# Patent

## Title
Genetic suppression and replacement

## Abstract

The invention relates to gene suppression and replacement. In particular, the invention relates to enhanced expression of suppression agents for suppressing gene expression in a cell and in vivo and replacement nucleic acids that are not inhibited by the suppression agent. Regulatory elements are included in expression vectors to optimize expression of the suppression agent and/or replacement nucleic acid.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 371 National Phase Entry Application of International Application No. PCT/GB2008/001310 filed Apr. 14, 2008, which designated the U.S., and which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional No. 60/923,067 filed Apr. 12, 2007, the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
The invention relates to mutation independent suppression and replacement of disease-causing mutant genes.
BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
The invention relates to gene suppression and replacement. In particular, the invention relates to enhanced expression of suppression agents for suppressing gene expression in a cell and in vivo and of replacement nucleic acids that are not inhibited and/or are partially inhibited by the suppression agent. Expression vectors used to express the suppression agent(s) and replacement nucleic acids comprise regulatory elements to optimize expression of the suppression agent(s) and or replacement nucleic acids.
The invention embodies use of replacement genes using sequences to enhance expression of replacement genes from viral and or non-viral vectors. In a further aspect the invection relates to enhanced expression of suppression agent(s) and or replacement genes from viral or and non-viral vectors. In a further embodiment the invention relates to enhanced expression of suppression agent(s) and or replacement genes and or genes encoding neurotrophic factors from viral and or non-viral vectors.
In one aspect the invention relates to use of conserved sequences from retinal genes to enhance expression of suppression agent(s) and or replacement genes and or genes encoding neurotrophic factors. The use of such conserved sequences has been found to result in surprisingly efficient expression. 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 and or genes encoding neurotrophic factors from adeno associated virus (AAV) vectors. In another aspect the invention provides vectors for expression of suppression agent(s) and or replacement gene(s) and or genes encoding neurotrophic factors using regulatory sequences from retinal gene(s) and or non-retinal gene(s) and or ubiquitously expressing genes to enhance expression from vectors.
In one aspect, the invention provides vectors for expressing a suppression agent for a disease causing gene and/or a replacement nucleic acid that is not recognized or is partially recognized by the suppression agent.
In an embodiment, the vector comprises an enhancer sequence, such as, for example, a sequence of SEQ ID NOs: 402-413 or functional variants or equivalents thereof. In another embodiment, the vector comprises at least one regulatory element selected from the group consisting of a promoter, a stuffer, an insulator, a silencer, an intron sequence, a post translational regulatory element, a polyadenylation site, and a transcription factor binding site.
In another embodiment, the vector comprises at least one of conserved regions A through I from the rhodopsin gene, as represented by SEQ ID NOs: 92-99, or functional variant or equivalent thereof. In another embodiment, the vector comprises at least one transcription factor binding site sequence selected from the group consisting of SEQ ID NOs: 100-401, or functional variant or equivalent thereof.
The suppression agent may be a nucleic acid, protein, amino acid(s), antibody, aptamer, or any such agent that can bind to and inhibit a DNA, RNA, or protein. In an embodiment, the suppression agent is a siRNA selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, 85, and 414-421 or functional variant or equivalent thereof.
The replacement nucleic acid is not recognized or is recognized partially by the suppression effector, because its sequence has been altered such that it cannot bind or binds less efficiently to the suppression agent but still encodes a normal or enhanced gene product. In an embodiment, the replacement nucleic acid is a siRNA selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functional variant or equivalent thereof.
In an embodiment, the invention provides vectors, such as viral vectors, that comprise a suppression agent and/or a replacement nucleic acid. For example, the vector comprises at least one suppression agent nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, and 85, or functional variant or equivalent thereof, and at least one replacement nucleic acid nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functional variant or equivalent thereof.
In another aspect, the invention provides therapeutic compositions comprising at least one vector comprising at least one suppression agent nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-67, 75, 77, 79, 81, 83, 85 and 414-421 or functional variant or equivalent thereof, and at least one replacement nucleic acid nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 76, 78, 80, 82, 84, and 86, or functional variant or equivalent thereof. In an embodiment, the vector of the therapeutic composition further comprises a regulatory element selected from the group consisting of an enhancer, a promoter, a stuffer, an insulator, a silencer, an antirepressor, an intron sequence, a post translational regulatory element, a polyadenylation signal (e.g. minimal poly A), a conserved region A through I, and a transcription factor binding site.
In another aspect the invention provides suppression and replacement in conjunction with provision of a gene encoding a neurotrophic/neuroprotective factor(s).
In another aspect, the invention provides cells comprising the nucleic acids and vectors of the invention.
In another aspect, the invention provides transgenic animals comprising the nucleic acids and vectors of the invention.
In yet another aspect, the invention provides methods of suppressing the expression of a mutant gene and replacing expression of the mutant gene with a replacement nucleic acid, the method comprising administering to a mammal a therapeutic composition of the invention.
In yet another aspect, the invention provides methods of suppressing the expression of a mutant gene and replacing expression of the mutant gene with a replacement nucleic acid in conjunction with a gene encoding a neurotrophic/neuropeotective factor(s), the method comprising administering to a mammal a therapeutic composition of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
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 by 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.
FIG. 3 illustrates replacement of RHO expression in conjunction with suppression in HeLa cells. Replacement RHO sequences were generated with altered degenerate nucleotides at siRNA target sites. HeLa cells were transiently co-transfected three times in triplicate with a replacement RHO expression vector (rBB, rCC or rQ1) and a RHO-targeting siRNA (siBB, siCC or siQ1) or a non-targeting siRNA (siNT). Replacement RHO mRNA levels were evaluated by real time RT-PCR. Error bars represent SD values.
FIG. 4 illustrates RHO suppression in retinal explants. Mouse retinas (n=6), dissected from newborn NHR+/− rho−/− pups (transgenic mice expressing a human rhodopsin transgene NHR on a mouse rhodopsin knockout background rho−/−), were electroporated with a construct co-expressing a shRNA targeting RHO or a non-targeting shRNA and EGFP (shBB-EGFP, shQ1-EGFP or shNT-EGFP). Negative control explants were not electroporated. Two week organotypic cultures were dissociated with trypsin and FACS analysed. Red and blue dots (right and left populations respectively in each of A1 and A2) represent gated and ungated populations of dissociated explants. Scatterplots of forward-(FSC) versus side-scatter (SSC) and histograms of EGFP fluorescence of the gated population of non-electroporated (A1 and A2, EGFP-negative) and electroporated (A3 and A4, EGFP-positive) retinas are given. FIG. 4B shows a bar chart which indicates RHO mRNA levels in retinal explant cells expressing sNT-EGFP, sBB-EGFP and sQ1-EGFP, quantified by real time RT-PCR. Error bars represent SD values.
FIG. 5 illustrates RHO suppression in photoreceptor cells in vivo. Adult transgenic NHR+/− rho−/− mice were subretinally injected with 3 μl 2×1012 vp/ml AAV co-expressing a RHO-targeting or non-targeting shRNA and EGFP (AAV-shBB-EGFP or AAV-shNT-EGFP). Retinas were analysed two weeks post-injection. Expression of the 21 nucleotide (nt) shRNA BB, detected by RNase protection in two transduced retinas, is depicted in lanes L1 and L2 (A). RHO RNA probes were labelled with P32-γATP and protected RNA separated on 15% denaturing acrylamide gels (A). M: size marker indicates 10, 20 and 30 nt. Bars represent RHO mRNA levels in FACS sorted cells from dissociated retinas (n=6) transduced with either AAV-shBB-EGFP or AAV-shNT-EGFP (B). Suppression levels were determined by real time RT-PCR. Error bars represent SD values. Rhodopsin immunocytochemistry (Cy3-labeled) and EGFP protein expression in cells from dissociated retinas, transduced with either AAV-shBB-EGFP (arrows) or AAV-shNT-EGFP (arrow heads), are depicted (C). Cell nuclei were counterstained with DAPI.
FIG. 6A-D illustrates retinal histology and ERG analysis of RHO-M mouse. Two month old rho+/+(wild type), rho+/−, NHR+/− rho−/− and RHO-M+/− rho−/− mice were analysed by retinal histology and ERG (n=8). A, B and C: rhodopsin immunocytochemistry (Cy3) showing similar rod outer segment (ROS) labelling in rho+/+, NHR+/− rho−/− and RHO-M+/− rho−/− retinas respectively. Nuclear layers were stained with DAPI. D: representative rod-isolated ERG responses. ONL: outer nuclear layer. INL: inner nuclear layer. GCL: ganglion cell layer.
FIG. 6E illustrates RNAi-mediated suppression of human rhodopsin in RHO-M mice. RHO-M mice were subretinally injected with AAV2/5 vectors carrying an shRNA-based suppressor and an EGFP reporter gene. Mice were sacrificed 14 days post-injection, retinas taken and retinal cells dissociated as in Palfi et al. 2006. RNAi-mediated suppression was evaluated using real-time RT-PCR assays. Retinal cells transduced with AAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP vs AAV-shNT-EGFP were FACS sorted from adult RhoM mouse retinas, 14 days post subretinal injection. Of note is that AAV-shCC-EGFP suppresses RHO less in RHO-M mice due to the presence of a 2 bp mismatch in the human rhodopsin transgenic in RHO-M animals. Levels of rhodopsin expression were shCC: 59.73%; shBB: 8.77%; shQ1: 20.6% when compared to the non-targeting control shNT which was set at 100% expression.
FIG. 6F illustrates depression of the ERG response in RHO-M eyes that have received AAV-shBB-EGFP or AAV-shQ1-EGFP when compared to eyes subretinally injected with AAV-shNT-EGFP. The top tracing in each panel represents the right eye which received the targeting AAV-shRNA vector and the bottom tracing in each panel represents the left eye which received the control non-targeting AAV-shNT vector. In contrast no reduction/depression of the ERG was observed in RHO-M mice subretinally injected with AAV-shCC-EGFP vector.
FIG. 7 illustrates the expression of replacement RHO in vivo. Ten day old rho−/− mice were subretinally injected with a 1:1 mixture of 2 μl 2×1012 vp/ml of two AAV vectors, AAV-EGFP (also termed AAV-CMV-EGFP) and AAV-shBB-rBB (also termed AAV-BB8). Rhodopsin, EGFP protein and nuclei were detected by Cy3-labeled immunocytochemistry, native fluorescence and nuclear DAPI staining respectively. Low magnification images show a cross section of a whole injected eye with arrowheads indicating the transduced area (A and B). High magnification laser scanning micrographs show transduced (C and D) and non-transduced (E and F) areas. INL: inner nuclear layer. GCL: ganglion cell layer. ROS: rod outer segments. ONL: outer nuclear layer. FIG. 7 provides evidence of rhodopsin protein expression from replacement genes in retinal sections obtained from rho−/− mice subretinally injected with AAV2/5 suppression and replacement vectors.
FIG. 8 illustrates the histology of AAV-transduced Pro23His retinas. Newborn Pro23His+/− rho+/− mice were subretinally injected with 1 μl 2×1012 vp/ml AAV-shBB-rBB or AAV-EGFP (n=6). Ten days post-transduction eyes were processed for semi-thin sectioning and stained with toluidine blue. Approximately 40 measurements in three layers per eye of outer nuclear layer (ONL) thickness (μm) were taken. A: bars represent ONL thickness, of the central meridian of the eye, of the lowest and highest 15% values (p<0.01). B and C: representative images of AAV-shBB-rBB- and AAV-EGFP- (control) injected sections corresponding to highest ONL thickness values. Yellow arrows indicate ONL thickness. INL: inner nuclear layer. GCL: ganglion cell layer. Error bars represent SD values.
FIG. 9 illustrates suppression and/or replacement constructs used to generate recombinant AAV2/5 viruses using the procedures provided in Example 1. RHO suppression and or replacement constructs, pAAV-BB8, pAAV-BB9, pAAV-BB10, pAAV-BB11, pAAV-BB12, pAAV-BB13, pAAV-BB18, pAAV-BB26/Q26, pAAV-BB16, pAAV-BB24 and pAAV-BB27. Illustrations of some control constructs are also provided (pAAV-rho-EGFP and pAAV-CMV-EGFP). Suppression constructs with EGFP reporter genes are also provided (pAAV-shBB-EGFP, pAAV-shQ1-EGFP, pAAV-shCC-EGFP). Suppressors were expressed from the H1 promoter and replacement RHO cDNAs from differently sized mouse rhodopsin promoter sequences. HGH int: human growth hormone intron. CRX-NRL indicates enhancer element SEQ ID NO: 94. 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. Notably, any combination of the elements and conserved regions outlined and indeed other elements that can modulate gene expression could be used in the invention to exert control over expression of suppression and or replacement components.
FIG. 10 illustrates a comparison of levels of expression from the Rho-M transgene versus that obtained from the suppression and replacement constructs in AAV2/5 and represented in FIG. 9, using RNAse protection. FIG. 10 illustrates that the suppression and replacement constructs (see FIG. 9) engineered into AAV2/5, AAV-BB8, AAV-BB10, AAV-BB11, AAV-BB12, AAV-BB13 and AAV-BB16 express the human rhodopsin replacement gene in RNA extracted from 129 wild type mice subretinally injected with suppression and or replacement constructs. (Lanes with material from mouse eyes injected with AAV-BB8 are indicated by BB8, AAV-BB10 by BB10, AAV-BB11 by BB11 etc. The plasmid constructs used to generate AAV vectors are written in the format pAAV as presented in FIG. 9). BB8, BB10 and BB11 express rhodopsin at lower levels than BB12, BB13 and BB16.
FIG. 11 provides a comparative analysis of rhodopsin expression from rAAV2/5 suppression and replacement vectors using real time RT-PCR. FIG. 11 illustrates replacement rhodopsin expression levels in RNA extracted from 129 wild type mice subretinally injected with suppression and/or replacement constructs. Expression levels were also determined in Rho-M transgenic mice which express a rhodopsin replacement construct rCC and display normal retinal function. Suppression and replacement constructs BB12, BB13, BB16 and BB18 express approximately in the same order of magnitude as levels of replacement rhodopsin transcript in Rho-M mice, indicating that enhanced replacement constructs with enhancer elements and conserved regions may express sufficient levels of rhodopsin to sustain a functional retina in vivo. (Lanes with material from mouse eyes injected with AAV-BB8 are indicated by BB8, AAV-BB10 by BB10, AAV-BB11 by BB11 etc.)
FIG. 12 illustrates retinal histology of adult wild type retinas were subretinally injected with 2 ul of 2×1012 particle/ml of different replacement-RHO AAV vectors (see FIG. 9). Two weeks post-injection transduced eyes were removed, fixed in 4% paraformaldehyde and cryosectioned (12 um). Subsequently, sections were stained with human specific anti-RHO antibody to visualize expression of replacement-RHO using Cy3 label (red) on the secondary antibody; cell nuclei were counterstained with DAPI (blue). A: AAV-BB8, B: AAV-BB13, C: AAV-BB24, D: AAV-Q8, E: AAV-Q26, F: retina from uninjected RhoM transgenic mouse expressing RHO (positive control). Sections indicate different levels of RHO expression in the sections. OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.
FIG. 13 illustrates retinal histology of adult NHR transgenic mice on a rho−/− background, therefore expressing normal human RHO but not mouse rho. These mice were transduced by subretinal injection of 2 ul of 2×1012 particle/ml of AAV-shQ1-EGFP (A) or AAV-shNT-EGFP (B). Two weeks after injection, eyes were removed, fixed in 4% paraformaldehyde and cryosectioned AAV-shQ1-EGFP expresses shRNA-Q1, which targets RHO, while AAV-shNT-EGFP expresses a non-targeting shRNA (FIG. 9 illustrates exemplary constructs). Both constructs express EGFP allowing tracking the transduced cell populations (green). Sections were counterstained DAPI (blue) to label position of the nuclear layers. A significant reduction in the photoreceptor cell number in the transduced part of the outer nuclear layer is apparent in the AAV-shQ1-EGFP injected (A) retinas compared to those of injected with AAV-shNT-EGFP (B). IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.
FIG. 14A-C illustrates retinal histology of adult RHO-347 transgenic mice carrying a dominant RHO mutation on a mouse rho+/+background causing retinal degeneration were subretinally injected with 2 ul of 2×1012 particle/ml of AAV-shNT-EGFP (A) or AAV-shQ1-EGFP (B) vectors. Two weeks post-injection transduced eyes were removed, fixed in 4% paraformaldehyde and cryosectioned (12 um). AAV-shQ1-EGFP expresses shRNA-Q1-EGFP, which targets RHO, while AAV-shNT-EGFP expresses a non-targeting shRNA. Both constructs express EGFP allowing tracking of the transduced part of the retina (green). Sections were counterstained with DAPI (blue) to indicate positions of the nuclear layers. A significant reduction of the photoreceptor cell numbers in the transduced part of the outer nuclear layer in the AAV-shNT-EGFP injected or the uninjected (C) retinas are apparent due to the degenerative effects of RHO-347 transgene. A significantly preserved outer nuclear layer is detected in the AAV-shQ1-EGFP transduced retinas, where shRNA-Q1-EGFP effectively suppresses the RHO-347 transcript therefore reducing retinal degeneration. Note, that mouse rho (expressed in these retinas) is refractory to suppression by shRNA-Q1-EGFP due to the presence of nucleotide changes at the target site for Q1 siRNA suppression. Suppression and replacement using the degeneracy of the genetic code provided therapeutic benefit at a histological level. FIG. 14D provides evidence of an improvement in the electroretinogram (ERG) in RHO-347 eyes treated with AAV-shQ1-EGFP versus eyes treated with AAV-shNT-EGFP. FIG. 14D provides a representative maximum ERG response of a RHO-347 mouse, containing a human rhodopsin transgene with a mutation at codon 347, subretinally injected with AAV2/5 constructs. This RHO-347 mouse normally displays a phenotype similar to autosomal dominant RP. The top panel in FIG. 14D is the response of the right eye, which received an injection of AAV-shQ1, a AAV2/5 vector containing suppressor siRNA Q1 driven by an H1 promoter (shQ1) and a CMV-driven EGFP gene. The left eye received an AAV-shNT, a AAV2/5 containing a non-targeting (control) siRNA driven by an H1 promoter (shNT) and a CMV-driven EGFP gene. As can be seen in FIG. 14D, the maximum response is significantly greater in the treated right eye than in the control left eye, indicating that suppression of the mutant rhodopsin transgene leads to some rescue at the ERG level.
FIG. 15 illustrates exemplary constructs utilising chromatin opening elements to optimise expression are presented. Components utilised to enhance expression may be cloned into vectors such as AAV vectors. Elements to optimise expression of a given gene may be combined with other promoter elements such as the rhodopsin promoter and/or enhancer sequences or alternatively sequences that modulate chromatin structures and drive gene expression may be utilised alone to facilitate optimisation of expression of a target gene.
FIG. 16 shows sequences of exemplary elements that can facilitate modulation of chromatin structures.
FIG. 17 shows nucleotide and amino acid sequences of a number of exemplary neurotrophic factors.
FIG. 18 illustrates exemplary suppression and replacement constructs containing other genetic elements which are beneficial for photoreceptor cell survival. In the example pAAV-BB18 has been combined with neurotrophic factor GDNF, driven by a small UCOE (chromatin opening element. A Thrasher, Abstract 36, British Society for Gene Therapy 5th Annual Conference 2008) promoter). Other neurotrophic factors such as, for example, Neurturin may also be used in combination with any of the suppression and replacement constructs described. In addition, other beneficial genes, other than neurotrophic factors may also be combined with suppression and replacement constructs such as for example, a second suppression element, a second replacement element, VEGF and others. In example A, the additional element, in this case GDNF is co-located with the suppression and replacement construct within the two AAV inverted terminal repeat sequences, ITR1 and ITR2. In the second example, B, the GDNF gene and its promoter are not co-located with the suppression and replacement elements within ITR1 and ITR2, but are located within the backbone of the plasmid used to generate AAV. Since a small proportion of the backbone is packaged during AAV production, this would result in a mixed population of AAVs with the majority containing the suppression and replacement elements and a minority the GDNF elements. In this case, other beneficial genes, other than neurotrophic factors may also be combined with suppression and replacement constructs such as for example, a second suppression element, a second replacement element, VEGF and others.



