Rhodopsin gene conserved regions in a viral vector enhance expression

#The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 3, 2010, is named 20100303_ReplacementSequenceListing_TextFile—740789—066400_US.txt and is 127,586 bytes in size.

#The invention relates to mutation independent suppression and replacement of disease-causing mutant genes.

#Many mutation-based diseases are more genetically diverse than can be predicted from clinical presentation. Some mutation-based diseases are Mendelian and involve the inheritance of a single mutant gene, others are polygenic or multifactorial and involve multiple genetic insults. In the case of some Mendelian disorders, many different mutations within the same gene can give rise to, or can predispose an individual to, a disease. Similarly, for some multifactorial disorders, many different mutations within one or more genes can predispose an individual to a disease or can act in an additive manner with other genetic and environmental influences to give rise to a disease. This mutational heterogeneity underlying the molecular etiologies of many diseases represents a significant barrier to the development of therapies for such diseases. Moreover, genetic strategies for suppressing and replacing a mutant protein face many challenges with regard to the effectiveness of the machinery used to deliver and regulate the expression of the suppressor and replacement nucleic acids in vivo. Therefore, a need exists for effective mutation-independent therapeutics that achieve effective suppression and replacement.

#The foregoing and other objects, features and advantages of the present invention, as well as the invention itself, will be more fully understood from the following description of preferred embodiments when read together with the accompanying drawings, in which:

#FIG. 1 illustrates RHO suppression and replacement constructs. FIG. 1A is a diagrammatic representation of a RHO suppressor-EGFP construct shBB-EGFP (shQ1-EGFP and shNT-EGFP have the same format). shRNAs were expressed from the H1 promoter and EGFP from the CMV immediate early promoter. The SV40 polyadenylation signal was located at the 3′ end of the EGFP gene. FIG. 1B illustrates a two component suppression and replacement construct shBB-rBB (shQ1-rQ1 and shNT-rBB have the same format). Suppressors were expressed from the H1 promoter and replacement RHO cDNAs from a 1.7 kb mouse rhodopsin promoter (rhoP). Polyadenylation signals of the RHO gene were included in the 1829 bp fragment. HGH int: human growth hormone intron. For tissue culture and retinal explant experiments these constructs were maintained in pEGFP-1 (A) or a CMV-promoterless derivative of pcDNA-3.1- (B) and for in vivo experiments in the AAV vector. Restriction enzyme sites used for cloning are indicated. Promoters were separated by spacer DNA fragments. Numbers indicate molecular sizes (bp) and arrows indicate direction of transcription.

#FIG. 2 illustrates RHO suppression in HeLa cells. HeLa cells were transiently co-transfected three times in triplicate with wild type RHO and RHO-targeting siRNAs (siB, siBB, siC, siCC, siQ1 or siQ2) or control siRNAs (siEGFP or siNT). Following transfection, RHO mRNA and protein levels were evaluated by real time RT-PCR (A), ELISA (A) and Alexa Fluor 568-labeled immunocytochemistry (B). Cell nuclei were counterstained with DAPI. Error bars represent SD values.

#The instant invention utilises efficient gene suppression in conjunction with gene replacement to overcome the challenge of mutational heterogeneity. The suppression agent does not necessarily target a mutation (although it can encompass the site of a mutation), but is rather mutation independent. Suppression can involve one or both alleles of an endogenous gene. In conjunction with suppression, a replacement gene is provided that has been modified such that the replacement gene is refractory or partially refractory to suppression. The invention uses the degeneracy of the genetic code to modify the replacement gene. Alteration of “wobble” bases makes it possible for replacement nucleic acids to escape suppression at least in part, but does not change the protein product expressed from the replacement nucleic acids. Alternatively, replacement genes are modified in such a way that they encode altered amino acids but still encode a functional or partially functional protein that does not lead to pathology (e.g., because the amino acid changes are silent mutations or polymorphisms). Replacement has been demonstrated using rhodop sin nucleic acids, however, other genes or combinations of genes can be made and used in the practice of the invention. In particular, the invention relates to modulating and optimizing the expression levels of the suppression agents and/or replacement nucleic acids using one or more of the untranslated regions (UTRs) of a gene, intronic sequences, the degeneracy of the genetic code and/or polymorphisms to alter the sequence of replacement nucleic acids such that they are refractory or partially refractory to suppression.

#In one aspect, the invention provides methods for preparing and using a suppression agent and replacement nucleic acid. The suppression agent binds to a coding region of a mature RNA or DNA encoding a mutant allele and inhibits expression of the mutant allele. The replacement nucleic acid encodes a wild-type or non-disease causing allele and 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 of one or both alleles of a gene.

#The invention provides for replacement genes using sequences to enhance expression of replacement genes from viral and or non-viral vectors. In particular the invention relates to enhanced expression of suppression agent(s) and or replacement genes from viral or and non-viral vectors. The invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes. In a particular aspect the invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes from adeno associated virus (AAV) vectors. In another aspect the invention provides vectors for expression of suppression agent(s) and or replacement gene(s) using regulatory sequences from retinal gene(s) and or non-retinal gene(s) and or ubiquitously expressing genes such as those provided in the Tables below to enhance expression from viral and non-viral vectors.