or partially specific to one allele, for example, to the allele carrying a deleterious mutation. Suppressors are targeted to coding regions of a gene or to a combination of coding and non-coding regions of a gene. Suppressors can be targeted to a characteristic of one allele of a gene such that suppression is specific or partially specific to one allele of a gene (PCT/GB97/00574). The invention further provides for use of replacement nucleic acids with altered coding sequences such that replacement nucleic acids will not be recognised (or will be recognised less effectively) by suppression nucleic acids that are targeted specifically or partially specifically to one allele of the gene to be suppressed. Replacement nucleic acids provide the wild type gene product, an equivalent gene product or an improved gene product but are protected completely or partially from suppression effectors targeted to coding sequences.

In a further embodiment of the invention, replacement nucleic acids are provided such that replacement nucleic acids will not be recognised by naturally occurring suppressors found to inhibit or reduce gene expression in one or more individuals, animals or plants. The invention provides for use of replacement nucleic acids that have altered sequences targeted by suppressors of the gene such that suppression by naturally occurring suppressors is completely or partially prevented.

In an additional embodiment of the invention, there is provided replacement nucleic acids with altered nucleotide sequence in coding regions such that replacement nucleic acids code for a product with one or more altered amino acids. Replacement nucleic acids provide a gene product that is equivalent to or improved compared with the naturally occurring endogenous wild type gene product.

In an additional embodiment of the invention there is provided a strategy to suppress a gene where the gene transcript or gene product interferes with the action of an administered compound.

The invention further provides the use of a vector or vectors containing suppression effectors in the form of nucleic acids, said nucleic acids being directed towards coding sequences or combinations of coding and non-coding sequences of the target gene and vector(s) containing genomic DNA or cDNA encoding a replacement gene sequence to which nucleic acids for suppression are unable to bind (or bind less efficiently), in the preparation of a combined medicament for the treatment of an autosomal dominant or polygenic disease. Nucleic acids for suppression or replacement gene nucleic acids may be provided in the same vector or in separate vectors. Nucleic acids for suppression or replacement gene nucleic acids may be provided as a combination of nucleic acids alone or in vectors.

The invention further provides a method of treatment for a disease caused by an endogenous mutant gene, said method comprising sequential or concomitant introduction of (a) nucleic acids to the coding regions of a gene to be suppressed and/or nucleic acids to coding regions and any combination of 5' and/or 3' untranslated regions, intronic regions, control regions or regions adjacent to a gene to be suppressed (b) replacement nucleic acids with sequences that allow the replacement gene to be expressed.

The nucleic acid for gene suppression can be administered before, after or at the same time as the replacement gene is administered.

The invention further provides a kit for use in the treatment of a disease caused by a deleterious mutation in a gene, the kit comprising nucleic acids for suppression able to bind to the gene to be suppressed and if required a replacement nucleic acid to replace the mutant gene having sequence that allows it to be expressed and completely or partially escape suppression.

Nucleotides can be administered as naked DNA or RNA. Nucleotides can be delivered in vectors. Naked nucleic acids or nucleic acids in vectors can be delivered with lipids or other derivatives which aid gene delivery. Nucleotides may be modified to render them more stable, for example, resistant to cellular nucleases while still supporting RNase H mediated degradation of RNA or with increased binding efficiencies. Antibodies or peptides can be generated to target the protein product from the gene to be suppressed.

The strategy described herein has applications for alleviating autosomal dominant diseases. Complete silencing of a disease allele may be difficult to achieve using antisense, PNA, ribozyme and triple helix approaches or any combination of gene silencing approaches. However small quantities of mutant product may be tolerated in some autosomal dominant disorders. In others a significant reduction in the proportion of mutant to normal product may result in an amelioration of disease symptoms. Hence, this invention may be applied to any autosomal dominantly or polygenically inherited disease in man where the molecular basis of the disease has been established or is partially understood. This strategy enables the same therapy to be used to treat a range of different disease mutations within the same gene. The development of such approaches is important to future therapies for autosomal dominant and polygenic diseases, the key to a general strategy being that it circumvents the need for a specific therapy for every mutation causing or predisposing to a disease. This is particularly relevant in some disorders, for example, rhodopsin linked autosomal dominant RP, in which to date about one hundred different mutations in the rhodopsin gene have been observed in adRP patients. Likewise, hundreds of mutations have been identified in the human type I Collagen 1A1 and 1A2 genes in autosomal dominant osteogenesis imperfecta. Costs of developing therapies for each mutation are prohibitive at present. Inventions such as this using a general approach for therapy will be required. General approaches may be targeted to the primary defect, as is the case with this invention, or to secondary effects such as apoptosis.

This invention may be applied in gene therapy approaches for biologically important polygenic disorders affecting large proportions of the world's populations such as age related macular degeneration, glaucoma, manic depression, cancers having a familial component and indeed many others. Polygenic diseases require inheritance of more than one mutation (component) to give rise to the disease state. Notably an amelioration in disease symptoms may require reduction in the presence of only one of these components, that is, suppression of one genotype which, together with others leads to the disease phenotype, may be sufficient to prevent or ameliorate symptoms of the disease. In some cases suppression of more than one component may be required to improve disease symptoms. This invention may be applied in possible future interventive therapies for common polygenic diseases to suppress a particular genotype(s) using suppression and, when necessary, replacement steps.

The present invention is exemplified using four genes: human rhodopsin, mouse rhodopsin, human peripherin and human collagen 1A2. The first of these genes are retinal specific. In contrast, collagen 1A2 is expressed in a range of tissues including skin and bone. While these four genes have been used as examples there is no reason why the invention could not be deployed in the suppression of many other genes in which mutations cause or predispose to a deleterious effect. Many examples of mutant genes that give rise to disease phenotypes are available from the prior art--these genes all represent targets for the invention. The present invention is exemplified using hammerhead ribozymes with antisense arms to elicit RNA cleavage. There is no reason why other suppression effectors directed towards genes, gene transcripts or gene products could not be used to achieve gene suppression. Many examples from the prior art detailing use of suppression effectors such as, inter alia, antisense RNA/DNA, triple helix forming DNA, PNAs and peptides to achieve suppression of gene expression are reported. The present invention is exemplified using hammerhead ribozymes with antisense arms to elicit sequence specific cleavage of transcripts from genes implicated in dominant disorders and non-cleavage of transcripts from replacement genes containing sequence modifications in wobble positions such that the replacement gene still codes for wild type protein. The present invention is exemplified using suppression effectors targeting sites in coding regions of the human and mouse rhodopsin, human peripherin and human collagen 1A2 genes. Target sites, which include sequences from transcribed but untranslated regions of genes, introns, regions involved in the control of gene expression, regions adjacent to the gene or any combination of these, could be used to achieve gene suppression. Multiple suppression effectors, for example, shotgun ribozymes could be used to optimise efficiency of suppression when necessary. Additionally, when required, expression of a modified replacement gene such that the replacement gene product is altered from the wild type product such that it provides a beneficial effect may be used to provide gene product.
 

Claim 1 of 59 Claims

1. A method for preparing a suppression effector and replacement nucleic acid, said method comprising: a) preparing a suppression effector that binds to a coding region of a mature RNA encoding a mutant allele, thereby to inhibit the expression of the mutant allele, wherein the suppression effector is a nucleic acid or a peptide nucleic acid; and b) preparing a replacement nucleic acid that encodes a wild-type or non-disease causing allele and that comprises at least one degenerate/wobble nucleotide that is altered so that the replacement nucleic acid is not suppressed, or is only partially suppressed, by the suppression effector.

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