DETAILED DESCRIPTION OF THE INVENTION
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 rhodopsin 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.
In another aspect, the invention provides a composition comprising a suppression agent that binds to the coding region of a mature and/or immature RNA or DNA encoding a mutant allele to inhibit expression of the mutant allele and a replacement nucleic acid that 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 agent.
In yet another aspect, the invention provides a kit comprising a suppression agent that suppresses the expression of a mature and or immature RNA or DNA encoding a mutant allele and a replacement nucleic acid that encodes a wild-type or non-disease causing allele that is not Suppressed, or is only partially suppressed, by the suppression agent and differs from the mutant allele in at least one degenerate/wobble nucleotide.
Suppression is achieved using a wide variety of molecular tools, such as, for example, RNA interference (RNAi) including non-coding RNAs such as small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNAs (miRNA), or other nucleotide-based molecules. In an embodiment, siRNAs in the order of 14-27 nucleotides in length are used for gene suppression. ShRNAs can be used to express functional siRNAs intracellularly and to achieve suppression in vitro and in vivo. Other suppression molecules include, for example, sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins.
In an embodiment, the invention relates to vector(s) for supplying an endogenously generated suppression agent, such as, for example, a dsRNA in the form of a short hairpin (shRNA) which can be processed intracellularly into siRNA. dsRNA may be locally or systemically delivered. Expression vectors are used to generate functional siRNAs in cells and in animals typically using polymerase III promoters to drive expression, although polymerase II promoters are also used. For example, miRNA structures can be used to express double stranded RNAs from polymerase II promoters to enable tissue specific expression of double stranded RNA or polymerase II promoters can be juxtaposed to shRNA sequences to be expressed.
Suppression agents may be modified to alter the potency of the suppression agent, the target affinity of the suppression agent, the safety profile of the suppression agent and/or the stability of the suppression agent, for example, to render them resistant or partially resistant to intracellular degradation. Modifications, such as phosphorothioates, for example, can be made to oligonucleotides to increase resistance to nuclease degradation, binding affinity and/or uptake. In addition, hydrophobization and bioconjugation enhances siRNA delivery and targeting (De Paula et al., RNA. 13(4):431-56, 2007) and siRNAs with ribo-difluorotoluyl nucleotides maintain gene silencing activity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006). siRNAs with amide-linked oligoribonucleosides have been generated which are more resistant to Si nuclease degradation (Iwase R et al. 2006 Nucleic Acids Symp Ser 50: 175-176). In addition, modification of siRNA at the 2′-sugar position and phosphodiester linkage confers improved serum stability without loss of efficacy (Choung et al., Biochem. Biophys. Res. Commun. 342(3):919-26, 2006). In one study, 2′-deoxy-2′-fluoro-beta-D-arabinonuclecic acid (FANA)-containing antisense oligonucleotides compared favourably to phosphorothioate oligonucleotides, 2′-O-methyl-RNA/DNA chimeric oligonucleotides and siRNAs in terms of suppression potency and resistance to degradation (Ferrari N et al. 2006 Ann N Y Acad Sci 1082: 91-102.)
Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia. 6(11):1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res. 55(1):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998). For example, neoplastic reversion was obtained using a ribozyme targeted to an H-ras mutation in bladder carcinoma cells (Feng et al., Cancer Res. 55(10):2024-8, 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9, 1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29):121-2, 1993).
Triple helix approaches have also been investigated for sequence-specific gene suppression. Triplex forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Haney et al., Antisense Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83, 1997). Minor groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al., Chem. Biol. 3(5):369-77, 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppression strategies have lead to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.
The diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target, for example, a protein of interest such as rhodopsin. In the case of age related macular degeneration (AMD), anti-VEGF aptamers have been generated and have been shown to provide clinical benefit in some AMD patients (Ulrich H, et al. Comb. Chem. High Throughput Screen 9: 619-632, 2006). Suppression and replacement using aptamers for suppression in conjunction with a modified replacement gene and encoded protein that is refractory or partially refractory to aptamer-based suppression could be used in the invention.
Recent evidence suggests that control of gene expression occurs endogenously in part by the activity of small non-coding RNAs, one broad category of which is termed microRNAs (miRNAs). miRNAs are expressed from polymerase II promoters, but can also be expressed from polymerase III promoters. miRNAs are processed intracellularly from larger transcripts to form small molecules approximately 20 nucleotides in length. miRNA structures can be used to express small double stranded RNAs and thus can be used to express the double stranded RNAs of the current invention.
Suppression targeted to coding sequence holds the advantage that such sequences are present in both precursor and mature RNAs, thereby enabling suppressor effectors to target all forms of RNA. A combined approach using a number of suppression effectors directed to multiple targets on an RNA or to multiple RNAs may also be used in the invention. As with suppression, multiple replacement nucleic acids can be used in the invention. 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. The invention can thus provide partial or complete suppression.
In one embodiment of the invention, suppressors are targeted to genes that are involved in the regulation of other genes. Suppression of these genes therefore may lead to up- or down-regulation of other genes.
In another embodiment, the invention relates to suppression of the expression of mutated genes that give rise to a dominant or deleterious effect or disease. A suppression effector may target either the disease allele or the normal allele. In another embodiment, the suppression effector targets both the disease allele and the normal allele.
In an embodiment of the invention, a replacement nucleic acid is provided that is altered at one or more degenerate or wobble bases from the endogenous wild type gene but that encodes the identical amino acids as the wild type or a non-disease causing gene. In another embodiment, the replacement nucleic acid encodes a beneficial replacement nucleic acid (e.g., a more active or stable product than that encoded by the wild-type gene). The replacement nucleic acid provides expression of a normal or non-disease causing protein product when required to ameliorate pathology associated with reduced levels of wild type protein. The same replacement nucleic acid can be used in conjunction with the suppression of many different disease mutations within a given gene. In addition, multiple replacement nucleic acids can be used in the invention.
Although the instant application provides numerous exemplary suppression agents and replacement nucleic acid sequences, these are only examples and other such sequences can be determined as described herein for the same targets or for any desired target. “Functional variant” includes any variant nucleic acid or other suppression agent that may have one or more nucleic acid substitutions but that does not have a materially different function than, or that can still hybridize under stringent hybridization conditions (0.2×SCC, 0.1% SDS) to, or that shares at least 70% identity, for example 80%, such as at least 90% or at least 95% sequence identity with the nucleic acid indicated.
In another embodiment of the invention, suppression effectors are targeted to the untranslated regions (either 5′UTR or 3′UTR) of at least one allele of a gene. In another embodiment of the invention replacement nucleic acids are provided that have been altered at the suppression site, such that replacement nucleic acids provide functional or partially functional protein and escape or partially escape from suppression by suppressors.
In another embodiment of the invention, suppression effectors are targeted to intronic sequences. In another embodiment, replacement nucleic acids are provided which have been altered at one or more nucleotides of the targeted site of the intron so that transcripts from the replacement nucleic acids escape or partially escape suppression by suppressors. In another embodiment the whole targeted intron may not be present in replacement nucleic acids.
In another embodiment of the invention, suppression effectors are targeted to polymorphic sites and at least one allele of the gene is suppressed or partially suppressed. In another embodiment, replacement nucleic acids are provided for the alternative polymorphic variant such that replacement nucleic acids encode functional or partially functional protein and escape or partially escape from suppression by suppressors.
In another embodiment of the invention the suppression agent and/or replacement nucleic acid is expressed from one or more promoter sequences. The invention provides promoter sequences that have been demonstrated to promote ubiquitous expression of nucleotides and/or promoters that have been demonstrated to exert tissue specific, temporal, inducible, and/or quantitative control of gene expression. The invention also provides enhancer sequences (Table 1) and/or post-translational regulatory elements and/or other regulatory elements and/or epigenetic elements that provide optimized expression of suppression agents and/or replacement nucleic acids.







TABLE 1







Exemplary Enhancer Elements








Enhancer Element
Reference





Chicken ovalbumin upstream promoter
Eguchi et al., Biochimie


transcription factor II
89(3): 278-88, 2007


Mouse dystrophin muscle promoter/
Anderson et al., Mol. Ther.


enhancer
14(5): 724-34, 2006


Tobacco eIF4A-10 promoter elements
Tian et al., J. Plant Physiol.



162(12): 1355-66, 2005


Immunoglobulin (Ig) enhancer element
Frezza et al., Ann. Rheum.


HS1, 2A
Dis. Mar. 28, 2007


Col9a1 enhancer element
Genzer and Bridgewater



Nucleic Acids Res.



35(4): 1178-86, 2007


Gata2 intronic enhancer
Khandekar et al.,



Development Mar. 29,



2007


TH promoter enhancer
Gao et al., Brain Res.



1130(1): 1-16, 2007


CMV enhancer
InvivoGen cat# pdrive-cag



05A13-SV


Woodchuck hepatitis virus posttran-
Donello et al., J. Virol.


scriptional regulatory element
72(6): 5085-92, 1998


Woodchuck hepatitis virus posttran-
Schambach et al., Gene


scriptional regulatory element
Ther. 13(7): 641-5, 2006


IRBP
Ying et al., Curr. Eye Res.



17(8): 777-82, 1998


CMV enhancer and chicken β-actin
InvivoGen cat# pdrive-cag


promoter
05A13-SV


CMV enhancer and chicken β-actin
InvivoGen cat# pdrive-cag


promoter and 5′UTR
05A13-SV


CpG-island
Antoniou et al., Genomics



82: 269-279, 2003









In a particular embodiment, sequences that influence chromatin structure, such as but not exclusive to insulator, antirepressor, cis-acting modulators of nucleosome positioning and/or silencer elements, sometimes termed epigenetic elements, are used to modulate expression of suppression agents and/or replacement nucleic acids. Exemplary epigenetic elements such as insulator and antirepressor sequences are provided in Table 2. It is clear that chromatin structures influence gene expression, for example, chromatin structures influence the ability of the transcriptional machinery to access promoter and/or enhancer elements amongst other sequence motifs. The inclusion of sequences which influence chromatin structures in viral and/or non-viral vectors and/or administered in conjunction with suppression and/or replacement nucleic acids can be used to optimize expression of either or both suppressors and replacement nucleic acids. In addition, chemical entities which influence chromatin structures can be used to optimize expression such as histone deacetylase (HDAC) inhibitors and/or DNA methyl transferase inhibitors and/or histone methyl transferase inhibitors. Such entities can be supplied in the form of DNA and/or RNA and/or protein amongst other forms. Similarly attracting enzymes and/or supplying enzymes (in the form of DNA and/or RNA and or protein) involved in chromatin remodelling such as but not exclusive to histone acetyl transferases to nucleic acids to be expressed and their associated regulatory regions can be used to optimize expression of suppression and/or replacement nucleic acids.







TABLE 2







Exemplary Epigenetic Elements








Epigenetic elements
Reference





Mcp Insulators
Kyrchanova et al., Mol. Cell



Biol. 27(8): 3035-43, 2007


CpG-island region of the HNRPA2B1
Williams et al., BMC


locus
Biotechnol. 5: 17, 2005


Chicken b-globin 5′hypersensitive site 4
Kwaks and Otte 2006 Trends in


(cHS4)
Biotechnology 24: 137-142


Ubiquitous chromatin opening elements
Kwaks and Otte 2006 Trends in


(UCOEs)
Biotechnology 24: 137-142


Matrix associated regions (MARs)
Kwaks and Otte 2006 Trends in



Biotechnology 24: 137-142


Stabilising and antirepressor elements
Kwaks and Otte 2006 Trends in


(STAR)
Biotechnology 24: 137-142


Human growth hormone gene silencer
Trujillo MA et al. 2006 Mol



Endocrinol 20: 2559


S/MAR
Liebich et al., Nucleic



Acids Res. 30: 3433-42, 2002









In another embodiment, expression of a suppression agent and/or replacement nucleic acid is optimized to enable efficient suppression in conjunction with sufficient replacement. In an additional embodiment, suppression and/or replacement nucleic acids are provided with agents that aid vector transfection, transduction, and/or expression of suppression and replacement nucleic acids.
The invention circumvents the need for a specific therapy for every disease-causing mutation within a given gene. Notably, the invention has the advantage that the same suppression agents can be used to suppress many mutations in a gene. This is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology. The compositions and methods of the invention allow greater flexibility in choice of target sequence for suppression of expression of a disease allele. Furthermore, the compositions and methods of the invention allow greater flexibility in terms of controlling expression of the suppression and/or replacement of a given gene and or allele of a gene.
Suppression and replacement can be undertaken in conjunction with each other or separately. Suppression and replacement utilizing the degeneracy of the genetic code may be undertaken in test tubes, in cells, in animals, or in plants and may be used for experimental research (e.g., for the study of development or gene expression) or for therapeutic purposes. Suppression and replacement may be used in conjunction with agents to promote cell transfection or cell transduction such as, for example, lipids and polymers. Suppression and replacement may be provided to consumers in a kit.
The suppression and replacement agents of the invention can be delivered to a target cell and or tissue and or animal and or plant using ‘naked’ reagents such as DNA, RNA, peptides or other reagents. Alternatively viral and or non-viral vectors can be used with or without ‘naked’ reagents.
In an embodiment, suppression and/or replacement construct(s) can be delivered to a cell using an AAV2/5 recombinant virus, however, other viral and non-viral vectors, such as other AAV serotypes, adenovirus, herpes virus, SV40, HIV, SIV and other lentiviral vectors, RSV and non-viral vectors including naked DNA, plasmid vectors, peptide-guided gene delivery, terplex gene delivery systems, calcium phosphate nanoparticles, magnetic nanoparticles, colloidal microgels and/or the integrase system from bacteriophage phiC31 may be utilised in the invention, for example. Suppression and replacement components may be found on separate vectors or may be incorporated into the same vector. Viral vectors useful in the invention include, but are not limited to, those listed in Table 3. Non-viral vectors useful in the invention include, but are not limited to, those listed in Table 4. Cationic lipid-based non-viral vectors can include glycerol-based (e.g. DOTMA, DOTAP, DMRIE, DOSPA), non-glycerol-based (e.g. DOGS, DOTIM) and/or cholesterol-based cationic lipids (e.g. BGTC, CTAP; Karmali P P and Chaudhuri A 2006 Med Res Rev). Viral and non-viral vector delivery may be accompanied by other molecules such as cationic lipids and/or polymers and/or detergents and/or agents to alter pH, such as, for example, polyethelene glycol (PEG), to enhance cellular uptake of vectors and/or to enhance expression from vectors and/or to evade the immune system. For example, polycationic molecules have been generated to facilitate gene delivery including but not exclusive to cationic lipids, poly-amino acids, cationic block co-polymers, cyclodextrins amongst others. Pegylation of vectors with polyethelene glycol (PEG) can shield vectors from, for example, the extracellular environment. Vectors may be used in conjunction with agents to avoid or minimise cellular immune responses such as PEG or as a Polyplex with Poly(L-Lysine) Vector delivery may be undertaken using physical methodologies such as electroporation, nucleofection and/or ionotophoresis, either alone or in combination with molecules to enhance delivery. Vectors may be used in conjunction with agents to promote expression of suppression and/or replacement components incorporated into vectors, for example, using histone deacetylase inhibitors (HDAC) and/or DNA methyl transferase inhibitors and/or histone methyl transferase inhibitors to modulate chromatin structures thereby aiding expression. HDAC inhibitors include but are not exclusive to short chain fatty acids such as valproic acid and sodium butyrate, ketones, benzamides, cyclic and non-cyclic hydroxamates such as suberoyl anilide hydroxamic acids (SAHA), trichostatin A (TSA), cyclic peptides or tetrapeptides amongst others (Liu T et al. 2006 Cancer Treatment Reviews 32: 157-165). DNA methyl transfease inhibitors including, for example, 5-AC, decitabine and zebularine can be used to modulate chromatin structures. In addition, histone methyl transferase inhibitors can influence chromatin states, for example, BIX-01294 (diazepin-quinazolin-amine derivative). In addition, to the chemical entities referred to above, nucleic acids-based inhibitors can be used to suppress expression of proteins and/or non-coding RNAs involved in chromatin remodelling. In one embodiment of the invention vectors are optimized to specifically transduce target cell type(s) or target tissue type(s). Viral and/or non-viral vectors may be modified to target specific cell types and/or to prevent targeting of some cell types. For example, the inclusion of the capsid from AAV serotype 5 in an AAV2/5 hybrid virus facilitates transduction of photoreceptor cells. Similarly, for example, peptides may be included in viral vectors to facilitate targeting. Synthetic non-viral vectors can be modified to include ligands to facilitate targeting of vectors to specific cell and/or tissue types, for example, folate can be conjugated to liposomes to target tumour cells which over express the folate receptor (Hattori Y et al. 2005; Curr Drug Deliv 3: 243-52). In another embodiment of the invention, suppression and replacement vectors are designed to optimize the generation and/or production of vector, for example, to optimize viral titre and/or to optimize the number or type of nucleotides incorporated into vector(s). For example, vector genomes may be modified such that large transgenes may be incorporated into vectors, for example, ‘gutless’ adenovirus vectors have an increased capacity in terms of size than previous generations of adenovirus vectors. Components of vectors can be modified to optimize generation and production of vectors, for example, genes involved in replication of AAV can be modified to optimize replication and/or self complementary AAV vectors can be used to optimize rates of transgene expression. In an additional embodiment, vectors are designed to optimize suppression in conjunction with replacement, to enable optimal expression of all components of a therapeutic. For example, to optimize expression of both elements of suppression and replacement from a given vector, additional sequences can be included in the vector. For example, inclusion of nucleotides to separate the ITRs of AAV and the shRNA sequences of an RNAi-based suppression agent can result in optimisation of expression of the suppression component. Nucleotides encoding suppressors and/or replacement nucleic acids can be juxtaposed or separated from each other and/or can be in the same orientation or opposing orientations. In addition, the suppressor(s) can be 5′ and/or 3′ to the replacement nucleic acids. Nucleotides encoding suppressors and/or replacement nucleic acids can be juxtaposed to nucleotides comprising vector(s) or can be separated from nucleotides comprising vector(s). Nucleotides encoding suppressors and/or replacement nucleic acids may be cloned within the backbone of the plasmid used to generate AAV and or may be cloned between the AAV ITRs and not within the plasmid backbone of the plasmid, and/or may be cloned in a combination of these positions. Additional sequences, such as, for example, stuffer sequences can be included in vectors to optimize vector design. In addition, multiple suppressors and/or replacement nucleic acids may be used in one vector.







TABLE 3







Exemplary Viral Vectors









Delivery Method
Serotype
Reference





AAV
All serotypes,
Lebkowski et al., Mol. Cell



including but
Biol. 8(10): 3988-96, 1988



not limited to
Flannery et al., Proc. Natl.



1, 2, 3, 4, 5,
Acad. Sci. U.S.A.



6, 7, 8, 9,
94(13): 6916-21, 1997



10, 11, 12,



Lentivirus (for example but
VSV-G
Pang et al., Mol. Vis.


not exclusively Feline -
Rabies-G
12: 756-67, 2006


FIV, Equine - EIAV,
Further
Takahashi Methods Mol.


Bovine - BIV and Simian -
serotypes**
Biol. 246: 439-49, 2004


SIV).

Balaggan et al., J. Gene




Med. 8(3): 275-85, 2006


Adenovirus
Various
Bennett et al., Nat. Med.




2(6): 649-54, 1996


Simian papovirius SV40
Various
Kimchi-Sarfaty et al., Hum.




Gene Ther. 13(2): 299-310,




2002


Semliki Forest Virus
Various
DiCiommo et al., Invest.




Ophthalmol. Vis. Sco.




45(9): 3320-9, 2004


Sendai Virus
Various
Ikeda et al., Exp. Eye Res.




75(1): 39-48, 2002









The list provided is not exhaustive; other viral vectors and derivatives, natural or synthesized could be used in the invention.







TABLE 4







Exemplary Non-Viral Vectors or Delivery Methods








Delivery Method
Reference





Cationic liposomes
Sakurai et al., Gene Ther. 8(9): 677-86, 2001


HVJ liposomes
Hangai et al., Arch. Ophthalmol.



116(3): 342-8, 1998


Polyethylenimine
Liao and Yau Biotechniques 42(3): 285-6, 2007


DNA nanoparticles
Farjo et al., PloS ONE 1: e38, 2006


Dendrimers
Marano et al., Gene Ther. 12(21): 1544-50, 2005


Bacterial
Brown and Giaccia Cancer Res.



58(7): 1408-16, 1998


Macrophages
Griffiths et al., Gene Ther. 7(3): 255-62, 2000


Stem cells
Hall et al., Exp. Hematol. 34(4): 433-42, 2006


Retinal transplant
Ng et al., Chem. Immunol. Allergy 92: 300-16,



2007


Marrow/Mesenchymal
Kicic et al., J. Neurosci. 23(21): 7742-9, 2003


stromal cells
Chng et al., J. Gene Med. 9(1): 22-32, 2007


Implant (e.g.,
Montezuma et al., Invest. Ophthalmol. Vis.


Poly(imide) uncoated
Sci. 47(8): 3514-22, 2006


or coated)



Electroporation
Featherstone A. Biotechnol. Lab. 11(8): 16,



1993


Targeting peptides
Trompeter et al., J. Immunol Methods.


(for example but not
274(1-2): 245-56, 2003


exclusively Tat)



Lipid mediated
Nagahara et al., Nat. Med. 4(12): 1449-52,


(e.g., DOPE, PEG)
1998



Zeng et al., J. Virol. 81(5): 2401-17, 2007



Caplen et al., Gene Ther. 2(9): 603-13,



1995Manconi et al., Int. J. Pharm. 234(1-2):



237-48, 2006



Amrite et al., Invest. Ophthalmol. Vis. Sci.



47(3): 1149-60, 2006



Chalberg et al., Invest. Ophthalmol. Vis.



Sci. 46(6): 2140-6, 2005









The list provided is not exhaustive. Other non-viral vectors and derivatives, natural or synthesized and other delivery methods could be used with the invention.
In an embodiment, the replacement nucleic acid encodes mammalian rhodopsin, collagen 1A1, collagen 1A2, collagen 7A1, or peripherin. In another embodiment, the replacement nucleic acid encodes a protein that has been mutated to cause an autosomal or X-linked dominant retinitis pigmentosa, such as those listed in Table 5. Suppression agents and replacement nucleic acids may be generated for one or more of these genes, for example.







