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Genetic suppression and replacement

a gene and gene technology, applied in the field of gene suppression and replacement, can solve the problems of difficult targeting of disease mutations, inability to develop specific gene therapies for each of them, and inability to differentiate strategies,

Inactive Publication Date: 2006-06-15
OPTIGEN PATENTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new strategy for treating inherited disorders by targeting the genetic code and using gene suppression and replacement techniques. This approach allows for flexibility in targeting specific mutations and can be used for many disease mutations in a gene. The invention also relates to a medicament for suppressing deleterious alleles that contribute to disease states. The suppression effectors that can be used include nucleic acids, ribozymes, peptide nucleic acids, triple helix forming oligonucleotides, peptides, and antibodies. Overall, the invention provides a novel way to target disease-causing genes and offers potential for improved treatment outcomes.

Problems solved by technology

Strategies to differentiate between normal and disease alleles and to selectively switch off the disease allele using suppression effectors such as antisense DNA / RNA, PNAs, ribozymes, or triple helix forming DNA, targeted towards the disease mutation may be difficult in many cases—frequently disease and normal alleles differ by only a single nucleotide.
A further difficulty inhibiting development of gene therapies is the heterogeneous nature of some dominant disorders—many different mutations in the same gene give rise to a similar disease phenotype.
Development of specific gene therapies for each of these may be prohibitive in terms of cost.

Method used

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  • Genetic suppression and replacement
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Examples

Experimental program
Comparison scheme
Effect test

example 1

A: Human Rhodopsin

[0111] The unadapted human rhodopsin CDNA (SEQ ID NO:1) and the human rhodopsin CDNA with a single nucleotide substitution in the coding sequence (SEQ ID NO:2) were cut with BstEII and expressed in vitro. The single base change occurs at the third base position or wobble position of the codon (at position 477) (nucleotide 271 of SEQ ID NO:2) and therefore does not alter the amino acid coded by this triplet. The Rz10 clone was cut with XbaI and expressed in vitro. Resulting ribozyme and human rhodopsin RNAs were mixed with varying concentrations of MgCl2 to optimise cleavage of template RNA by Rz10. A profile of human rhodopsin RNA cleavage by Rz10 over time is given in FIG. 2A. The MgCl2 curve profile used to test if adapted human rhodopsin transcripts could be cleaved by Rz10 is given in FIG. 3B. Unadapted and adapted human rhodopsin cDNAs were cut with FspI and BstEII respectively, expressed and mixed together with Rz10 RNA to test for cleavage (FIG. 2B) over t...

example 2

Mouse Rhodopsin

[0114] Rz33 was cut with XbaI and expressed in vitro. Similarly the mouse rhodopsin cDNA was cut with Eco47III and expressed in vitro. Resulting RNAs were mixed and incubated with varying concentrations of MgCl2. All expressed products and cleavage products were the correct size. FIG. 6A demonstrates specific cleavage of the mouse rhodopsin RNA over various MgCl2 concentrations incubated at 37° C. for 3 hours. Using a replacement gene with a sequence change around the Rz33 cleavage site (TTT-->TCT) (nucleotides 189-191 of SEQ ID NO:7) which is at a wobble position we demonstrated that transcripts from the replacement gene remain intact due to absence of the Rz33 target site (FIG. 6B). Hence Rz33 could be used to cleave mutant transcripts in a manner independent of the disease mutation itself (that is, using this site) while transcripts from the replacement gene which code for the correct protein would remain intact and therefore could supply the wild type protein.

example 3

Human Peripherin

[0115] The unadapted human peripherin cDNA and two human peripherin DNA fragments generated by PCR mutagenesis with a single nucleotide substitution in the coding sequence were cut with BglII and AvrII respectively and expressed in vitro. The single base changes in the adapted DNAs occur at third base positions or wobble positions of the codon (at position 257 and 359) (nucleotide 468 of SEQ ID NO:13 and nucleotide 332 of SEQ ID NO:10, respectively) and therefore do not alter the amino acid coded by these triplets. The Rz30 and Rz31 clones were cut with XbaI and expressed in vitro. Resulting ribozymes and unadapted human rhodopsin RNAs were mixed with varying concentrations of MgCl2 to optimise cleavage of template RNA by Rz30 and Rz31 . Profiles of human peripherin RNA cleavage by Rz30 over various MgCl2 concentrations and over time are given in FIG. 7. Similarly profiles of human peripherin RNA cleavage by Rz31 over various MgCl2 concentrations and over time are g...

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Abstract

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.

Description

REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 09 / 155,708, which was filed under 35 U.S.C. §371 for, and claims priority to, PCT / GB97 / 00929, filed Apr. 2, 1997, which claims priority to GB9606961.2, filed Apr. 2, 1996, the disclosures of which are incorporated by reference herein.FIELD OF THE INVENTION [0002] The present invention relates to a strategy for suppressing a gene. In particular the invention relates to suppression of a mutated gene that gives rise to a dominant or deleterious effect, either monogenically or polygenically. BACKGROUND OF THE INVENTION [0003] Studies of degenerative hereditary ocular conditions, including Retinitis Pigmentosa (RP) and various macular dystrophies, have resulted in a substantial elucidation of the molecular basis of these debilitating human retinal degenerations. Applying the approach of genetic linkage, x-linked RP (xlRP) genes have been localised to the short arm of the X chromosome ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00A61K38/00C12N15/113
CPCA61K38/00A61K48/00A61K48/005C12N15/113C12N2310/111C12N2310/121C12N2310/15C12N2310/3181
Inventor FARRAR, GWENYTHHUMPHRIES, PETERKENNA, PAUL
Owner OPTIGEN PATENTS
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