TABLE 5







Genes known to be involved in retinitis pigmentosa (Table adapted 


from RETNET) (http://www.sph.uth.tmc.edu/Retnet/)










Symbols;





OMIM





Numbers
Location
Diseases; Protein
References





LCA9
1p36
recessive Leber 
Keen et al., Hum. 




congenital amaurosis
Mol. Genet. 3: 367-





368 (1994)


NPHP4,
1p36.31
recessive Senior-
Mollet et al., Nat.


SLSN4;

Loken syndrome; 
Genet. 32: 300-305




recessive 
(2002); Otto et al., 




nephronophthisis, 
Am. J. Hum. Genet.




juvenile; protein: 
71: 1161-1167 




nephronophthisis 4 
(2002); Schuermann 




protein
et al., Am. J. Hum. 





Genet. 70: 1240-





1246 (2002)


RP32;
1p34.3-p13.3
recessive RP, severe
Zhang et al., Hum.





Genet. 118: 356-365





(2005)


RPE65,
1p31.2
recessive Leber 
Acland Nat. Genet.


LCA2, 

congenital amaurosis; 
28: 92-95 (2001)


RP20;

recessive RP; protein:





retinal pigment 





epithelium-specific 65 





kD protein



ABCA4,
1p22.1
recessive Stargardt 
Lewis et al., Am. J.


ABCR, 

disease, juvenile and 
Hum. Genet. 64: 


RP19,

late onset; recessive 
422-434 (1999)


STGD1;

MD; recessive RP; 





recessive fundus 





flavimaculatus; 





recessive cone-rod 





dystrophy; protein:





ATP-binding cassette 





transporter-retinal



COL11A1,
1p21.1
dominant Stickler 
Annunen et al., Am. 


STL2;

syndrome, type II; 
J. Hum. Genet. 65: 




dominant Marshall 
974-983 (1999)




syndrome; protein: 





collagen, type XI, 





alpha 1



GNAT2,
1p13.3
recessive 
Aligianis et al., J. 


ACHM4;

achromatopsia; 
Med. Genet. 39: 




protein: guanine 
656-660 (2002)




nucleotide binding 





protein (G protein) 





cone-specifc 





transducin alpha 





subunit



PRPF3,
1q21.2
dominant RP; protein: 
Chakarova et al., 


HPRP3,

human homolog of 
Hum. Mol. Genet. 


PRP3, 

yeast pre-mRNA 
11: 87-92 (2002)


RP18;

splicing factor 3



SEMA4A,
1q22
dominant RP; 
Abid et al., J. Med.


SEMAB;

dominant cone-rod 
Genet. 43: 378-381




dystrophy; protein: 
(2005)




semaphorin 4A



CORD8;
1q23.1-q23.3
recessive cone-rod 
Ismail et al., J. Hum.




dystrophy
Genet. 51: 827-831





(2006)


AXPC1
1q31-q32
recessive ataxia, 
Higgins et al., 




posterior column with 
Neurol. 52: 146-150 




RP
(1999)


ARMD1,
1q31.1
dominant MD, age-
Schultz et al., Hum.


FIBL6,

related; protein: 
Mol. Genet. 12: 


FBLN6;

hemicentin 1 
3315-3123 (2003)




(fibulin 6)



CFH, HF1;
1q31.3
age-related macular 
Edwards et al., 




degeneration, 
Science 308: 421-




complex etiology; 
424 (2005)




protein: complement 





factor H



CRB1, 
1q31.3
recessive RP with 
Jacobson et al., 


RP12

para-arteriolar 
Hum. Mol. Genet. 9: 




preservation of the 
1073-1078 (2003)




RPE (PPRPE); 





recessive RP; 





recessive Leber 





congenital amaurosis; 





dominant pigmented 





paravenous 





chorioretinal atrophy; 





protein: crumbs 





homolog 1



RD3,
1q32.3
recessive Leber 
Friedman et al., Am. 


C1ORF36;

congenital amaurosis; 
J. Hum. Genet. 79: 




protein: RD3 protein
1059-1070 (2006)


USH2A;
1q41
recessive Usher 
Seyedahmadi et al.,




syndrome, type 2a; 
Exp. Eye. Res. 79: 




recessive RP; protein: 
167-173 (2004)




usherin



RP28;
2p16-p11
recessive RP
Kumar et al., Mol. 





Vis. 10: 399-402 





(2004)


EFEMP1,
2p16.1
dominant radial, 
Kermani et al., 


DHRD,

macular drusen; 
Hum. Genet. 104: 


MTLV,

dominant Doyne 
77-82 (1999)


FBLN3;

honeycomb retinal 





degeneration 





(Malattia 





Leventinese); protein: 





EGF-containing 





fibrillin-like extra-





cellular matrix protein 





1 (fibulin 3)



ALMS1,
2p13.1
recessive Alstrom 
Hearn et al., Nat.


ALSS

syndrome; protein: 
Genet. 31: 79-83 




ALMS1 protein
(2002)


RP33
2cen-q12.1
dominant RP
Zhao et al., Hum.





Genet. 119: 617-623





(2006)


LOC619531
2q11
recessive cone-rod 
Michaelides et al., J.




dystrophy and 
Med. Genet. 41: 




amelogenesis 
468-473 (2004)




imperfecta



CNGA3,
2q11.2
recessive 
Nishiguchi et al., 


ACHM2,

achromatopsia; 
Hum. Mutat. 25: 


CNCG3,

protein: cone 
248-258 (2005)


RMCH2

photoreceptor cGMP-





gated cation channel 





alpha subunit



MERTK
2q13
recessive RP; protein: 
Vollrath et al., Proc.




c-mer protooncogene 
Natl. Acad. Sci. 




receptor tyrosine
USA 98: 12584-




kinase
12589 (2001)


NPHP1,
2q13
recessive Senior-
Hildebrandt et al., 


JBTS4,

Loken syndrome; 
Nat. Genet. 17: 149-


SLSN1

recessive 
153 (1997)




nephronophthisis, 





juvenile; recessive 





Joubert syndrome;





protein: 





nephronophthisis 3





protein



BBS5
2q31.1
recessive Bardet-
Li et al., Cell. 117: 




Biedl syndrome; 
541-552 (2004)




protein: flagellar 





apparatus-basal body 





protein 





DKFZp7621194



CERKL,
2q31.3
recessive RP; protein: 
Tuson et al., Am. J.


RP26

ceramide kinase-like 
Hum. Genet. 74: 




protein
128-138 (2004)


SVD
2q36
dominant vitreoretinal
Jiao et al., Invest.




degeneration, 
Ophthalmol. Vis. 




snowflake
Sci. 45: 4498-503 





(2004)


SAG
2q37.1
recessive Oguchi 
Nakazawa et al., 




disease; recessive RP; 
Arch. Ophthalmol. 




protein: arrestin (s-
116: 498-501 (1998)




antigen)



USH2B
3p24.2-p23
recessive Usher 
Hmani et al., Eur. J.


DFNB6

syndrome, type 2; 
Hum. Genet. 7: 363-




recessive 
367 (1999)




sensorineural deafness 





without RP



CRV,
3p21.3-p21.1
dominant hereditary 
OPhoff et al., Am. J.


HERNS, 

vascular retinopathy 
Hum. Genet. 69: 


HVR

with Raynaud 
447-453 (2001)




phenomenon and 





migraine



GNAT1
3p21.31
dominant CSNB, 
Dryja et al., Nat. 




Nougaret type; 
Genet. 13: 358-360 




protein: rod 
(1996)




transducin alpha 





subunit



ATXN7,
3p14.1
dominant spinocere-
Aleman et al., Exp.


ADCA2,

bellar ataxia w/ MD 
Eye. Res. 74: 737-


OPCA3,

or retinal degenera-
745 (2002)


SCA7

tion; protein: ataxin 7



ARL6, 
3q11.2
recessive Bardet-
Fan et al., Nat. 


BBS3

Biedl syndrome; 
Genet. 36: 989-993 




protein: ADP-ribo-
(2004)




sylation factor-like 6



IQCB1,
3q13.33
recessive Senior-
Otto et al., Nat. 


NPHP5,

Loken syndrome; 
Genet. 37: 282-288 


SLSN5

protein: IQ motif 
(2005)




containing B1 protein



NPHP3,
3q22.1
recessive Senior-
Olbrich et al., Nat.


SLSN3

Loken syndrome; 
Genet. 34: 455-459




recessive 
(2003)




nephronophthisis, 





adolescent; protein: 





nephronophthisis 3





protein



RHO, RP4
3q22.1
dominant RP; 
Dryja et al., Nat. 




dominant CSNB; 
Genet. 4: 280-283 




recessive RP; protein: 
(1993)




rhodopsin



RP5
same as RHO
not distinct from 
Farrar et al., Hum. 




RHO/RP4
Mol. Genet. 1: 769-





771 (1992)


USH3A,
3q25.1
recessive Usher 
Joensuu et al., Am. 


USH3

syndrome, type 3;
J. Hum. Genet. 69: 




protein: clarin-1
673-684 (2001)


OPA1
3q29
dominant optic 
Aung et al., Hum.




atrophy, Kjer type; 
Genet. 110: 52-56




dominant optic 
(2002)




atrophy with sensori-





neural hearing loss; 





protein: OPA1 protein



STGD4
4p
dominant Stargardt-
Kniazeva et al., Am. 




like macular 
J. Hum. Genet. 64: 




dystrophy
1394-1399 (1999)


MCDR2
4p16.3-p15.2
dominant MD, bull's-
Michaelides et al.,




eye
Invest. Ophthalmol.





Vis. Sci. 44: 1657-





1662 (2003)


PDE6B,
4p16.3
recessive RP; 
Pearce-Kelling et 


CSNB3

dominant CSNB; 
al., Mol. Vis. 7: 42-




protein: rod cGMP 
47 (2001)




phosphodiesterase 





beta subunit



WFS1,
4p16.1
recessive Wolfram 
Hum. Mol. Genet.


DFNA38

syndrome; dominant 
10: 2501-2508 




low frequency 
(2001)




sensorineural hearing 





loss; protein: 





wolframin



PROML1
4p15.32
recessive retinal 
Maw et al., Hum. 




degeneration; protein: 
Mol. Genet. 9: 27-34 




prominin (mouse)-like 
(2000)




1



CNGA1,
4p12
recessive RP; protein: 
Dryja et al., Proc. 


CNCG,

rod cGMP-gated 
Natl. Acad. Sci. 


CNCG1

channel alpha subunit
USA 92: 10177-





10181 (1995)


WFS2
4q22-q24
recessive Wolfram 
El-Shanti et al., Am. 




syndrome; dominant
J. Hum. Genet. 66: 





1229-1236 (2000)


MTP, ABL
4q23
recessive 
Narcisi et al., Am. J.




abetalipoproteinemia;
Hum. Genet. 57: 




protein: microsomal 
1298-1310 (1995)




triglyceride transfer 





protein



BBS7,
4q27
recessiveBardet Biedl 
Badano et al., Am. J.


BBS2L1

syndrome; protein: 
Hum. Genet. 72: 




BBS7 protein
650-658 (2003)


BBS12,
4q27
recessive Bardet-
Stoetzel et al., Am. 


FLJ35630

Biedl syndrome; 
J. Hum. Genet. 80: 




protein: BBS12 
1-11 (2007)




protein



RP29
4q32-q34
recessive RP
Hameed et al., 





Invest. Ophthalmol. 





Vis. Sci. 42: 1436-





1438 (2001)


LRAT
4q32.1
recessive RP, severe 
Thompson et al., 




early-onset; recessive 
Nat. Genet. 128: 




Leber congenital 
123-124 (2001)




amaurosis; protein: 





lecithin retinol 





acyltransferase



CYP4V2,
4q35.2
recessive Bietti 
Li et al., Am. J. 


BCD

crystalline corneo-
Hum. Genet. 74: 




retinal dystrophy; 
817-826 (2004)




protein: cytochrome 





P450 4V2



MCDR3
5p15.33-p13.1
dominant MD
Michaelides et al.,





Invest. Ophthalmol.





Vis. Sci. 44: 2178-





2183 (2003)


CSPG2,
5q14.3
dominant Wagner 
Kloeckener-


WGN1,

disease and erosive 
Gruissem et al., 


ERVR

vitreoretinopathy; 
Mol. Vis. 12: 350-




protein: chondroitin 
355 (2006)




sulfate proteoglycan 2





(versican)



MASS1,
5q14.3
recessive Usher 
Weston et al., Am. J.


USH2C,

syndrome, type 2; 
Hum. Genet. 74: 


VLGR1

dominant/recessive 
357-366 (2004)




febrile convulsions; 





protein: monogenic





audiogenic seizure 





susceptibility 1 





homolog



BSMD
5q21.2-q33.2
dominant MD, 
den Hollander et al., 




butterfly-shaped
J. Med. Genet. 41: 





699-702 (2004)


PDE6A
5q33.1
recessive RP; protein: 
Dryja et al., Invest.




cGMP 
Ophthalmol. Vis. 




phosphodiesterase 
Sci. 40: 1859-1865 




alpha subunit
(1999).


GRM6
5q35.3
recessive CSNB; 
Dryja et al., Proc. 




protein: metabotropic 
Natl. Acad. Sci. 




glutamate receptor 6
USA 102: 4884-





4889 (2005)


C2
6p21.32
age-related macular 
Gold et al., Nat. 




degeneration, 
Genet. 38: 458-462 




complex etiology; 
(2006)




protein: complement 





component 2



CFB, BF,
6p21.32
age-related macular 
Gold et al., Nat. 


BFD

degeneration, 
Genet. 38: 458-462 




complex etiology; 
(2006)




protein: complement 





factor B, properdin



TULP1, RP14
6p21.31
recessive RP; 
Banerjee et al., Nat.




recessive Leber 
Genet. 18: 177-179




congenital amaurosis; 
(1998)




protein: tubby-like 





protein 1



RDS, RP7
6p21.2
dominant RP; 
Hum. Mutat. 10: 




dominant MD; 
301-309 (1997)




digenic RP with 





ROM1; dominant 





adult vitelliform MD;





protein: peripherin 2



GUCA1A,
6p21.1
dominant cone 
Payne et al., Am. J.


COD3,

dystrophy; dominant 
Hum. Genet. 61: 


GCAP1

cone-rod dystrophy; 
A290 (1997)




protein: guanylate 





cyclase activating 





protein 1A



GUCA1B,
6p21.1
dominant RP; 
Sato et al., Graefes


GCAP2

dominant MD; 
Arch. Clin. Exp.




protein: guanylate 
Ophthalmol. 243: 




cyclase activating 
235-242 (2004)




protein 1B



BCMAD
6p12.3-q16
dominant MD, benign 
van Lith-Verhoeven 




concentric annular
et al., Invest. 





Ophthalmol. Vis. 





Sci. 45: 30-35 





(2004)


RP25
6cen-q15
recessive RP
Abd El-Aziz et al.,





Ann. Hum. Genet.





(2006)


LCA5
6q11-q16
recessive Leber 
Dharmaraj et al., 




congenital amaurosis
Am. J. Hum. Genet. 





66: 319-326 (2000)


COL9A1
6q13
recessive Stickler 
Van Camp et al., 




syndrome; dominant 
Am. J. Hum. Genet. 




multiple epiphyseal 
79: 449-457 (2006)




dysplasia (MED); 





protein: collagen, type 





IX, alpha-1



RIMS1,
6q13
dominant cone-rod 
Kelsell et al., Am. J.


CORD7,

dystrophy; protein: 
Hum. Genet. 63: 


RIM1

regulating synaptic 
274-279 (1998)




membrane exocytosis 





protein 1or rab3A-





interacting molecule



MCDR1,
6q14-q16.2
dominant MD, North 
Small et al., Mol. 


PBCRA

Carolina type; 
Vis. 5: 38 (1999)




dominant progressive





bifocal chorioretinal 





atrophy



ELOVL4,
6q14.1
dominant MD, 
Edwards et al., 


STGD3

Stargardt-like; 
Invest. Ophthalmol. 




protein: elongation of 
Vis. Sci. 42: 2652-




very long fatty acids 
2663 (2001)




protein



AHI1, JBTS3
6q23.3
recessive Joubert 
Parisi et al., J. Med.




syndrome; protein: 
Genet. 43: 334-339




Abelson helper 
(2006)




integration site 1



PEX7,
6q23.3
recessive Refsum 
van den Brink 0et 


PTS2R,

disease, adult form; 
al., Am. J. Hum. 


RCDP1,

protein: peroxisome
Genet. 72: 471-477 




biogenesis factor 7
(2003)


RCD1
6q25-q26
dominant retinal-cone 
OMIM 07




dystrophy 1



MDDC,
7p21-p15
dominant MD, cystoid
Inglehearn et al., 


CYMD


Am. J. Hum. Genet. 





55: 581-582 (1994)


PTHB1,
7p14.3
recessive Bardet Biedl 
Nishimura et al., 


BBS9, 

syndrome; protein: 
Am. J. Hum. Genet. 


PHTB1

parathyroid hormone-
77: 1021-1033 




responsive B1 protein
(2005)


RP9, PAP1,
7p14.3
dominant RP; protein: 
Sullivan et al., 


PIM1K

RP9 protein or PIM1-
Invest. Ophthalmol. 




kinase associated
Vis. Sci. 47: 3052-




protein 1
3064 (2006)


PEX1, IRD
7q21.2
recessive Refsum 
Portsteffen et al., 




disease, infantile 
Nat. Genet. 17: 449-




form; protein: 
452 (1997)




peroxisome 





biogenesis factor 1



IMPDH1,
7q32.1
dominant RP; 
Mortimer et al.,


RP10

dominant Leber 
Biochem. J. 390: 41-




congenital amaurosis; 
47 (2005)




protein: inosine 





monophosphate





dehydrogenase 1



OPN1SW,
7q32.1
dominant tritanopia; 
Fitzgibbon et al., 


BCP, CBT

protein: blue cone 
Hum. Genet. 93: 79-




opsin
80 (1994)


CORD9
8p11
recessive cone-rod 
Danciger et al., 




dystrophy
Invest. Ophthalmol. 





Vis. Sci. 42: 2458-





2465 (2001)


RP1
8q12.1
dominant RP; 
Bowne et al., Hum.




recessive RP; protein: 
Mol. Genet. 11: 




RP1 protein
2121-2128 (1999)


TTPA
8q12.3
recessive RP and/or 
Yokota et al., New




recessive or dominant 
Eng. J. Med. 335: 




ataxia; protein: alpha-
1770-1771 (1996)




tocopherol-transfer 





protein



ROA1
8q21-q22
recessive optic 
Barbet et al., Eur. J.




atrophy
Hum. Genet. 11: 





966-971 (2003)


PXMP3,
8q21.13
recessive Refsum 
Gartner et al., Nat.


PAF1, 

disease, infantile 
Genet. 1: 16-23 


PEX2, 

form; protein: 
(1992)


PMP35

peroxisomal 





membrane protein 3



CNGB3,
8q21.3
recessive achromatop-
Kohl et al., Eur. J.


ACHM3

sia Pingelapese; 
Hum. Genet. 13: 




recessive, progressive 
302-308 (2005)




cone dystrophy;





protein: cone cyclic 





nucleotide-gated 





cation channel beta 3





subunit



VMD1
not 8q24
dominant MD, 
Sohocki et al., Am. 




atypical vitelliform
J. Hum. Genet. 61: 





239-241 (1997)


RP31
9p22-p13
dominant RP
Papaioannou et al.,





Hum. Mut. 118: 





501-503 (2005)


KCNV2
9q24.2
recessive cone 
Wu et al., Am. J. 




dystrophy with super-
Hum. Genet. 79: 




normal rod 
574-579 (2006)




electroretinogram; 





protein: potasium 





channel subfamily V





member 2



INVS, 
9q31.1
recessive Senior-
O'Toole et al., 


NPHP2

Loken syndrome; 
Nephrol. Dial. 




recessive 
Transplant. 21: 




nephronophthisis; 
1989-1991 (2006)




protein: inverson



DFNB31
9q32
recessive Usher 
Ebermann et al., 




syndrome, type 2; 
Hum. Genet. (2006)




recessive deafness 





without RP; protein: 





whirlin



TLR4
9q33.1
age-related macular 
Zareparsi et al., 




degeneration, 
Hum. Mol. Genet. 




complex etiology; 
14: 1449-1455 




protein: toll-like 
(2005)




receptor 4



TRIM32,
9q33.1
recessive Bardet-
Chiang et al., Proc.


BBS11, 

Biedl syndrome; 
Natl. Acad. Sci. 


HT2A

recessive limb-girdle 
USA 103: 6287-




muscular dystrophy; 
6292 (2006)




protein: tripartite 





motif-containing 





protein 32



RP21, RP8
not 9q34-qter
dominant RP with 
Mansergh et al., 




sensorineural deafness
Am. J. Hum. Genet. 





64: 971-985 (1999)


JBTS1,
9q34
recessive Joubert 
Saar et al., Am. J. 


CORS1

syndrome
Hum. Genet. 65: 





1666-1671 (1999)


PHYH,
10p13
recessive Refsum 
Jansen et al., Nat.


PAHX, 

disease, adult form; 
Genet. 17: 190-193


RDPA

protein: phytanoyl-
(1997)




CoA hydroxyase



RNANC
10q21
recessive nonsyn-
Ghiasvand et al., 




dromal congenital 
Am. J. Med. Genet. 




retinal nonattachmen
90: 165-168 (2000)


PCDH15,
10q21.1
recessive Usher 
Ahmed et al., Hum.


DFNB23,

syndrome, type 1f; 
Mol. Genet. 12: 


USH1F

recessive deafness 
3215-3223




without RP; protein: 
(2003)




protocadherin 15



CDH23,
10q22.1
recessive Usher 
Astuto et al., Am. J.


DFNB12,

syndrome, type 1d; 
Hum. Genet. 71: 


USH1D

recessive deafness 
262-275 (2002)




without RP; protein: 





cadherin-like gene 23



RGR
10q23.1
recessive RP; 
Morimura et al., 




dominant choroidal 
Nat. Genet. 23: 393-




sclerosis; protein: 
394 (1999)




RPE-retinal G 





protein-coupled 





receptor



RBP4
10q23.33
recessive RPE 
Seeliger et al., 




degeneration; protein: 
Invest. Ophthalmol. 




retinol-binding 
Vis. Sci. 40: 3-11 




protein 4
(1999)


PAX2, 
10q24.31
dominant renal-
Favor et al., Proc. 


ONCR

coloboma syndrome; 
Natl. Acad. Sci. 




protein: paired home-
USA 93: 13870-




otic gene 2 protein
13875 (1996)


HTRA1,
10q26.13
age-related macular 
DeWan et al., 


PRSS11

degeneration, 
Science 314: 989-




complex etiology; 
992 (2006)




protein: HtrA serine 





peptidase 1



LOC387715
10q26.13
age-related macular 
Jakobsdottir et al., 




degeneration, com-
Am. J. Hum. Genet. 




plex etiology; protein: 
77: 389-407 (2005)




hypothetical protein 





with Entrez ID 





387715



OAT
10q26.13
recessive gyrate 
D Valle, O Simell. 




atrophy; protein:
In ‘The Metabolic 




ornithine 
and Molecular Bases 




aminotransferase
of Inherited 





Disease’, 8th Ed. CR 





Schriver, et al. eds., 





McGraw-Hill. 





(2000)


TEAD1, 
11p15.3
dominant atrophia 
Fossdal et al., Hum.


AA, TCF13, 

areata; protein: TEA 
Mol. Genet. 13: 975-


TEF1

domain family 
981 (2004)




member 1 [Entrez]



USH1C,
11p15.1
recessive Usher 
Ahmed et al., Hum.


DFNB18

syndrome, Acadian; 
Genet. 110: 527-531




recessive deafness 
(2002)




without RP; protein: 





harmonin



EVR3
11p13-p12
dominant familial 
Downey et al., Am. 




exudative 
J. Hum. Genet. 68: 




vitreoretinopathy
778-781 (2001)


CORS2,
11p12-q13.3
recessive Joubert 
Valente et al., Ann.


JBTS2

syndrome
Neurol. 57: 513-519





(2005)


ROM1
11q12.3
dominant RP; digenic 
Dryja et al., Invest.




RP with RDS; 
Ophthalmol. Vis. 




protein: retinal outer
Sci. 18: 1972-1982 




segment membrane 
(1997)




protein 1



VMD2
11q12.3
dominant MD, Best 
Weber et al., Am. J.




type; dominant vitreo-
Hum. Genet. 55: 




retinochoroidopathy;
1182-1187 (1994a)




protein: bestrophin



BBS1
11q13
recessive Bardet-
Mykytyn et al., Nat.




Biedl syndrome;
Genet. 31: 435-438




protein: BBS1 protein
(2002)


VRNI
11q13
dominant neovascular
Stone et al., Hum. 




inflammatory 
Mol. Genet. 1: 685-




vitreoretinopathy
689 (1992)


CABP4
11q13.1
recessive CSNB; 
Zeitz et al., Am. J.




protein: calcium 
Hum. Genet. 79: 




binding protein 4
657-667 (2006)


LRP5, 
11q13.2
dominant familial 
Jiao et al., Am. J. 


EVR4,

exudative vitreo-
Hum. Genet. 75: 


HBM, 

retinopathy; dominant 
878-884 (2004)


OPPG

high bone mass trait; 





recessive osteo-





porosis-pseudoglioma





syndrome; recessive 





FEVR; protein: low 





density lipoprotein





receptor-related 





protein 5



MYO7A,
11q13.5
recessive Usher 
Gibbs et al., Natl.


DFNB2,

syndrome, type 1; 
Acad. Sci. USA


USH1B

recessive congenital 
100: 6481-6486 




deafness without RP; 
(2003)




recessive atypical 





Usher syndrome 





(USH3-like); protein: 





myosin VIIA



FZD4, 
11q14.2
dominant familial 
Muller et al., 


EVR1,

exudative vitreo-
Genomics 20: 317-


FEVR

retinopathy; protein: 
319 (1994)




frizzled-4 Wnt 





receptor homolog



C1QTNF5,
11q23.3
dominant MD, late 
Ayyagari et at., 


CTRP5

onset; dominant MD 
Invest. Ophthalmol. 




with lens zonules; 
Vis. Sci. 46: 3363-




protein: C1q and 
3371 (2005)




tumor necrosis-related 





protein 5 collagen



COL2A1,
12q13.11
dominant Stickler 
Snead et al., J. Med.


AOM, STL1

syndrome, type I; 
Genet. 36: 353-659




dominant Wagner 
(1999)




syndrome; dominant 





epiphyseal dysplasia;





protein: collagen, type 





II, alpha 1



RDH5, 
12q13.2
recessive fundus 
Cideciyan et al., Vis.


RDH1

albipunctatus; 
Neurosci. 17: 667-




recessive cone 
678 (2000)




dystrophy, late onset; 





protein: 11-cis retinol





dehydrogenase 5



BBS10,
12q21.2
recessive Bardet-
Stoetzel et al., Nat.


FLJ23560

Biedl syndrome; 
Genet. 38: 521-524




protein: BBS10 
(2006)




(C12orf58) 





chaperonin



CEP290,
12q21.32
recessive Senior-
Chang et al., Hum.


JBTS5,

Loken syndrome; 
Mol. Genet. 15: 


NPHP6,

recessive Joubert 
1847-1857 (2006)


SLSN6

syndrome; recessive 





Leber congenital 





amaurosis; protein:





centrosomal protein 





290 kDa



RB1
13q14.2
dominant germline or 
Lohmann et al., Am. 




somatic retino-
J. Hum. Genet. 58: 




blastoma; benign 
940-949 (1996)




retinoma; pinealoma; 





osteogenic sarcoma;





protein: retino-





blastoma protein 1



GRK1,
13q34
recessive CSNB, 
Cideciyan et al., 


RHOK, RK

Oguchi type; protein: 
Proc. Natl. Acad. 




rhodopsin kinase
Sci. USA 95: 328-





333 (1998)


STGD2
not 13q34
dominant MD, 
Zhang et al., Nat.




Stargardt type
Genet. 27: 89-93 





(2001)


ACHM1,
14
recessive rod 
Pentao et al., Am. J.


RMCH

monochromacy or
Hum. Genet. 50: 




achromatopsia
690-699 (1992)


RP16
not 14
recessive RP
Bruford et al., Am. 





J. Hum. Genet. 55: 





A181 (1994)


MCDR4
14q
dominant MD, North 
Francis et al., Br. J.




Carolina-like with 
Ophthalmol. 87: 




progressive sensori-
893-898 (2003)




neural hearing loss



NRL, RP27
14q11.2
dominant RP; 
Mears et al., Nat.




recessive RP; protein: 
Genet. 29: 447-452




neural retina lucine 
(2001)




zipper



RPGRIP1,
14q11.2
recessive Leber 
Mellersh et al.,


LCA6

congenital amaurosis; 
Genomics 88: 293-




protein: RPGR-
301 (2006)




interacting protein 1



LCA3
14q24
recessive Leber 
Stockton et al., 




congenital amaurosis
Hum. Genet. 103: 





328-333 (1998)


RDH12
14q24.1
recessive Leber 
Janecke et al., Nat.




congenital amaurosis 
Genet. 36: 850-854




with severe childhood 
(2004)




retinal dystrophy; 





protein: retinol 





dehydrogenase 12



USH1A,
not 14q32
recessive Usher 
Gerber et al., Am. J.


USH1

syndrome, French
Hum. Genet. 78: 





357-359 (2006)


TTC8, 
14q32.11
recessive Bardet-
Ansley et al., Nat.


BBS8

Biedl syndrome; pro-
425: 628-633 (2003)




tein: tetratricopeptide 





repeat domain 8



FBLN5
14q32.12
familial MD, age-
Arch. Ophthalmol.




related; protein: 
112: 765-772 (1994)




fibulin 5



NR2E3,
15q23
recessive enhanced S-
Sharon et al., Arch.


ESCS, PNR

cone syndrome; 
Ophthalmol. 121: 




recessive RP in 
1316-1323 (2003)




Portuguese Crypto 





Jews; Goldmann-





Favre syndrome; pro-





tein: nuclear receptor





subfamily 2 group E3



MRST
15q24
recessive retardation, 
Mitchell et al., Am. 




spasticity and retinal 
J. Hum. Genet. 62: 




degeneration
1070-1076 (1998)


BBS4
15q24.1
recessive Bardet-
Katsanis et al., Nat.




Biedl syndrome; 
Genet. 26: 67-70 




protein: BBS4 protein
(2000)


RLBP1,
15q26.1
recessive RP; 
Burstedt et al., 


CRALBP

recessive Bothnia 
Invest. Ophthalmol. 




dystrophy; recessive 
Vis. Sci. 40: 995-




retinitis punctata 
1000 (1999)




albescens; recessive 





Newfoundland rod-





cone dystrophy; pro-





tein: retinaldehyde-





binding protein 1



ABCC6,
16p13.11
recessive pseudo-
Bergen et al., Nat.


ARA, 

xanthoma elasticum; 
Genet. 25: 228-231


MRP6, PXE

dominant pseudo-
(2000)




xanthoma elasticum;





protein: ATP-binding 





casette, subfamily C, 





member 6



RP22
16p12.3-p12.1
recessive RP
Finckh et al., 





Genomics





48: 341-345 (1998)


CLN3, 
16p11.2
recessive Batten 
Kremmidiotis et al.,


JNCL

disease (ceroid-
Hum. Mol. Genet.




lipofuscinosis, 
8: 523-531 (1999)




neuronal 3), juvenile; 





protein: Batten 





disease protein



BBS2
16q12.2
recessive Bardet-
Beales et al., Am. J.




Biedl syndrome;
Hum. Genet. 68: 




protein: BBS2 protein
606-616 (2001)


CNGB1,
16q13
recessive RP; protein: 
Bareil et al., Hum.


CNCG2,

rod cGMP-gated 
Genet. 108: 328-334


CNCG3L,

channel beta subunit
(2001)


GAR1, 





GARP





CDH3,
16q22.1
recessive MD, 
Indelman et al., J.


CDHP, 

juvenile with hypotri-
Invest. Dermatol.


PCAD

chosis; protein: 
119: 1210-1213 




cadherin 3, type 1, 
(2002)




placental



FHASD
16q23.2-q24.2
recessive foveal hypo-
Pal et al., J. Med.




plasia and anterior 
Genet. 41: 772-777




segment dysgenesis
(2004)


CACD
17p13
dominant central 
Hughes et al., J. 




areolar choroidal 
Med. Genet. 35: 




dystrophy
770-772 (1998)


PRPF8,
17p13.3
dominant RP; protein: 
Kojis et al., Am. J.


PRPC8, 

human homolog of 
Hum. Genet. 58: 


RP13

yeast pre-mRNA 
347-355 (1996)




splicing factor C8



AIPL1, 
17p13.2
recessive Leber 
Hanein et al., Hum.


LCA4

congenital amaurosis; 
Mutat. 23: 306-317




dominant cone-rod 
(2004)




dystrophy; protein:





arylhydrocarbon-





interacting receptor 





protein-like 1



GUCY2D,
17p13.1
recessive Leber 
Hanein et al., Hum.


CORD6,

congenital amaurosis; 
Mutat. 23: 306-317


LCA1,

dominant cone-rod 
(2004)


RETGC,

dystrophy; protein: 



RETGC1

retinal-specific 





guanylate cyclase



CORD5,
same as
dominant cone-rod 
Udar et al., Hum. 


RCD2
GUCY2D
dystrophy, progress-
Mut. 21: 170-171 




ive; recessive cone-
(2003)




rod dystrophy



CORD4
17q
cone-rod dystrophy
Klystra et al.,


UNC119,
17q11.2
dominant cone-rod 
Kobayashi et al.,


HRG4

dystrophy; protein: 
Invest. Ophthalmol.




human homolog of C. 
Vis. Sci. 41: 3268-




elegans unc119 
3277 (2000)




protein



CA4, RP17
17q23.2
dominant RP; protein: 
Rebello et al., Proc.




carbonic anhydrase IV
Natl. Acad. Sci. 





USA 101: 6617-





6622 (2004)


USH1G,
17q24-q25
recessive Usher 
Kikkawa et al., 


SANS

syndrome; protein: 
Hum. Mol. Genet. 




human homolog of 
12: 453-461 (2003)




mouse scaffold 





protein containing





ankyrin repeats and 





SAM domain



RGS9
17q24.1
recessive delayed 
Nishiguchi et al.,




cone adaptation; 
Nature 427: 75-78




protein: regulator of
(2004)




G-protein signalling 9



PRCD
17q25.1
recessive RP; protein: 
Zangerl et al.,




progressive rod-cone 
Genomics (2006)




degneration protein



FSCN2, 
17q25.3
dominant RP; 
Wada et al., Arch.


RP30

dominant MD; protein: retinal fascin 
Ophthalmol. 121: 




homolog 2, actin 
1613-1620 (2003)




bundling protein



OPA4
18q12.2-q12.3
dominant optic 
Kerrison et al., 




atrophy, Kjer type
Arch. Ophthalmol. 





117: 805-810 (1999)


CORD1
18q21.1-q21.3
cone-rod dystrophy; 
Manhant et al., Am. 




de Grouchy syndrome
J. Hum. Genet. 57: 





A96 (1995)


R9AP
19q13.12
recessive delayed 
Nishiguchi et al.,




cone adaptation; 
Nature 427: 75-78




protein: regulator of
(2004)




G-protein signalling 





9-binding protein



MCDR5
19q13.31-
dominant macular 
Yang et al., Science



q13.32
dystrophy
314: 992-993 (2006)


CRX, 
19q13.32
dominant cone-rod 
Hanein et al., Hum.


CORD2

dystrophy; recessive, 
Mutat. 23: 306-317




dominant and de novo 
(2004)




Leber congenital 





amaurosis; dominant 





RP; protein: cone-rod





otx-like photoreceptor 





homeobox 





transcription factor



OPA3, 
19q13.32
recessive optic 
Anikster et al., Am. 


MGA3

atrophy with ataxia and 3-methylgluta-
J. Hum. Genet. 69: 




conic aciduria; 
1218-1224 (2001)




protein: OPA3 protein



PRPF31,
19q13.42
dominant RP; protein: 
Sullivan et al., 


PRP31, 

human homolog of 
Invest. Ophthalmol. 


RP11

yeast pre-mRNA
Vis. Sci. 47: 4579-




splicing factor 31
4588 (2006)


JAG1, AGS
20p12.2
dominant Alagille 
Li et al., Nat. Genet.




syndrome; protein: 
16: 243-251 (1997)




Jagged protein 1



MKKS, 
20p12.2
recessive Bardet-
Beales et al., Am. J.


BBS6

Biedl syndrome; 
Hum. Genet. 68: 




protein: McKusick-
606-616 (2001)




Kaufman syndrome 





protein



PANK2,
20p13
recessive HARP
Hartig et al., Ann.


HARP, 

(hypoprebetalipo-
Neurol. 59: 248-256


PKAN

proteinemia, acantho-
(2006)




cytosis, RP, and 





palladial degenera-





tion); recessive 





Hallervorden-Spatz 





syndrome; protein: 





pantothenate kinase 2



USH1E
21q21
recessive Usher 
Chaib et al., Hum. 




syndrome, type 1
Mol. Genet. 6: 27-31 





(1997)


OPA5
22q12.1-q12.3
dominant optic 
Rozet et al., Invest.




atrophy
Ophthalmol. Vis. 





Sci. 46: E-Abstract 





2292 (2005)


TIMP3, 
22q12.3
dominant Sorsby's 
Felbor et al., Am. J.


SFD

fundus dystrophy; 
Hum. Genet. 60: 57-




protein: tissue 
62 (1997)




inhibitor of 





metalloproteinases-3



RP23
Xp22
X-linked RP
Hardcastle et al.,





Invest. Ophthalmol.





Vis. Sci. 41: 2080-





2086 (2000)


RS1, XLRS1
Xp22.13
retinoschisis; protein:
Grayson et al., Hum.




retinoschisin
Mol. Genet. 9: 1873-





1879 (2000)


(—)
Xp21-q21
RP with mental 
Aldred et al., Am. J.




retardation
Hum. Genet. 55: 





916-922 (1994)


RP6
Xp21.3-p21.2
X-linked RP
Breuer et al., Invest.





Ophthalmol. Vis. 





Sci. 41: S191 (2000)


DMD
Xp21.2-p21.1
Oregon eye disease 
D'Souza et al., Hum.




(probably); protein: 
Mol. Genet. 5: 837-




dystrophin
842 (1995)


AIED, OA2
Xp11.4-q21
Åland island eye 
Wutz et al., Eur. J.




disease
Hum. Genet. 10: 





449-456 (2002)


COD4
Xp11.4-q13.1
X-linked progressive 
Jalkanen et al., J. 




cone-rod dystrophy
Med. Genet. 40: 





418-423 (2003)


OPA2
Xp11.4-p11.2
X-linked optic 
Assink et al., Am. J.




atrophy
Hum. Genet. 61: 





934-939 (1997)


NYX, 
Xp11.4
X-linked CSNB; 
Bech-Hansen et al.,


CSNB1

protein: nyctalopin
Nat. Genet. 26: 319-





323 (2000)


CSNB4
same as NYX
X-linked CSNB
Pusch et al., Nat.





Genet. 26: 324-327





(2000)


RPGR, RP3
Xp11.4
X-linked RP, 
Bader et al., Invest.




recessive; X-linked 
Ophthalmol. Vis. 




RP, dominant; X-
Sci. 44: 1458-1463 




linked CSNB; X-
(2003)




linked cone dystrophy 





1; X-linked atrophic 





MD, recessive; 





protein: retinitis 





pigmentosa GTPase 





regulator



COD1
same as 
X-linked cone 
Demirci et al., Am. 



RPGR
dystrophy 1
J. Hum. Genet. 70: 





1049-1053 (2002)


RP15
same as 
X-linked RP, 
Mears et al., Am. J.



RPGR
dominant
Hum. Genet. 67: 





1000-1003 (2000)


PRD
Xp11.3-
retinal dysplasia, 
Ravia et al., Hum. 



p11.23
primary
Mol. Genet. 8: 1295-





1297 (1993)


NDP, EVR2
Xp11.3
Norrie disease; 
Black et al., Hum. 




familial exudative 
Mol. Genet. 11: 




vitreoretinopathy; 
2021-2035 (1999)




Coats disease; protein:





Norrie disease protein



CACNA1F,
Xp11.23
X-linked CSNB, 
Nakamura et al., 


CSNB2,

incomplete; ÅIED-
Arch. Ophthalmol. 


CSNBX2

like disease; severe 
121: 1028-1033 




CSNB; protein: L-
(2003)




type voltage-gated 





calcium channel 





alpha-1 subunit



RP2
Xp11.23
X-linked RP; protein: 
Hardcastle et al., 




novel XRP2 protein 
Am. J. Hum. Genet. 




similar to human 
64: 1210-1215 




cofactor C
(1999)


PGK1
Xq21.1
RP with myopathy; 
Tonin et al., Neurol.




protein: phospho-
43: 387-391 (1993)




glycerate kinase,



CHM
Xq21.2
choroideremia; 
van den Hurk et al.,




protein: geranyl-
Hum. Mutat. 9: 110-




geranyl transferase 
117 (1997)




Rab escort protein 1



TIMM8A,
Xq22.1
optic atrophy with 
Koehler et al., Proc.


DDP, 

deafness-dystonia 
Natl. Acad. Sci USA


DDP2, 

syndrome; protein: 
96: 2141-1246 


DFN1

inner mitochondrial 
(1999)




membrane translocase 





8 homolog A



RP24
Xq26-q27
X-linked RP
Gieser et al., Am. J.





Hum. Genet. 63: 





1439-1447 (1998)


COD2,
Xq27
X-linked progressive 
Bergen et al.,


XLPCD

cone dystrophy, 2



RP34
Xq28-qter
X-linked RP
Melamud et al., J. 





Med. Genet. 43: e27 





(2006)


OPN1LW,
Xq28
deuteranopia and rare 
Ayyagari et al., Mol.


GCP, CBD

macular dystrophy in 
Vis. 58: 98-101 




blue cone mono-
(1999)




chromacy with loss of 





locus control element; 





protein: green cone 





opsin



OPN1MW,
Xq28
protanopia and rare 
Ayyagari et al., Mol.


RCP, CBP

macular dystrophy in 
Vis. 58: 98-101 




blue cone mono-
(1999)




chromacy with loss of 





locus control element; 





protein: red cone 





opsin



KSS
mito-
Kearns-Sayre 
al., Science 283: 



chondrion
syndrome including 
1482-1488 (1999)




retinal pigmentary 





degeneration; protein: 





several mitochondrial





proteins



LHON,
mito-
Leber hereditary optic
Brown et al., Am. J.


MTND1,
chondrion
neuropathy; protein: 
Hum. Genet. 60: 


MTND4,

complex I, III or IV 
381-387 (1997)


MTND6

proteins



MTTL1,
mito-
macular pattern 
Bonte et al., Retina


DMDF,
chondrion
dystrophy with type II 
17: 216-221 (1997)


TRNL1

diabetes and deafness;





protein: leucine tRNA 





1 (UUA/G), nt 3230-





3304



MTATP6,
mito-
RP with develop-
White et al., J. 


ATP6, 
chondrion
mental and neuro-
Inherit. Metab. Dis. 


NARP

logical abnormalities; 
22: 899-914 (1999)




Leigh syndrome; 





LHON; protein: 





complex V ATPase 6 





subunit, nt 8527-9207



MTTH,
mito-
pigmentary retin-
Crimi et al., 


TRNH
chondrion
opathy and sensori-
Neurology 60: 1200-




neural hearing loss; 
1203 (2003)




protein: histidine 





tRNA, nt 12138-





12206



MTTS2,
mito-
RP with progressive 
Mansergh et al., 


TRNS2
chondrion
sensorineural hearing 
Am. J. Hum. Genet. 




loss; protein: serine 
64: 971-985 (1999)




tRNA 2 (AGU/C), nt 





12207-12265









In an embodiment of the invention, suppression agents are siRNAs or shRNAs targeting human rhodopsin. Exemplary siRNAs and replacement rhodopsin sequences are provided in Table 6A.







TABLE 6A







Exemplary siRNA Sequences Targeting Human Rhodopsin and


Replacement Rhodopsin Sequences











SEQ ID

SEQ ID


siRNA Target Site
NO
Replacement Site
NO













1. TACGTCACCGTCCAGCACAAG
1
TATGTGACGGTGCAACATAA
2


 


2. CTCAACTACATCCTGCTCAAC
3
CTGAATTATATTTTATTGAAT
4


 


3. CAGCTCGTCTTCACCGTCAAG
5
CAATTGGTGTTTACGGTGAAA
6


 


4. ATCTATATCATGATGAACAAG
7
ATTTACATTATGATGAATAAA
8


 


5. GCCTACATGTTTCTGCTGATC
9
GCTTATATGTTCTTATTAATT
10


 


6. TACATGTTTCTGCTGATCGTG
11
TATATGTTCTTATTAATTGTC
12


 


7. CTGCGCACGCCTCTCAACTAC
13
TTACGGACCCCCTTGAATTAT
14


 


8. CGCACGCCTCTCAACTACATC
15
CGGACCCCCTTGAATTATATT
16


 


9. CTCAAGCCGGAGGTCAACAAC
17
TTGAAACCCGAAGTGAATAAT
18


 


10. CAGCTCGTCTTCACCGTCA
19
CAATTGGTGTTTACGGTGA
20


 


11. TACGCCAGCGTGGCATTCTAC
21
TATGCTTCTGTCGCCTTTTAC
22


 


12. CCAGCGTTCTTTGCCAAGA
23
CCCGCCTTTTTCGCTAAAA
24


 


13. GTCATCTATATCATGATGAAC
25
GTGATTTACATTATGATGAAT
26


 


14. AACTGCATGCTCACCACCATC
27
AATTGTATGTTGACGACGATT
28


 


15. ACCATCTGCTGCGGCAAGA
29
ACGATTTGTTGTGGGAAAA
30


 


16. GACGATGAGGCCTCTGCTA
31
GAGGACGAAGCTAGCGCCA
32


 


17. CACCTCTCTGCATGGATACT
33
CACGAGCTTACACGGGTATT
34


 


siRNAs Targeting 5′ UTR





18. AGCTCAGGCCTTCGCAGCA
35




 


19. CAGGCCTTCGCAGCATTCT
36




 


siRNAs Targeting 3′ UTR





20. TCACTTTCTTCTCCTATAA
37




 


21. TAGTTAATGTTGTGAATAA
38




 


22. GCTCCTATGTTGGTATTAA
39




 


23. AGTCACATAGGCTCCTTAA
40




 


24. GATTCTTGCTTTCTGGAAA
41




 


25. ACAGTAGGTGCTTAATAAA
42




 


26. GAACATATCTATCCTCTCA
43




 


27. CTGTACAGATTCTAGTTAA
44




 


28. TGTGAATAACATCAATTAA
45




 


29. CAATTAATGTAACTAGTTA
46




 


30. TGATTATCACCTCCTGATA
47




 


31. GCAGTCATCAGACCTGAAA
48




 


32. TGTCATCCTTACTCGAAGA
49




 


33. GAATTAAGCTGCCTCAGTA
50




 


34. GCCAGAAGCTCTAGCTTTA
51




 


35. AGCTCTGCCTGGAGACTAA
52




 


siRNAs Targeting an Intron





36. GATCTTATTTGGAGCAATA
53




 


37. TGGCTGTGATCCAGGAATA
54




 


38. GATGCATTCTTCTGCTAAA
55




 


39. GCAATATGCGCTTGTCTAA
56




 


40. TTGTCTAATTTCACAGCAA
57




 


41. TGTTTGTTGCATTCAATAA
58




 


42. CCAGAGCGCTAAGCAAATA
59




 


43. GTCTTGCATTTAACAGGAA
60




 


44. GGCTGTGATCCAGGAATAT
61




 


45. TGCAGGAGGAGACGCTAGA
62




 


46. CTTTCACTGTTAGGAATGT
63




 


47. TTTGGTTGATTAACTATAT
64




 


48. TTAACTATATGGCCACTCT
65




 


49. AGATGTTCGAATTCCATCA
66




 


siRNAs Targeting a Polymorphism





50. TCTTCACCGTCAAGGAGGTAT
67
TGTTTACGGTGAAAGAAGTAC
68









siRNA sequences 1-17 target the human rhodopsin coding sequence. siRNA sequences 18 and 19 target the human rhodopsin 5′UTR. siRNA sequences 20-35 target the human rhodopsin 3′UTR. siRNA sequences 36-49 target human rhodopsin intronic sequence. The sequence of the sense strand of the siRNA is given. Notably, siRNAs may also target a combination of these. For example, an siRNA target site may be in the 5′UTR and exon 1. Or an siRNA target site may be in the coding region and an intron. Or an siRNA target site may be in an exon and the 3′UTR. siRNA sequence 50 is an example of an siRNA that has a target site that spans Exon 3/intron 3 of the human rhodopsin gene. The site contains a known polymorphism in intron 3. If this site was used as an siRNA target, the replacement gene would have the wildtype base at the polymorphic site but degeneracy of the genetic code could be used to change other bases at the replacement site. The siRNA(s) may comprise all or part of the sequence provided. The sequences of replacement human rhodopsin nucleic acids over the target for siRNA-mediated suppression are provided for siRNA sequences 1-17. Replacement nucleic acids include at least one altered nucleotide(s) at degenerate position(s) over the siRNA target site (highlighted in bold print). Thus, replacement sequences here provide one of multiple replacement options. Some replacement constructs contain nucleotide changes in the coding sequence. These replacement constructs while altered in nucleotide sequence encode the same amino acids as the wild type rhodopsin protein. Other replacement constructs are altered at either silent or non-silent polymorphic sites. These replacement constructs encode wild type protein, with wild type function. For siRNAs targeting the UTRs or intronic sequence, no replacement constructs have been suggested because the number of base changes within the site is not limited to degenerate positions (as is the case for sequence coding for amino acids).

It is notable that suppression of a given gene such as rhodopsin may be evaluated in a variety of animal species. The siRNA sequences provided in Table 6B represent examples of RNAi sequences that are homologous between porcine and human rhodopsin. In some transgenic animal models the presence of the human transgene enables direct evaluation of sequences that target the human gene in that animal model. In other instances suppressor sequences may be chosen to maximise the homology between the human gene (for example, rhodopsin) and the endogenous gene in the animal under evaluation.







TABLE 6B







Exemplary siRNA Sequences Targeting Homologous


Sequences Between Human and Porcine Rhodopsin













SEQ

Suppression




ID
Position in
levels in HeLa


siRNA
Sequence
NO:
NM_000539.2
Cells





P1
ACCTCTCTGCATGG
414
384-403
69%



ATAGT-TT





 


P2
CATGTTCGTGGTCC
415
713-732
81%



ACTTC-TT









siRNA can be expressed in miR vectors using polymerase II promoters. For this purpose pcDNA6.2-GW/EmGFP-miR from Invitrogen is used where the cloned miR-155 gene is recombined in order to express the choice of siRNA. The antisense strand of the siRNA is kept intact followed by a modified terminal loop and the sense strand, which is modified by introducing a deletion of 2 central nucleotides in order to form an internal loop. See Catalogue no K4936-00, Block-IT, POLII, miR RNAi expression vector kits catalogue, Invitrogen, page 7 for figure showing the native miR-155 sequence and the converted sequence of siRNA-lacZ in the form of miR-lacZ.
Exemplary miRNA Sequences Targeting Human Rhodopsin:







CC miRNA oligos:


(SEQ ID NO: 416)


Top strand: 5′-


TGCTGCTTCTTGTGCTGGACGGTGACGTTTTGGCCACTGACTGACGTCAC


CGTAGCACAAGAAG-3′


 


Bottom strand: 5′-


(SEQ ID NO: 417)


CCTGCTTCTTGTGCTACGGTGACGTCAGTCAGTGGCCAAAACGTCACCGT


CCAGCACAAGAAGC-3′


 


Q1 miRNA oligos:


(SEQ ID NO: 418)


Top strand: 5′-


TGCTGGTAGTAGTCGATTCCACACGAGTTTTGGCCACTGACTGACTCGTG


TGGTCGACTACTAC-3′


 


Bottom strand: 5′-


(SEQ ID NO: 419)


CCTGGTAGTAGTCGACCACACGAGTCAGTCAGTGGCCAAAACTCGTGTGG


AATCGACTACTACC-3′


 


BB miRNA oligos:


(SEQ ID NO: 420)


Top strand: 5′-


TGCTGGTAGAGCGTGAGGAAGTTGATGTTTTGGCCACTGACTGACATCAA


CTTTCACGCTCTAC-3′


 


Bottom strand: 5′-


(SEQ ID NO: 421)


CCTGGTAGAGCGTGAAAGTTGATGTCAGTCAGTGGCCAAAACATCAACTT


CCTCACGCTCTACC-3′






In an embodiment of the invention, suppression agents and replacement genes are expressed in photoreceptor cells to alleviate disease pathology. In a further embodiment, replacement nucleic acids encode a gene which when mutated may cause retinal degeneration other than retinitis pigmentosa, for example, Stargarts Syndrome, glaucoma, cod-rod dystrophy, corneal dystrophy or Age-related Macular Degeneration (AMD) (Table 5).
In another aspect, the invention provides cells expressing a suppression effector such as a dsRNA, either transiently or stably, for experimental or therapeutic use. In an embodiment, the cells express an siRNA that targets rhodopsin. In another embodiment, the cells express a replacement nucleic acid expressing rhodopsin that is not targeted by the siRNA. In another embodiment, the cells comprise a vector encoding at least one or more siRNAs. In another embodiment, the cells comprise a vector encoding a replacement nucleic acid. In an additional embodiment, the cells comprise one or more vectors encoding siRNA(s) and replacement nucleic acid(s).
In another aspect, the invention provides transgenic animals and their experimental or therapeutic use. In an embodiment, the transgenic animal is a model for Retinitis Pigmentosa, for example, an animal with a mutation observed in humans such as the Pro23His and or Pro347ser mutations. In another embodiment, the transgenic animal expresses a dsRNA that targets human rhodopsin. In another embodiment, the transgenic animal expresses a replacement nucleic acid transgene that has been altered at one or more wobble position(s) such that it escapes suppression.
Suppression agents and replacement nucleic acids of the invention can be administered to cells, tissues, plants and/or animals, either separately or together. In yet another aspect administration of suppression agent and/or replacement nucleic acid may be systemic or local. In yet another aspect, administration of suppression agent and replacement nucleic acid may be used in conjunction with chemical and/or physical agents to aid administration. In another aspect, the invention provides methods for suppressing rhodopsin expression in an animal by intraocular (e.g., subretinal or intravitreal) injection of a suppression agent into the animal. In another aspect intraocular administration (e.g., subretinal injection, intravitreal) is used to administer a suppression agent and/or replacement nucleic acid to an animal. In another embodiment, ionthophoresis or electroporation is used to administer suppression agents and/or replacement nucleic acids. In another embodiment, suppression agents and/or replacement nucleic acids are administered using nanotechnology (Kawasaki and Player Nanomedicine 1(2):101-9, 2005; Silva Surg. Neurol. 67(2):113-6, 2007; Andrieu-Solar et al., Mol. Vis. 12:1334-47, 2006) or bacteria (Daudel et al., Expert Rev. Vaccines 6(1):97-110, 2007).
Suppression agents and replacement nucleic acids may be optimally combined with conserved regions A-I and/or transcription factor binding sites identified within conserved regions A-I and/or with enhancer elements and/or other regulatory elements (see Tables 1 and 2 above and Tables 9-12 below).
In one aspect of the invention, there is provided a vector for expression of a suppression agent for a disease causing gene and/or a replacement nucleic acid that is not recognized by the suppression agent, wherein the vector comprises at least one of the conserved regions selected from: conserved region B from the rhodopsin gene represented by SEQ ID NO: 93, or a variant or equivalent thereof; conserved region C from the rhodopsin gene represented by SEQ ID NO: 94, or a variant or equivalent thereof; conserved region F and G from the rhodopsin gene represented by SEQ ID NO: 97 or a variant or equivalent thereof; and conserved region A from the rhodopsin gene represented by SEQ ID NO: 92, or a variant or equivalent thereof. In a particular embodiment, the vector comprises at least one of the conserved regions selected from: conserved region B from the rhodopsin gene represented by SEQ ID NO: 93; conserved region C from the rhodopsin gene represented by SEQ ID NO: 94; conserved region F and G from the rhodopsin gene represented by SEQ ID NO: 97; and conserved region A from the rhodopsin gene represented by SEQ ID NO: 92.
In one embodiment of the invention the use of suppression and replacement constructs in combination with one or more factors to facilitate cell survival, cell viability and/or cell functioning is contemplated. In relation to neurons, a range of neurotrophic and/or neuroprotective factors may be used inter glia brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), neurturin, ciliary derived neurotrophic factor (CNTF), nerve growth factor (NGF), fibroblast growth factors (FGF), insulin-like growth factors (IGF), pigment epithelium-derived factor (PEDG), hepatocyte growth factor (HGF), thyrotrophin releasing hormone (TRH) and rod derived cone viability factor (RDCVF) amongst others. There is substantial evidence in the literature that such factors may increase cell viability and/or cell survival for a range of cell types. For example, these factors have been shown to provide beneficial effects to a wide range of neuronal cell types including, for example, photoreceptors, when delivered either in protein or DNA forms (Buch et al., Mol. Ther., 2006; 14(5):700-709). The use of GDNF to augment gene-based therapies for recessive disease has been demonstrated in mice (Buch et al., Mol. Ther., 2006; 14(5):700-709). Genes encoding neurotrophic/neuroprotective factors may be expressed from general promoters such as the CBA promoter (Buch et al., Mol. Ther., 2006; 14(5):700-709) or from tissue specific promoters. Sequences to optimise expression of neurotrophic/neuroprotective factors such as those sequences identified in Tables 1, 2, 9-13 may be included in constructs.
Sequences of a number of exemplary neurotrophic factors are provided in FIG. 17. DNA encoding one or more neurotrophic and/or neuroprotective factors may be utilised in conjunction with suppression and replacement. FIG. 18 provides examples of constructs incorporating suppression and replacement sequences together with sequence encoding a factor promoting cell viability and/or cell functioning such as GDNF, neurturin, CNTF and/or RDCVF amongst others as described above. Well established art known methods involving DNA restriction digestion, DNA ligation into plasmids, bacterial transformation, characterization of transfected bacterial colonies, plasmid purification and DNA sequencing may be used to clone suppression and replacement and neuroprotection/neurotrophic sequences into DNA-based vectors. Examples of the design of such constructs are provided in FIG. 18
Constructs incorporating suppression and replacement and neurotrophic/neuroprotective factor(s) may be delivered using viral and/or non-viral vectors using art known methods (Andrieu-Soler et al., Mil. Vis., 2006; 12:1334-47). Naked DNA, lipids, polymers, nanoparticles, electrotransfer amongst other methods have been used to achieve gene/nucleotide delivery in cells and animals. For example, lentiviral vectors and/or adenoassociated viral (AAV) vectors may be used to deliver constructs incorporating the 3 components defined above (suppression, replacement and neurotrophism/neuroprotection). 3-component constructs in some instances may require vectors that have significant capacity in terms of size of DNA inserts. Many viral and non-viral vectors have been characterised that can facilitate large DNA fragments including inter alia lentiviral vectors and some of adenoassociated viral serotypes. For example, AAV serotype 2 capsid 5 vectors (AAV2/5) have been shown to accommodate 8-9 kilobases of DNA (Alberto Aurrichio; British Society of Gene Therapy, 2008). One or more components (suppression, replacement, neurotrophism/neuroprotection) may, for example, in the case of AAV be cloned between the AAV ITRS and or one or more components may be cloned into the backbone of the plasmid used to generate AAV. FIG. 18 provides key elements of the construct design (B). Utilisation of backbone plasmid sequences to carry components of a multi-component construct can be used to optimise the population of AAV vectors generated using that plasmid. Moreover, in relation to eye disease, it is notable that there is significant evidence that AAV2/5 transduces photoreceptor efficiently. Generation of AAV vectors carrying suppression and replacement sequences in conjunction with sequences encoding neurotrophic and/or neuroprotective agents is contemplated. While AAV may be of value as a vector to deliver 3-component constructs for some target tissues, a range of additional viral and non-viral vectors are available for this purpose, such as those described above, and vectors that are well know in the art.
While utilisation of a single vector to deliver 3-component constructs involving suppression and replacement and a neurotrophic/neuroprotective sequence to a cell, a tissue and or an animal is contemplated, the use of multiple vectors in combination to deliver all 3 components is also contemplated. The multivalent approach involving suppression, replacement and neuroprotection may involve the use of 1 or more vectors for delivery. In addition, the 3 components may be delivered using a combination of a vector or vectors incorporating DNA sequences together with RNA and or dsRNA and or protein. In the current invention, delivery of protein, of RNA encoding protein and/or of DNA encoding protein or a combination thereof to achieve delivery of all 3 components, suppression, replacement and neuroprotection, is contemplated.
In another embodiment of the invention the size of the backbone of the AAV plasmid vector is either increased or decreased so as to increase expression from the virus. For example, it has been described in the art that increasing the AAV virus backbone in size such that it is larger than the insert cloned within ITSI and ITS2 favours AAV packaging of the insert over packaging of the backbone, thereby increasing expression of DNA cloned within the ITR regions (Bennet et al., Reversal of visual defects in animal models of LCA within weeks of treatment with an optimized AAV. Molecular Therapy Vol. 15, supplement 1, s286).
In a further embodiment of the invention the size of the backbone is increased with a gene which is therapeutically beneficial driven by a promoter. In this embodiment a portion of packaged AAV consists of the backbone and hence a portion of AAV particles will express the gene encoded within the backbone. In one embodiment the therapeutically beneficial gene cloned in the backbone is a neurotrophic factor such GDNF, Neurturin or others.
While the invention can be used for dominant and or polygenic disorders, it may also be practised for recessive disorders. For example, the art describes that when treating the recessive disorder phenylketonuria (PKU) with replacement genes, endogenous protein expressed from mutant genes interfered with protein from replacement genes (Described in a thesis submitted to the University of Florida in partial fulfillment of the requirements for the degree of Doctor of Philosophy, by Catherine Elisabeth Charron, August 2005 and entitled “Gene therapy for phenylketonuria: dominant-negative interference in a recessive disease”). Thus, suppression and replacement constructs may be targeted to recessive disorders which like PKU require suppression and replacement.
Suppression and replacement technology provides a strategy that may be applicable to a wide range of genetic disorders including disorders characterized by either a recessive, dominant, polygenic, multifactorial or a dominant negative pathology. In a further embodiment of the invention conserved regions identified in the promoter region of mammalian rhodopsin genes and/or enhancer elements and/or other regulatory elements and/or epigenetic elements such as listed in Table 5 may be combined with suppressors targeting genes with mutations other than rhodopsin and providing replacement genes other than rhodopsin. Osteogenesis imperfecta, epidermolysis bullosa, autosomal dominant early onset Alzheimer's disease, autosomal dominant polycystic kidney disease, Rett syndrome, familial platelet disorder, dominant negative diabetes insipidus, autosomal dominant Stargardt like macular dystrophy, nemaline myopathy, familial pulmonary arterial hypertension, APC and p53 related cancers and several other disorders (OMIM) may potentially benefit from a suppression and replacement therapeutic approach. Triplet repeat disorders, 14 of which have been characterised to date, including Huntington's disease, spinocerebellar ataxia and myotonic dystrophy may benefit from a suppression and replacement approach. For each disorder, promoters of the endogenous gene or constitutive promoters or promotes from other genes, or inducible promoters may be used to express the suppression agent or replacement nucleic acid.
In another embodiment of the invention, promoter and/or enhancer elements and/or other regulatory elements and/or epigenetic elements may be combined with other promoters than rhodopsin in combination with suppression and/or replacement elements. For example, but not exclusively, promoter and enhancer elements can be combined with the COL1A1 and or COL1A2 and or COL7A1 and or Keratin 5 and or Keratin 14 and or peripherin and/or IMPDH1 promoters and/or genes. Depending upon the tissue in which the suppression agent and/or replacement nucleic acid is administered or active in vivo, tissue specific regulatory elements are used to enhance expression of the suppression agent and/or replacement nucleic acid.
The suppressors and/or replacement nucleic acids of the invention can be targeted to suppress and replace a gene where mutations in the gene can give rise, predispose or work in combination with other genetic factors and/or environmental factors to cause disease pathology. For example, in the case of dominant retinopathies the rhodopsin geen may be suppressed and replaced. For example, siRNAs targeting RHO-(NM—000539.2) can be designed and provided commercially. Likewise control siRNAs, for example, targeting EGFP (U57608) and or other reporter genes and or other non-targeting siRNAs can be designed and synthesized. siRNAs are chosen to target sequences which differed by at least one and preferable many more nucleotides from any known gene in mouse and human databases (http://www.ncbi.nlm.nih.gov/blast, BLASTN2.2.6, Altschul et al., Nuc Acids Res. 25: (17:3389-402, 1997). siRNAs can be cloned downstream of, for example, polymerase III promoters such as the H1 or U6 promoters to generate short hairpin RNAs (shRNAs; Brummelkamp et al., Science 296: (5567:505-3, 2001). Alternatively, polymerase II promoters which drive expression in many or all cell or tissue types including the CMV promoter, ubquitin promoter and or the β-actin promoter, for example, may be used to express shRNAs. Likewise tissue specific promoters such as the rhodopsin promoter, peripherin promoter and or enolase promoter amongst others may be used to express shRNAs. shRNA sequences can be cloned into vectors with a reporter gene to facilitate monitoring expression from vectors, for example, shRNAs can be cloned in pEGFP-1 amongst other plasmids (BD Biosciences, Clontech, Palo Alto, Calif.). Suppressors can be delivered to cells, tissues and or animals with or without replacement nucleic acids.
Replacement nucleic acids with nucleotide sequence changes over the target site for siRNA-mediated suppression, for example, at degenerative nucleotides can be generated by primer directed mutagenesis and cloned into vectors such as pcDNA3.1-(Invitrogen). Replacement nucleic acids may also be modified at the UTRs and or at polymorphic sites within the target gene. Ubiquitous promoters such as the CMV promoter and or the ubiquitin promoter and or the β-actin promoter amongst others can be used to drive expression of replacement nucleic acids. Alternatively, tissue specific promoters such as the rhodopsin promoter, peripherin promoter, Col1A1 promoter, Col1A2 promoter, Col1A7 promoter, Keratin promoters and/or the enolase promoter amongst others and/or inducible promoters such as a tetracycline responsive promoter can be used to drive expression of replacement nucleic acids. Replacement human rhodopsin nucleic acids which have been altered in nucleotide sequence at degenerate positions over siRNA target sites for example, replacement nucleic acids for siRNA sequences 1-17 are provided in Table 5. Replacement nucleic acids can be delivered to cells, tissues and or animals with or without suppressor agents.
Suppression and Replacement in Cells and Tissues
Promoter driven replacement nucleic acids such as rhodopsin nucleic acids and siRNAs and/or shRNAs targeting rhodopsin can be co-transfected into cells, for example, HeLa and or Cos-7 cells amongst other cell types using art known methods. For example, 24 hours post-transfection of suppressor agents and/or replacement nucleic acids, RNA and cytoplasmic protein can be isolated from cells using well established methodologies. Additionally, suppression and replacement can be evaluated in tissues. In the case of retinal genes, for example, organotypic retinal explant cultures from mouse or rat, for example, can be prepared and maintained using art known methods and suppressor agents and or replacement nucleic acids can be delivered to organotypic cultures. For example, electroporation can be used to deliver siRNA and/or shRNA constructs and/or shRNA constructs and replacement nucleic acids to retinal explants as described in Palfi et al., Hum. Mutat. 27(3):260-8, 2006. Subsequent to electroporation of retinal explants, retinas can be treated with trypsin to expedite dissociation of cells. Retinal cell sub-populations within the dissociated cell population which have a particular feature, for example, that express a reporter gene such as EGFP can be identified. One method of identification that can be invoked is FACS (Palfi et al., Hum. Mutat. 27(3):260-8, 2006). Levels of suppression and replacement of a target gene can be evaluated in FACS isolated cell populations. For example, suppression and/or suppression and replacement can be evaluated in electroporated EGFP positive cells from retinal explants.
Evaluation of Suppression and Replacement Using RNA Assays
Suppression and replacement can be evaluated in cells, tissues and/or animals using RNA assays including real time RT-PCR, northern blotting, RNA in situ hybridisation and or RNAse protection assays. RNA expression levels of suppressors and/or of endogenous genes and or replacement nucleic acids can be assessed by real time RT-PCR using, for example, a 7300 Real Time PCR System (Applied Biosystems, Foster City, Calif., USA) and using, for example, a QuantiTect SYBR Green RT-PCR kit (Qiagen Ltd). RT-PCR assays are undertaken using levels of expression of housekeeping controls such as β-actin or GAPDH, for example, for comparative purposes. Levels of RNA expression can be evaluated using sets of primers targeting the nucleic acids of interest including suppressors, target genes and/or replacements, for example, the following primers can be used for the evaluation of levels of expression of human rhodopsin, β-actin and GAPDH.







TABLE 7







PCR Primers for measuring rhodopsin, β-actin, and


GAPDH











SEQ




ID


Primer
Sequence
NO





RHO forward
5′ CTTTCCTGATCTGCTGGGTG 3′
69


primer




 


RHO reverse
5′ GGCAAAGAACGCTGGGATG 3′
70


primer




 


β-actin forward
5′ TCACCCACACTGTGCCCATCTACGA 3′
71


primer




 


β-actin reverse
5′ CAGCGGAACCGCTCATTGCCAATGG 3′
72


primer




 


GAPDH forward
5′-CAGCCTCAAGATCATCAGCA-3′
73


primer:




 


GAPDH reverse
5′-CATGAGTCCTTCCACGATAC-3′
74


primer:









Expression of replacement constructs and/or shRNAs may be confirmed, for example, by Northern blotting. RNA may also be detected by in situ hybridisations using single stranded RNA probes that have been labelled with, for example, DIG. To evaluate levels of expression of suppression agents and/or replacement nucleic acids and/or endogenous target genes, RNase protections assays can be performed using art known methods, such as that described in the Ambion mirVanaTM Probe and Marker kit manual (catalogue number 1554) and the Ambion RPAIIITM Ribonuclease protection assay kit manual (catalogue number 1414). For example, RNA probes approximately 15-25 nucleotides in length specific for transcripts from, for example, an endogenous target gene and/or a suppressor and/or a replacement nucleic acid can be synthesized. For example, RNA probes targeting mouse rhodopsin and/or human rhodopsin and/or suppression agents targeting rhodopsin and/or rhodopsin replacement nucleic acids can be synthesized using companies such as Sigma-Proligo or Ambion. RNA probes and size standards can be labelled to aid visualization after separation of samples on denaturing polyacrylamide gels. For example, RNA probes and DecadeTM size marker (Ambion Inc) can be 5′ end-labelled with P32-γATP (GE Healthcare) using the mirVanaTM probe and marker kit according to the manufacturer's protocol (Ambion Inc.). RNase protection assays can be performed using art known methods, for example, using the RPA IIITM Ribonuclease Protection Assay Kit and the manufacturer's protocol (Ambion Inc.).
Expression of suppressors and/or replacement nucleic acids and/or endogenous genes can be undertaken and determined in cells, in tissues and or in animals using, for example, the assays and associated methodologies provided above.
Evaluation of Suppression and Replacement Using Protein Assays
Suppression and replacement can be evaluated in cells, tissues and/or animals using protein assays including ELISA, western blotting and immunocytochemistry assays. ELISAs can be undertaken to evaluate levels of suppression by assessing levels of expression of a target endogenous gene and/or can be used to evaluate levels expression of replacement nucleic acids—such proteins assays are well know in the art and methods are provided in, for example, Palfi et al., Hum. Mutat. 27(3):260-8, 2006. For example, in the case of retinal genes such as the rhodopsin gene, ELISA is undertaken using a rhodopsin primary antibody which is typically used in a diluted form, for example, using a 1/10-1/10000 dilution (but possibly outside of this range) of an antibody for the target protein. In addition, Western Blotting may be undertaken to determine relative quantities of a specific protein, for example rhodopsin. Briefly, protein samples are separated using SDS-PAGE and transferred to a membrane. The membrane is incubated with generic protein (for example milk proteins) to bind to “sticky” places on the membrane. A primary antibody is added to a solution which is able to bind to its specific protein and a secondary antibody-enzyme conjugate, which recognizes the primary antibody is added to find locations where the primary antibody bound.
In addition to the protein assays referred to above, assays using antibodies in conjunction with microscopy can be used to evaluate protein levels. For example, in the case of rhodopsin immunocytochemistry (for example, using a 1/10-1:1000 dilution of a primary rhodopsin antibody) and fluorescent microscopy can be carried out as has been documented in Kiang et al., 2005 Mol. Ther. 12(3):555-61, 2005. Immunocytochemistry can be undertaken on cells and/or tissues. In the case of the retina, various modes of sectioning can be implemented to evaluate retinal sections. For example, frozen sections, agar embedded sections and/or resin embedded sections can be used. To obtain thin sections, for example of the retina, epon embedding and semi-thin sectioning can be performed using art known methods such as those provided in McNally et al., Hum. Mol. Genet. 11(9):1005-16, 2002. Immunocytochemistry may be used to evaluate suppression of a target gene and or expression of replacement nucleic acids. Additionally, histological analyses can be used to evaluate the histological effect(s) associated with the administration of suppressors and or replacement nucleic acids. In animal models of retinal degenerations such as the rho−/−, rds, rhodopsin Pro23His, rhodopsin Pro2347Ser mice and others there is a degeneration of the photoreceptor cell layer over time. Histological analyses can be used to evaluate if this degeneration has been modulated subsequent to administration of suppression agents and/or replacement nucleic acids.
Delivery of Suppression and Replacement
Both non-viral and/or viral vectors can be used in the invention to deliver the suppression agents and/or replacement nucleic acids. For example, in the case of retina, recombinant adenoassociated virus (AAV) and more specifically AAV2/5 has previously been found to elicit efficient transduction of photoreceptopr cells. Other AAV serotypes may also be used to deliver to retina, for example, AAV2/2 elicits efficient delivery to the retinal pigment epithelium (RPE) as does AAV4. AAV vectors can be generated using protocols with and without helper virus. For example, a helper virus free protocol using a triple transfection approach is well documented (Xiao et al., J. Virol. 72(3):2224-32, 1998). Expression cassettes carrying suppression and/or replacement elements can be cloned into plasmids such as pAAV-MCS provided by Stratagene Inc. Suppressors and/or replacement nucleic acids are cloned between the inverted terminal repeats of AAV2 and transfected into 293 cells (Stratagene; ATACC cat no CRL-1573) with two other plasmids, hence the term triple transfection. For example, the pRep2/Cap5 plasmid (Hildinger et al., J. Virol. 75(13):6199-203, 2001) together with the pHelper plasmid (Stratagene), at, for example, a ratio of 1:1:2, can be used to generate AAV2/5 vectors. Virus can be generated using a variety of art known procedures including the method outlined below. For example, to generate virus fifty 150 mm plates of confluent HEK293 cells were transfected (50 μg DNA/plate) with polyethyleminine (Reed et al., J. Virol. Methods 138(1-2):85-98, 2006). 48 hrs post-transfection crude viral lysates were cleared (Auricchio et al., 2001) and purified by CsCl2 gradient centrifugation (Zolotukhin et al., Gene Ther. 6(6):973-85, 1999). The AAV containing fraction was dialyzed against PBS. Genomic titres, viral particles (vp/ml), were determined by quantitative real-time PCR using art known methods (Rohr et al., J. Virol. Methods 106(1):81-8, 2002). AAVs can be generated that contain, for example, either targeting shRNAs or control shRNAs and/or replacement nucleic acids such as rhodopsin and/or reporter nucleic acids such as EGFP and/or stuffer sequences and/or sequences aiding expression of suppression agents and/or replacement nucleic acids such as promoter and/or enhancer sequences and/or other regulatory sequences and/or epigenetic elements.
Administration of Suppression and Replacement Vectors
Animal models can be used to mirror human disorders. For example, animal models of human retinopathies or that express a human retinal gene have been generated, for example, rho−/− mice (Humphries et al., Nat. Genet. 15(2):216-9, 1997), NHR +/− mice (Olsson et al., Neuron 9(5):815-30, 1992), Pro23His mice (Olsson et al., Neuron 9(5):815-30, 1992), Pro347Ser mice (Li et al., Proc. Natl. Acad. Sci. U.S.A. 95(20):11933-8, 1998) and RHO-M mice (see below). Mice typically are maintained under specific pathogen free (SPF) housing conditions and in a controlled light environment. The suppression agents and/or replacement nucleic acids of the invention can be administered to animals either locally and/or systemically. Local administration can include direct injection to the target tissues and/or in the proximity of the target tissue as has been described in detail in the art in, for example, Xia et al. (ACS Chem. Biol. 1(3):176-83, 2004) delivered AAV vectors with shRNAs to brain to treat spinocerebellar ataxia. In the case of the retina, subretinal injection can be used to administer suppression agents and/or replacement nucleic acids according to the following procedure. For example, mice can be anaesthetised by intraperitoneal injection of Domitor and Ketalar (10 and 50 μg/g of body weight respectively). The pupils are dilated with phenylephrine and under local analgesia (amethocaine) a small puncture is made in the sclera. A micro-needle attached to a 10 μl syringe (Hamilton Company Europe) is inserted through the puncture to the subretinal space and 1-3 μl of vector is administered. For example, in the case of AAV 1-411 of a 1012-14 vp/ml AAV vector preparation in PBS is administered. A reverse anaesthetic (antisedan, 50 μg/g of body weight) can be applied by intraperitoneal injection post-delivery. Body temperature during the procedure is sustained using a homeothermic heating device. In addition newborn mice can be prepared for subretinal injection according to Matsuda and Cepko (Proc. Natl. Acad. Sci. U.S.A. 101(1):16-22, 2004).
Assay for Function
To evaluate if suppression and/or replacement modulates the function of a target tissue and/or cell type, one or more assays may be employed that are well described in the prior art. In the case of the retina, functional assays include but are not limited to electrophysiology, such as pattern electroretinogram (ERG), full field ERG, and visual evoked potentials. In addition, visual field assessments, color vision assessments, and pupilometry may be performed. For example, electroretinography can be used to evaluate the response of the retina to light. This can be performed using, for example, the following procedure or an adapted procedure. Animals can be dark-adapted overnight and prepared for ERG under dim red light. Pupils are dilated with 1% cyclopentalate and 2.5% phenylephrine. Animals are anesthetized with ketamine and xylazine (16 and 1.6 μg/10 g body weight respectively) injected intraperitoneally. Standardized flashes of light are presented to the animal, for example a mouse, in a Ganzfeld bowl. ERG responses are recorded simultaneously from both eyes by means of contact lens electrodes (Medical Workshop, Netherlands) using 1% amethocaine as topical anesthesia. Reference and ground electrodes are positioned subcutaneously, approximately one mm from the temporal canthus and anterior to the tail respectively. Responses are analysed using a RetiScan RetiPort electrophysiology unit (Roland Consulting Gmbh). The protocol is based on that approved by the International Clinical Standards Committee for human electroretinography. Rod-isolated responses are recorded using a dim white flash (−25 dB maximal intensity where maximal flash intensity was 3 candelas/m2/s) presented in the dark-adapted state. Maximal combined rod-cone responses to the maximal intensity flash are then recorded. Following a 10 minute light adaptation to a background illumination of 30 candelas/m2, cone-isolated responses are recorded to the maximal intensity flash presented initially as a single flash and subsequently as 10 Hz flickers. A-waves are measured from the baseline to the trough and b-waves from the baseline (in the case of rod-isolated responses) or from the a-wave to the trough.
The agents of the invention are administered in effective amounts. An effective amount is a dosage of the agent sufficient to provide a medically desirable result. An effective amount means that amount necessary to delay the onset of, inhibit the progression of or halt altogether the onset or progression of the particular condition or disease being treated. An effective amount may be an amount that reduces one or more signs or symptoms of the disease. When administered to a subject, effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight, concurrent treatment, frequency of treatment, and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
Actual dosage levels of active ingredients in the pharmaceutical compositions of the invention can be varied to obtain an amount of the agent(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level depends upon the activity of the particular agent, the route of administration, the severity of the condition being treated, the condition, and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the agent(s) at levels lower than required to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.
The agents and pharmaceutical compositions of the invention can be administered to a subject by any suitable route. For example, the compositions can be administered orally, including sublingually, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically and transdermally (as by powders, ointments, or drops), bucally, or nasally. The term “parenteral” administration as used herein refers to modes of administration other than through the gastrointestinal tract, which include intravenous, intramuscular, intraperitoneal, intrasternal, intramammary, intraocular, retrobulbar, intrapulmonary, intrathecal, subcutaneous and intraarticular injection and infusion. Surgical implantation also is contemplated, including, for example, embedding a composition of the invention in the body such as, for example, in the brain, in the abdominal cavity, under the splenic capsule, brain, or in the cornea.
Agents of the present invention also can be administered in the form of liposomes. As is known in the art, liposomes generally are derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any nontoxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33, et seq.
Dosage forms for topical administration of an agent of this invention include powders, sprays, ointments, and inhalants as described herein. The agent is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required. Ophthalmic formulations, eye ointments, powders, and solutions also are contemplated as being within the scope of this invention.
Pharmaceutical compositions of the invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water ethanol, polyols (such as, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such, as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions also can contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It also may be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of the agent, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This result can be accomplished by the use of a liquid suspension of crystalline or amorphous materials with poor water solubility. The rate of absorption of the agent then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug from is accomplished by dissolving or suspending the agent in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such a polylactide-polyglycolide. Depending upon the ratio of agent to polymer and the nature of the particular polymer employed, the rate of agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations also are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
The injectable formulations can be sterilized, for example, by filtration through a bacterial- or viral-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
The invention provides methods for oral administration of a pharmaceutical composition of the invention. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed., 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms for oral administration include capsules, tablets, pills, powders, troches or lozenges, cachets, pellets, and granules. Also, liposomal or proteinoid encapsulation can be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may include liposomes that are derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). In general, the formulation includes an agent of the invention and inert ingredients which protect against degradation in the stomach and which permit release of the biologically active material in the intestine.
In such solid dosage forms, the agent is mixed with, or chemically modified to include, a least one inert, pharmaceutically acceptable excipient or carrier. The excipient or carrier preferably permits (a) inhibition of proteolysis, and (b) uptake into the blood stream from the stomach or intestine. In a most preferred embodiment, the excipient or carrier increases uptake of the agent, overall stability of the agent and/or circulation time of the agent in the body. Excipients and carriers include, for example, sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, cellulose, modified dextrans, mannitol, and silicic acid, as well as inorganic salts such as calcium triphosphate, magnesium carbonate and sodium chloride, and commercially available diluents such as FAST-FLO®, EMDEX®, STA-RX 1500®, EMCOMPRESS® and AVICEL®, (b) binders such as, for example, methylcellulose ethylcellulose, hydroxypropyhnethyl cellulose, carboxymethylcellulose, gums (e.g., alginates, acacia), gelatin, polyvinylpyrrolidone, and sucrose, (c) humectants, such as glycerol, (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, starch including the commercial disintegrant based on starch, EXPLOTAB®, sodium starch glycolate, AMBERLITE®, sodium carboxymethylcellulose, ultramylopectin, gelatin, orange peel, carboxymethyl cellulose, natural sponge, bentonite, insoluble cationic exchange resins, and powdered gums such as agar, karaya or tragacanth; (e) solution retarding agents such a paraffm, (f) absorption accelerators, such as quaternary ammonium compounds and fatty acids including oleic acid, linoleic acid, and linolenic acid (g) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate, anionic detergent surfactants including sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate, cationic detergents, such as benzalkonium chloride or benzethonium chloride, nonionic detergents including lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose; (h) absorbents, such as kaolin and bentonite clay, (i) lubricants, such as talc, calcium sterate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, waxes, CARBOWAX® 4000, CARBOWAX® 6000, magnesium lauryl sulfate, and mixtures thereof; (j) glidants that improve the flow properties of the drug during formulation and aid rearrangement during compression that include starch, talc, pyrogenic silica, and hydrated silicoaluminate. In the case of capsules, tablets, and pills, the dosage form also can comprise buffering agents.
Solid compositions of a similar type also can be employed as fillers in soft and hard-filled gelatin capsules, using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They optionally can contain opacifying agents and also can be of a composition that they release the active ingredients(s) only, or preferentially, in a part of the intestinal tract, optionally, in a delayed manner. Exemplary materials include polymers having pH sensitive solubility, such as the materials available as EUDRAGIT® Examples of embedding compositions which can be used include polymeric substances and waxes.
The active compounds also can be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol ethyl carbonate ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydroflirfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions also can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, coloring, flavoring, and perfuming agents. Oral compositions can be formulated and further contain an edible product, such as a beverage.
Suspensions, in addition to the agent(s), can contain suspending agents such as, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.
Also contemplated herein is pulmonary delivery of the agent(s) of the invention. The agent(s) is delivered to the lungs of a mammal while inhaling, thereby promoting the traversal of the lung epithelial lining to the blood stream. See, Adjei et al., Pharmaceutical Research 7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology 13 (supp1.5): s.143-146 (1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine 3:206-212 (1989) (α1-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146 (1989) (α1-proteinase); Oswein et al., “Aerosolization of Proteins,” Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990 (recombinant human growth hormone); Debs et al., The Journal of Immunology 140:3482-3488 (1988) (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).
Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
Some specific examples of commercially available devices suitable for the practice of the invention are the ULTRAVENT® nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the ACORN II® nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the VENTOL® metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the SPINHALER® powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
All such devices require the use of formulations suitable for the dispensing of a agent(s) of the invention. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.
The composition is prepared in particulate form, preferably with an average particle size of less than 10 μm, and most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.
Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include lipids, such as DPPC, DOPE, DSPC and DOPC, natural or synthetic surfactants, polyethylene glycol (even apart from its use in derivatizing the inhibitor itself), dextrans, such as cyclodextran, bile salts, and other related enhancers, cellulose and cellulose derivatives, and amino acids.
Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
Formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise a compound of the invention dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation also can include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation also can contain a surfactant to reduce or prevent surface-induced aggregation of the inhibitor composition caused by atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the agent suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid also can be useful as a surfactant.
Formulations for dispensing from a powder inhaler device comprise a finely divided dry powder containing the agent and also can include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol, in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
Nasal delivery of the agent(s) and composition of the invention also is contemplated. Nasal delivery allows the passage of the agent or composition to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes also is contemplated.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the agent(s) of the invention with suitable nonirritating excipients or carriers, such as cocoa butter, polyethylene glycol, or suppository wax, which are solid at room temperature, but liquid at body temperature, and therefore melt in the rectum or vaginal cavity and release the active compound.
In order to facilitate delivery of the agent(s) across cell and/or nuclear membranes, compositions of relatively high hybrophobicity are preferred. The agent(s) can be modified in a manner which increases hydrophobicity, or the agent(s) can be encapsulated in hydrophobic carriers or solutions which result in increased hydrophobicity.
Practice of the invention will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.
EXEMPLIFICATION
Example 1
siRNAs Targeting Human Rhodopsin and Rhodopsin Replacement Nucleic Acids
siRNAs targeting human rhodopsin were synthesized and evaluated for RNAi-mediated suppression (listed in Table 8). Suppression and replacement constructs with suppressors targeting the human rhodopsin mRNA sequence and replacement rhodopsin genes that escape suppression by the suppressor due to subtle changes in the sequence were subsequently designed. These changes, while enabling replacement nucleic acids to escape suppression at least in part, did not change the protein product expressed from the replacement genes. Short hairpin RNAs (shRNAs) were used to demonstrate suppression in vivo (FIG. 1). The sequence of the sense and antisense strands of the shRNAs is the same as the sequence used for the siRNAs. An intervening loop is included between the sense and antisense strands in the same manner as Brummelkamp et al., Science 296(5567):550-3, 2001. Notably, the number of nucleotides and the make up of the nucleotides in the intervening loop can vary. The construct(s) were delivered using an AAV2/5 recombinant virus. Non-targeting siRNA can be used as controls, for example, a non-targeting siRNA directed towards an EGFP reporter gene can be used—for example.
siRNAs were designed according to the method of Elbashir et al., Nature 411(6836):494-8, 2001, or by using the HiPerformance siRNA design algorithm (Qiagen Ltd. Crawley, UK). siRNA target sequences differed by at least 4 nucleotides from any non-rhodopsin sequences in mouse and human databases (http://www.ncbi.nlm.nih.gov/blast, BLAST2.2.6 (Altschul et al., Nucleic Acids Res. 25(17):3389-402, 1997). siBB, siQ1 and a non-targeting siRNA siNT (5′ UUCUCCGAACGUGUCACGU 3′; SEQ ID NO:75) or EGFP (U57608), siEGFP (nt 256-277) were initially cloned downstream of the H1 promoter using BglII/BamH1 and Hind III restriction sites to generate shRNAs and subsequently in pEGFP-1 (BD Biosciences, Clontech, Palo Alto, Calif.) using EcoRI and Hind III sites generating shBB-EGFP, shQ1-EGFP and shNT-EGFP (FIG. 1A). The EGFP gene enabled viral transduction to be monitored. Six siRNAs sequences targeted the coding region of human rhodopsin. Replacement nucleic acids were cloned into pCDNA3.1-plasmid (Invitrogen, Karlsruhe, Germany). The CMV promoter was replaced with either the human ubiquitin C promoter (pUB6/V5-His, Invitrogen) or a 1.7 kb fragment of the mouse rho promoter (rhoP). Sequence alterations were introduced into replacement nucleic acids using primer directed PCR-based mutagenesis using art known methods. Replacement nucleic acids with sequence alterations over the target sites for siB, siBB, siC, siCC, siQ1 and siQ2 were termed rB, rBB, rC, rCC, rQ1, and rQ2. Altered nucleotides in the replacement rhodopsin sequences are at wobble positions (highlighted in bold print). These replacement genes were designed to avoid suppression by the siRNAs yet encode wild type protein. Table 8 provides one replacement example for each siRNA target site; however, in each case there are several alternative possible replacement sequences because some amino acids have as many as six codons and others have four or three codons.







TABLE 8







siRNA Sequence and Replacement Rhodopsin Sequence














SEQ

SEQ





ID
Replacement rhodopsin
ID
Position in


siRNA
Sequence
NO
sequence
NO
NM_000539.2





siB
TCAACTTCCTCACGCTCTA
75
ATAAATTTTTTGACCCTGTA
76
256-277





T




 


siBB
TCACCGTCCAGCACAAGAA
77
CTGTATGTGACGGTGCAGCA
78
254-274





C




 


siC
CGTGTGGAATCGACTACTA
79
AGCTGCGGTATAGATTATTA
80
270-292


 


siCC
CGCTCAAGCCGGAGGTCAA
81
ACCTTGAAACCCGAAGTGAA
82
274-294


 


siQ1
TCAACTTCCTCACGCTCTAC
83
CTGTATGTGACGGTGCAGCA
84
650-670



GT

C




 


siQ2
CTCTACGTCACCGTCCAGC
85
CTGTATGTGACGGTGCAGCA
86
671-694



ACAA

C









Suppression of RHO in HeLa Cells

RNAi-mediated suppression of RHO was initially evaluated in HeLa cells. siRNAs targeting RHO were co-transfected with a CMV promoter-driven wild type RHO. Transfections were carried out three times in quadruplicate using lipofectamine 2000 to aid transfections (Gibco-BRL). Real time RT-PCRs, performed on RNAs extracted from transfected cells 24 hours post-transfection, demonstrated up to 87% suppression (p<0.01, FIG. 2A) (see Table 7 for primer sequences). siRNAs siBB, siCC and siQ1 were selected for further analysis. Similar levels of rhodopsin protein suppression were quantified by ELISA (up to 88%, p<0.01, FIG. 2A) and demonstrated by immunocytochemistry 24 hours post-transfection (FIG. 2B). Subsequently, replacement RHO constructs, rBB, rCC and rQ1, were generated incorporating nucleotide changes at degenerate positions over the target sites for siRNAs, siBB, siQ1 and siCC as described above and shown in Table 8. Transfections were performed three times in quadruplicate in HeLa cells according to art known methods as described above. Results indicated that replacement RHO constructs were not suppressed by corresponding siRNAs, for example, rBB by siBB (FIG. 3). However, significant levels of suppression were obtained with other non-corresponding siRNAs, for example siQ1 suppressed rBB and rCC (FIG. 3).
Long Term Suppression of RHO in Retinal Explants
To provide long term RHO suppression, siBB and siQ1 were cloned as shRNAs into an EGFP expressing vector (shBB-EGFP and shQ1-EGFP, FIG. 1A). Plasmids were electroporated into retinal explants from newborn NBR+/− rho−/− mice using the methods described in Matsuda and Cepko (2004) Proc. Natl. Acad. Sci. USA 101: 16-22. NHR+/− mice express a wild type human RHO gene and display a wild type phenotype. Cells from retinal explants (n=6) were dissociated two weeks post-electroporation and EGFP-positive cells isolated by FACS (FIG. 4A). Real time RT-PCR was performed on RNA extracted from EGFP-positive FACS-isolated cells using the primers described in Table 8 and results obtained in explants mirrored those found in HeLa cells. Results indicated that RHO suppression of greater than 85% was achieved (p<0.001, FIG. 4B).
Long Term Suppression Using AAV Vectors
Long-term expression of therapies will be required for a progressive retinopathy such as adRP. To achieve long-term suppression in vivo, shBB-EGFP and the non-targeting shNT-EGFP were engineered into AAV vectors (AAV-shBB-EGFP and AAV-shNT-EGFP) (FIG. 1A). Recombinant AAV2/5 viruses were generated using a helper virus free system. Expression cassettes were cloned into pAAV-MCS (Stratagene, La Jolla, Calif., USA), between the inverted terminal repeats of AAV2, and transfected into HEK-293 cells (ATCC no. CRL-1573) with pRep2/Cap5 and pHelper (Stratagene), at a ratio of 1:1:2. Fifty 150 mm plates of confluent cells were transfected (50 μg DNA/plate) with polyethyleminine. Forty eight hours post-transfection crude viral lysates were cleared and purified by CsCl2 gradient centrifugation. AAV-containing fractions were dialysed against PBS. Genomic titres, i.e., viral particles (vp/ml), were determined by quantitative real time PCR. AAVs generated contained the shBB-EGFP and shNT-EGFP constructs (AAV-shBB-EGFP and AAV-shNT-EGFP, FIG. 1A).
The EGFP gene enabled viral transduction to be monitored. Three μl of AAV-shBB-EGFP (2×1012 vp/ml) or AAV-shNT-EGFP (3×1012 vp/ml) were subretinally injected into adult NHR+/− rho−/− mice. Two weeks post-injection two animals were sacrificed and expression of the 21 nucleotide shBB shown in two retinas using RNase protection (FIG. 5A). Retinas were dissociated and EGFP-positive cells collected by FACS. RNAi-mediated suppression of RHO, as evaluated by real time RT-PCR (see Table 8 for primer sequences) two weeks post-injection (n=6), was approximately 90% (p<0.001) in AAV-shBB-EGFP-transduced photoreceptor cells (FIG. 5B). Four retinas were dissociated and significant suppression of rhodopsin protein expression was demonstrated in vivo in EGFP-positive transduced cells by immunocytochemistry (FIG. 5C).
Suppression in Transgenic Animals
A transgenic mouse expressing a sequence-modified RHO gene was generated (RHO-M). RHO-M+/− rho−/− were evaluated at two months of age for rescue of the retinal pathology present in rho−/− mice by histology (FIG. 6A-C) and ERG (FIG. 6D). Rhodopsin immunolabeling in rod outer segments and the thickness of outer nuclear layers were similar in wild type rho+/+ (FIG. 6A), NHR+/− rho−/− (FIG. 6B) and RHO-M+/− rho−/− (FIG. 6C) mice. Additionally, ERG responses were similar in wild type rho+/+, rho+/−, NHR+/− rho−/− and RHO-M+/− rho−/− mice. ERG b-waves of rod-isolated responses of 500-700 μV were observed in mice of all genotypes (FIG. 6D). The amplitudes and timings of the combined rod and cone responses to the maximal intensity flash presented in the dark-adapted state, as well as the light adapted cone-isolated responses both to single flash and 10 Hz flickers, were equivalent in all the genotypes examined (data not shown). These results validate the use of the degeneracy of the genetic code to engineer codon-modified human RHO genes which can provide functional human rhodopsin protein.
AAV-Delivered Suppression and Replacement of Human RHO In Vivo
Having established shBB and shQ1 as potent suppressors and rBB and rQ1 as being refractory to their corresponding suppressors, shBB-rBB and shQ1-rQ1 were cloned into AAV vectors using the triple plasmid system detailed above and viruses containing both elements of the therapeutics were generated (AAV-shBB-rBB (also termed AAV-BB8) and AAV-shQ1-rQ1 (also termed AAV-Q1)) using the method detailed above. Three μl of AAV-shBB-rBB was subretinally injected into adult wild type rho+/+ mice (n=12) and replacement RHO mRNA expression confirmed by RT-PCR and RNase protection using RNA extracted 10 days post-injection (data not shown). To demonstrate that AAV-delivered rBB is translated into protein, 2 μl of a 1:1 mix of AAV-shBB-rBB and AAV-CMV-EGFP was subretinally injected into 10 day old rho−/− mice (n=6). Two weeks post-injection rhodopsin and EGFP protein expression were determined using fluorescent microscopy. Marked rhodopsin expression, overlapping with EGFP, was observed in transduced areas (FIG. 7).
Subsequently, 1 μl of AAV-shBB-rBB or AAV-shQ1-rQ1 was subretinally injected into newborn Pro23His+/− rho+/− mice (n=10) that present with a retinal degeneration resulting in complete loss of photoreceptors by two weeks of age. In all animals one eye was injected with therapeutic virus (either AAV-shBB-rBB or AAV-shQ1-rQ1) and the other with a control virus (AAV-EGFP). The early onset and rapid nature of the retinopathy in young Pro23His pups precluded use of ERG as a readout for benefit. However, at ten days of age retinal histology was evaluated in semi-thin resin embedded sections cut at approximately 50 μm intervals throughout the central meridian of the eye (n=10). From each section approximately 40 measurements of ONL thickness were taken. Since only a part of the retina is transduced by a single subretinal injection of AAV (particularly in newborn pups), to identify the transduced area ONL measurements were ordered by thickness and the 15% highest and lowest values grouped for analysis. Lowest values represent thinnest ONL readings, most likely corresponding to peripheral areas of the retina and thus not in close proximity to injection sites. Highest values represent thickest ONL readings, most likely corresponding to central areas of the retina and thus in closer proximity to injection sites. Significant differences in ONL thickness between AAV-shBB-rBB- and AAV-EGFP-treated eyes were observed. The ONL of treated eyes was found to be approximately 33% (p<0.001) thicker than control injected counterparts for the highest value groupings (FIG. 8A-C). In the lowest value groupings a difference of approximately 10% was observed (FIG. 8A). These data provide evidence at the histological level that AAV2/5-delivered RNAi in conjunction with provision of a codon-modified replacement gene can beneficially modulate the retinopathy in Pro23His+/− rho−/− mice.
RNAi-mediated suppression was evaluated in retinal tissue after sub-retinal injection of AAV vectors expressing either a suppressor targeting rhodopsin (AAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP) or a non-targeting control (AAV-shNT-EGFP). Mice expressing a human rhodopsin replacement gene (referred to as RHO-M mice and detailed in the section on suppression in transgenic animals) were subretinally injected with AAV vectors (AAV2/5), containing shRNA sequences for BB, CC and Q1 and an EGFP reporter gene (AAV-shBB-EGFP, AAV-shCC-EGFP and AAV-shQ1-EGFP). The presence of the EGFP reporter gene enabled isolation of the population of retinal cells that are EGFP positive and therefore have received AAV using FACS to isolate these cell populations. AAV-delivered RNAi-mediated suppression with each suppressor (BB, CC and Q1) was evaluated using real-time RT-PCR in cell populations characterised by FACS and was compared to suppression obtained using AAV with non-targeting control shRNA sequences (AAV-shNT-EGFP). Significant rhodopsin suppression was obtained with BB and Q1 suppressors, however, significantly lower levels of suppression were obtained with the CC suppressor (FIG. 6E). The replacement gene in RHO-M mice was partially protected from suppression due to the presence of two nucleotide mismatches between the CC suppressor sequence and the target site for suppression in the human rhodopsin replacement gene. The replacement gene is partially protected from siRNA CC-based suppression by the introduction of two nucleotide changes at degenerate sites in the replacement gene. FIG. 6F illustrates depression of the ERG response in RHO-M eyes that have received AAV-shBB-EGFP (panel 1) or AAV-shQ1-EGFP (panel 2) when compared to eyes subretinally injected with AAV-shNT-EGFP. The top tracing in each panel represents the right eye which received the targeting AAV-shRNA vector and the bottom tracing in each panel represents the left eye which received the control non-targeting AAV-shNT vector. In contrast no reduction/depression of the ERG was observed in RHO-M mice subretinally injected with AAV-shCC-EGFP (panel 3) vector; this is likely due to the reduced levels of rhodopsin suppression observed with AAV-shCC-EGFP (see FIG. 6E above).
Example 2
Optimization of Expression of Suppression Agents and Replacement Nucleic Acids
Expression of suppression and/or replacement vectors was optimized by including in the vectors sequences that enhanced and/or modulate expression levels at the RNA and/or protein level. A list of exemplary sequence elements is provided in Table 1, however, the enhancing and/or modulating elements of the invention are not exclusive to this list. For example, one or more of a promoter, a stuffer, an insulator, a silencer, a chromatin remodelling sequence, an intron sequence, a poly adenylation signal, a post translational regulatory element, and a transcription factor binding site can be included in suppression and/or replacement constructs to modulate expression of suppression and/or replacement components relating to the invention. Such elements and derivatives thereof can be used to modulate levels of expression, tissue specificity, timing of expression, and/or induction of expression. Table 9 provides some exemplary sequences that can be used to modulate expression of suppression and/or replacement constructs relating to the invention. The sequences provided are within conserved regions as evaluated by comparison of sequences from multiple species. At any one position a nucleotide may not be conserved between all species the sequences represent regions where overall there is a high degree of conservation. Such conserved sequences from any species such as human, mouse, rat, bacteria, virus and/or indeed a hybrid sequence from more than one species could be used in the invention.







TABLE 9





Exemplary Enhancer Sequences















CMV enhancer element amplified from pCDNA3.1 Invitrogen nt 


308-734 http://www.invitrogen.com/ (SEQ ID NO: 87)


CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG


 


CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA


 


TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT


 


GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG


 


GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA


 


TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA


 


TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT


 


TTG TTTTGGCACC AAAATCAACG GGAC


 


pAAV.BB11 The WPR element from pSin11 CMV GFPpre mut FL (Gene 


Therapy (7): 641-5 (2006)) (SEQ ID NO: 88)


GAGCAT CTTACCGCCATTTATTCCCA TATTTGTTCT GTTTTTCTTG ATTTGGGTAT


 


ACATTTAAATGTTAATAAAA CAAAATGGTG GGGCAATCAT TTACATTTTT


 


AGGGATATGTAATTACTAGT TCAGGTGTAT TGCCACAAGA CAAACATGTT


 


AAGAAACTTTCCCGTTATTT ACGCTCTGTT CCTGTTAATC AACCTCTGGA


 


TTACAAAATTTGTGAAAGAT TGACTGATAT TCTTAACTAT GTTGCTCCTT


 


TTACGCTGTGTGGATATGCT GCTTTATAGC CTCTGTATCT AGCTATTGCT


 


TCCCGTACGGCTTTCGTTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTC


 


TCTTTTAGAGGAGTTGTGGC CCGTTGTCCG TCAACGTGGC GTGGTGTGCT


 


CTGTGTTTGCTGACGCAACC CCCACTGGCT GGGGCATTGC CACCACCTGT


 


CAACTCCTTTCTGGGACTTT CGCTTTCCCC CTCCCGATCG CCACGGCAGA


 


ACTCATCGCCGCCTGCCTTG CCCGCTGCTG GACAGGGGCT AGGTTGCTGG


 


GCACTGATAATTCCGTGGTG TTGTC


 


pAAV.BB13 The WPR element from pBSK11 (Donello JE, et al. J. 


Virol. 1998 72(6): 5085-92.) (SEQ ID NO: 89)


AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT


 


GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC


 


CCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGG


 


AGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAA


 


CCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT


 


CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGAC


 


AGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTC


 


CTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGC


 


TACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC


 


TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGC


 


CGCCTCCCC





(Wild type woodchuck hepatitis B virus genome sequence ACCESSION J04514)






Example 3
Comparison of Rhodopsin Genes
In addition to adding enhancing and/or modulating elements to suppression and/or replacement vectors, the rhodopsin promoter was studied in detail. A comparison of rhodopsin genes present in different mammals resulted in identification of 9 highly conserved regions in the rhodopsin gene (conserved regions A though I, Sequence 1, Table 10). Regions A, B, C and D are in the rhodopsin promoter region, conserved region E is in intron 2 of the gene and conserved regions F, G, H and I are in the 3′ region.
The following sequence (Sequence 1; Table 10) shows the conserved regions within the mouse promoter human intronic and exonic and 3′ sequence. Notably, conserved sequences in the mouse promoter are nearly the same in the human rhodopsin promoter and it is contemplated that the human or other mammalian rhodopsin promoters and/or derivatives and/or hybrids thereof may be used in suppression and replacement constructs. Additionally, it is contemplated that other promoters could be combined with some or all of conserved regions A though I and used in suppression and/or replacement constructs, for example, other retinal promoter sequences may be used.







TABLE 10





Conserved Regions of Rhodopsin















GTTCCAGGGC CCAGGGGCTT CCAGCCATGA GGGCACCTAG ACTTGTAATC


 


CCTAGAGTCC TCCTGATGCC ACTGCCCAGG GACAGACAGC ACACAGCACC


 


CCTCCCCCAC TCTCTTAACA GGCAGAAGCA GGGAGATGGA GGCATGCTGA


 


AGATGTCCAT GTGAGGCTGG TGGTAGCATG CCCACTGCTG GGATGAAGAG


 


ATGGGGGCAA AGTGAGTGGC AGAGGCCAGG CCAGGTCCAG GCCCTTCCAG


 


GCTTCCTCTG CCACTGTGGA GATGAAAGAG GGAGCCAGGC AAGGTCCAGG


 


CCCTCCCCAC CCCCTCTGCC TCTATGGAGA TGAAGGGGGA ATGAAGAAGG


 


GAGCCAGACA GTTGTGCCAA CACAACTCCT CCGTCGAGTG TCTAATTGCT


 


TATGATCATG CATGCTCTCT CTCCCACTAA ACATTTATTA ATGTGTTAGG cons reg A


 


ATTTCCATTA GCGCGTGCCT TGAACTGAAA TCATTTGCAT ATGGCTGGGA


 


AAAAGTGGGG TGAGGGAGGA AACAGTGCCA GCTCCCCAAC AGGCGTCAAT


 


CACAGTGACA GATCAGATGG TTTCTGGCTG GAGGCAGGGG GGCTGTCTGA


 


GATGGCGGCA TGCATCCTTT CAGTGCATAT CACAGAAATT CAGGTGACTC


 


CTGCTGGGAG CCAAGACCCT GAGGCTGAGC CTGGCCACAG CTCCAATAGC


 


TGCTGGATAT CATCATGTCT GGGCTGAGCA GCCTCTAGAG GTACCCTTTT


 


ACAGATAGTA AAACTGAGGC TCAGTGACTG CTGAGCCAAA GTTGGACCCA


 


CCCACACTCA TTTGCAGACT GCCGTGGGCC ATGTTCTGAT CTCTTCCCTA


 


CCTGGACTCA GCCCAGCACA CTCGGCACAC AAGGCCCTTC TTCAGCTTGA


 


ATACAGCGTC CTCAGCTATA GCCAGCATCT ATGAATGGAG CTCAGTGACC


 


CTGACTGGAG GAAGTTAGGA CAGGGATTTT TTCTGGAGTT TTGGCAGGAA


 


GAGGCCAGGG TCAGGTGACT GCTGGAGCAC ACAGCTTGGT AAGACTAGTC


 


AGGACCTGCG TCCTGAGGCT ACATGTCATA TCCACAGTAA GGAAGTGGAA


 


GATGGGAGAT GACTGGCTGG GCCACAACCA GTGAGTGGAA TGTCCTTGTG


 


CATCTTTGTT TCCTAACCTT CCCCTCTGTA GCTGCTGAAA CACACACACA


 


CCCCATGCTC TGTTATGCCT CTTCCCTGGC CTGGGATTTC CATGGCTGAG


 


GTGATGGGGC ACTGAGGCAC CGCCAGGAAA GGCTGTAACC CATCTGCTCC


 


CCCATCCTTC ACCAGACTTC AAGCACCTAC CTAGAGCACA GGTGCAATTT


 


TGTACCCTCC CTGTCTGGGA CCCACAGTGG TTCCTCAATG CCGGCCAACC


 


AGACTCATAG GCCTGCCCAC AAGGCCCTTG GGGCTATCTG TCTGAGGCCT


 


GCAGGTGCCC TCCTGGCCAC CTAGGCTCCT GTGAGACTTA GACTTCCATA


 


GATTCTTCCT GAAAGACTAC TGAGGGCAGG AGCCCCCAAG CCTCAGGGTT


 


AGCTTTCCTC AGCCCTGCCT CTTTGCTAGC TCCGTTTCCA CATTGAAGGC


 


AGGGCTGAGC AGGGCAGGCG CAGCGAGGAG CTAACTGCTG CTTCTCTCTC


 


GTTCATTTGT CTGCTGCCCT GAGACGCCAC AGCACCTAAT AAGAGCATGT


 


TATGTGTAGC AAACATTAGG CCTGTAAGGA AGGAAAGGAG TGACGTCCCT


 


TGACGTCCTC AGCTAGGCTG TGGTGACACA AGCAAGAGGA CTAAGCCACA


 


GGTGAGGAGA AAGGGGGGGG GGGGTCTGCT GACCCAGCAA CACTCTTTCC cons reg B


 


TTCTGAGGCT TAAGAGCTAT TAGCGTAGGT GACTCAGTCC CTAATCCTCC


 


ATTCAATGCC CTGTGACTGC CCCTGCTTCT GAAGGGCCAA CATGGCTACA


 


GCTAGCTCCA GAGACAGCTT TTCAGGGCCC CAGCATCCAA GCATCTCACA


 


GTTCTCCACT GACCACACTC CTGTGCAGCA CTGGGCTTTT CAATGCCCCT


 


GACTTGAAGA GAACTCAAAC TGCAGGTCAA CTAGACTCTG CAAACTTCAC


 


CTGTGCTGGG GGTTCCTAGC CTGTGGGGAC AGTGTATCTT GAATACCTGC


 


TGCTATGGAC CAAGAGCTGA ACACACAGAC AAACAGGCTC AGCTGGCCGG


 


CATTCTGGAA CCACAAATGA GTGTGGATGA GCAGGAGGGC AACAAAATGG


 


TCTGGGTGTT GTCAACACAG TCAGTAAACA ATGCACGCAG TGGGGCTGGG


 


CCCTGATGTG GAGCTAGGTG GGGTTGGCTC TCCTTGGAAA CCTGAAGGGA


 


GAAGGAGAGG GAGCGAGATG ATGAGGTTTA TCAGCCTGCA GAGGCAGGGG


 


GTCAGGAAGG AGTGCCACTG TACTGACCCA GGACCTCTGT GGGACATCAA


 


GCCATGCCAA GGAGCCATGG AGCCTCGATT GCACTGGCAG GGACAGGTTG


 


TGATGCCCCA GAGTCCCCAG ACCCAGCAAA CAGAGGCCCA GAGTGGGAAG


 


TGGAGCTTTC CAGGGTATCG GGGTGACTCA GAGACACAGG GTAGAATCTG


 


CCTTGGGTGC TCACTGCCCT ATCTGAGTCC ACATGGCTCA GTCCCCAGGC


 


CCTGTTCTCT AGTGACTGTT GCTTTGATGA GGTAGAGACA GGCAGCCCTC


 


TTCTAAGAAC TATGTTTTGA TGGGGGACTC AGAGTTGGGG TGGGGTGGCA


 


ATGAAATTCT GTAGACTGTG TGGTTATAAC CCTGGCTGTT ACTAGCTAGT


 


TCTGTGACCT TGGTGACCCA CTTCAGACTC TAGGCCTCAG CCTCTGTAAG


 


TGCAGATACA CAGCGCCAAT CAGCCGATGA CTTCTAACAA TACTCTTAAC


 


TCACACAGAG CTTGTCTCAC TGAGCCAACA CCCTGTACCC TCAGCTCAGT


 


GACGGCTTTC AACCTGTGGG GCTGCCTCTG TTACCCAAGT GAGAGAGGGC


 


CAGTGCTCCC AGAGGTGACC TTGTTTGCCC ATTCTCTCCC TGGGTCAGCC


 


AGTGTTTATC TGTTGTATAC CCAGTCCACC CTGCAGGCTC ACATCAGAGC


 


CTAGGAGATG GCTAGTGTCC CCGCGGAGAC CACGATGAAG CTTCCCAGCT HindIII


 


GTCTCAAGCA CAAGCTGGCT GCAGAGGCTG CTGAGGCACT GCTAGCTGGG start 1.734kb


 


GATGGGGGCA GGGTAGATCT GGGGCTGACC ACCAGGGTCA GAATCAGAAC


 


CTCCACCTTG ACCTCATTAA CGCTGGTCTT AATCACCAAG CCAAGCTCCT cons reg C


 


TAAACTGCTA GTGGCCAACT CCCAGGCCCT GACACACATA CCTGCCCTGT


 


GTTCCCAAAC AAGACACCTG CATGGAAGGA AGGGGGTTGC TTTTCTAAGC


 


AAACATCTAG GAATCCCGGG TGCAGTGTGA GGAGACTAGG CGAGGGAGTA


 


CTTTAAGGGC CTCAAGGCTC AGAGAGGAAT ACTTCTTCCC TGGTTAGCCT


 


CGTGCCTAGG CTCCAGGGTC TTTGTCCTGC CTGGATACCT ATGTGGCAAG


 


GGGCATAGCA TTTCCCCCAC CATCAGCTCT TAGCTCAACC TTATCTTCTC


 


GGAAAGACTG CGCAGTGTAA CAACACAGCA GAGACTTTTC TTTTGTCCCC


 


TGTCTACCCC TGTAACTGCT ACTCAGAAGC ATCTTTCTCA CAGGGTACTG


 


GCTTCTTGCA TCCAGAGTTT TTTGTCTCCC TCGGGCCCCC AGAATCAAAT


 


TCTTCCTCTG GGACTCAGTG GATGTTTCAC ACACGTATCG GCCTGACAGT


 


CATCCTGGAG CATCCTACAC AGGGGCCATC ACAGCTGCAT GTCAGAAATG


 


CTGGCCTCAC ATCCTCAGAC ACCAGGCCTA GTGCTGGTCT TCCTCAGACT


 


GGCGTCCCCA GCAGGCCAGT AGGATCATCT TTTAGCCTAC AGAGTTCTGA


 


AGCCTCAGAG CCCCAGGTCC CTGGTCATCT TCTCTGCCCC TGAGATTTTT


 


CCAAGTTGTA TGCCTTCTAG GTAAGGCAAA ACTTCTTACG CCCCTCCTCG


 


TGGCCTCCAG GCCCCACATG CTCACCTGAA TAACCTGGCA GCCTGCTCCC


 


TCATGCAGGG ACCACGTCCT GCTGCACCCA GCAGGCCATC CCGTCTCCAT


 


AGCCCATGGT CATCCCTCCC TGGACAGGAA TGTGTCTCCT CCCCGGGCTG


 


AGTCTTGCTC AAGCTAGAAG CACTCCGAAC AGGGTTATGG GCGCCTCCTC


 


CATCTCCCAA GTGGCTGGCT TATGAATGTT TAATGTACAT GTGAGTGAAC


 


AAATTCCAAT TGAACGCAAC AAATAGTTAT CGAGCCGCTG AGCCGGGGGG


 


CGGGGGGTGT GAGACTGGAG GCGATGGACG GAGCTGACGG CACACACAGC


 


TCAGATCTGT CAAGTGAGCC ATTGTCAGGG CTTGGGGACT GGATAAGTCA


 


GGGGGTCTCC TGGGAAGAGA TGGGATAGGT GAGTTCAGGA GGAGACATTG


 


TCAACTGGAG CCATGTGGAG AAGTGAATTT AGGGCCCAAA GGTTCCAGTC


 


GCAGCCTGAG GCCACCAGAC TGACATGGGG AGGAATTCCC AGAGGACTCT


 


GGGGCAGACA AGATGAGACA CCCTTTCCTT TCTTTACCTA AGGGCCTCCA


 


CCCGATGTCA CCTTGGCCCC TCTGCAAGCC AATTAGGCCC CGGTGGCAGC


 


AGTGGGATTA GCGTTAGTAT GATATCTCGC GGATGCTGAA TCAGCCTCTG


 


GCTTAGGGAG AGAAGGTCAC TTTATAAGGG TCTGGGGGGG GTCAGTGCCT


 


GGAGTTGCGC TGTGGGAGCC GTCAGTGGCT GAGCTCGCCA AGCAGCCTTG


 


GTCTCTGTCT ACGAAGAGCC CGTGGGGCAG CCTCGAG      XhoI


 


ggatcctgag tacctctcct ccctgacctc aggcttcctc ctagtgtcac cttggcccct conserved region D


 


cttagaagcc aattaggccc tcagtttctg cagcggggat taatatgatt atgaacaccc


 


ccaatctccc agatgctgat tcagccagga gcttaggagg gggaggtcac tttataaggg


 


tctggggggg tcagaaccca gagtcatcca gctggagccc tgagtggctg agctcaggcc


 


ttcgcagcat tcttgggtgg gagcagccac gggtcagcca caagggccac agccatgaat


 


ggcacagaag gccctaactt ctacgtgccc ttctccaatg cgacgggtgt ggtacgcagc


 


cccttcgagt acccacagta ctacctggct gagccatggc agttctccat gctggccgcc


 


tacatgtttc tgctgatcgt gctgggcttc cccatcaact tcctcacgct ctacgtcacc


 


gtccagcaca agaagctgcg cacgcctctc aactacatcc tgctcaacct agccgtggct


 


gacctcttca tggtcctagg tggcttcacc agcaccctct acacctctct gcatggatac


 


ttcgtcttcg ggcccacagg atgcaatttg gagggcttct ttgccaccct gggcggtatg


 


agccgggtgt gggtggggtg tgcaggagcc cgggagcatg gaggggtctg ggagagtccc


 


gggcttggcg gtggtggctg agaggccttc tcccttctcc tgtcctgtca atgttatcca


 


aagccctcat atattcagtc aacaaacacc attcatggtg atagccgggc tgctgtttgt


 


gcagggctgg cactgaacac tgccttgatc ttatttggag caatatgcgc ttgtctaatt


 


tcacagcaag aaaactgagc tgaggctcaa aggccaagtc aagcccctgc tggggcgtca


 


cacagggacg ggtgcagagt tgagttggaa gcccgcatct atctcgggcc atgtttgcag


 


caccaagcct ctgtttccct tggagcagct gtgctgagtc agacccaggc tgggcactga


 


gggagagctg ggcaagccag acccctcctc tctgggggcc caagctcagg gtgggaagtg


 


gattttccat tctccagtca ttgggtcttc cctgtgctgg gcaatgggct cggtcccctc


 


tggcatcctc tgcctcccct ctcagcccct gtcctcaggt gcccctccag cctccctgcc


 


gcgttccaag tctcctggtg ttgagaaccg caagcagccg ctctgaagca gttccttttt


 


gctttagaat aatgtcttgc atttaacagg aaaacagatg gggtgctgca gggataacag


 


atcccactta acagagagga aaactgaggc agggagaggg gaagagactc atttagggat


 


gtggccaggc agcaacaaga gcctaggtct cctggctgtg atccaggaat atctctgctg


 


agatgcagga ggagacgcta gaagcagcca ttgcaaagct gggtgacggg gagagcttac


 


cgccagccac aagcgtctct ctgccagcct tgccctgtct cccccatgtc caggctgctg


 


cctcggtccc attctcaggg aatctctggc cattgttggg tgtttgttgc attcaataat


 


cacagatcac tcagttctgg ccagaaggtg ggtgtgccac ttacgggtgg ttgttctctg


 


cagggtcagt cccagtttac aaatattgtc cctttcactg ttaggaatgt cccagtttgg


 


ttgattaact atatggccac tctccctatg aaacttcatg gggtggtgag caggacagat


 


gttcgaattc catcatttcc ttcttcttcc tctgggcaaa acattgcaca ttgcttcatg


 


gctcctagga gaggccccca catgtccggg ttatttcatt tcccgagaag ggagagggag


 


gaaggactgc caattctggg tttccaccac ctctgcattc cttcccaaca aggaactctg


 


ccccacatta ggatgcattc ttctgctaaa cacacacaca cacacacaca cacacaacac


 


acacacacac acacacacac acacacacac aaaactccct accgggttcc cagttcaatc


 


ctgaccccct gatctgattc gtgtccctta tgggcccaga gcgctaagca aataacttcc


 


cccattccct ggaatttctt tgcccagctc tcctcagcgt gtggtccctc tgccccttcc


 


ccctcctccc agcaccaagc tctctccttc cccaaggcct cctcaaatcc ctctcccact


 


cctggttgcc ttcctagcta ccctctccct gtctaggggg gagtgcaccc tccttaggca


 


gtggggtctg tgctgaccgc ctgctgactg ccttgcaggt gaaattgccc tgtggtcctt


 


ggtggtcctg gccatcgagc ggtacgtggt ggtgtgtaag cccatgagca acttccgctt


 


cggggagaac catgccatca tgggcgttgc cttcacctgg gtcatggcgc tggcctgcgc


 


cgcaccccca ctcgccggct ggtccaggta atggcactga gcagaaggga agaagctccg


 


ggggctcttt gtagggtcct ccagtcagga ctcaaaccca gtagtgtctg gttccaggca


 


ctgaccttgt atgtctcctg gcccaaatgc ccactcaggg taggggtgta gggcagaaga


 


agaaacagac tctaatgttg ctacaagggc tggtcccatc tcctgagccc catgtcaaac conserved region E


 


agaatccaag acatcccaac ccttcacctt ggctgtgccc ctaatcctca actaagctag


 


gcgcaaattc caatcctctt tggtctagta ccccgggggc agccccctct aaccttgggc


 


ctcagcagca ggggaggcca caccttccta gtgcaggtgg ccatattgtg gccccttgga


 


actgggtccc actcagcctc taggcgattg tctcctaatg gggctgagat gagactcagt


 


ggggacagtg gtttggacaa taggactggt gactctggtc cccagaggcc tcatgtccct


 


ctgtctccag aaaattccca ctctcacttc cctttcctcc tcagtcttgc tagggtccat


 


ttctacccct tgctgaattt gagcccaccc cctggacttt ttccccatct tctccaatct


 


ggcctagttc tatcctctgg aagcagagcc gctggacgct ctgggtttcc tgaggcccgt


 


ccactgtcac caatatcagg aaccattgcc acgtcctaat gacgtgcgct ggaagcctct


 


agtttccaga agctgcacaa agatccctta gatactctgt gtgtccatct ttggcctgga


 


aaatactctc accctggggc taggaagacc tcggtttgta caaacttcct caaatgcaga


 


gcctgagggc tctccccacc tcctcaccaa ccctctgcgt ggcatagccc tagcctcagc


 


gggcagtgga tgctggggct gggcatgcag ggagaggctg ggtggtgtca tctggtaacg


 


cagccaccaa acaatgaagc gacactgatt ccacaaggtg catctgcatc cccatctgat


 


ccattccatc ctgtcaccca gccatgcaga cgtttatgat ccccttttcc agggagggaa


 


tgtgaagccc cagaaagggc cagcgctcgg cagccacctt ggctgttccc aagtccctca


 


caggcagggt ctccctacct gcctgtcctc aggtacatcc ccgagggcct gcagtgctcg


 


tgtggaatcg actactacac gctcaagccg gaggtcaaca acgagtcttt tgtcatctac


 


atgttcgtgg tccacttcac catccccatg attatcatct ttttctgcta tgggcagctc


 


gtcttcaccg tcaaggaggt acgggccggg gggtgggcgg cctcacggct ctgagggtcc


 


agcccccagc atgcatctgc ggctcctgct ccctggagga gccatggtct ggacccgggt


 


cccgtgtcct gcaggccgct gcccagcagc aggagtcagc caccacacag aaggcagaga


 


aggaggtcac ccgcatggtc atcatcatgg tcatcgcttt cctgatctgc tgggtgccct


 


acgccagcgt ggcattctac atcttcaccc accagggctc caacttcggt cccatcttca


 


tgaccatccc agcgttcttt gccaagagcg ccgccatcta caaccctgtc atctatatca


 


tgatgaacaa gcaggtgcct actgcgggtg ggagggcccc agtgccccag gccacaggcg


 


ctgcctgcca aggacaagct actcccaggg caggggaggg gctccatcag ggttactggc


 


agcagtcttg ggtcagcagt cccaatgggg agtgtgtgag aaatgcagat tcctggcccc


 


actcagaact gctgaatctc agggtgggcc caggaacctg catttccagc aagccctcca


 


caggtggctc agatgctcac tcaggtggga gaagctccag tcagctagtt ctggaagccc


 


aatgtcaaag tcagaaggac ccaagtcggg aatgggatgg gccagtctcc ataaagctga


 


ataaggagct aaaaagtctt attctgaggg gtaaaggggt aaagggttcc tcggagaggt


 


acctccgagg ggtaaacagt tgggtaaaca gtctctgaag tcagctctgc cattttctag


 


ctgtatggcc ctgggcaagt caatttcctt ctctgtgctt tggtttcctc atccatagaa


 


aggtagaaag ggcaaaacac caaactcttg gattacaaga gataatttac agaacaccct


 


tggcacacag agggcaccat gaaatgtcac gggtgacaca gcccccttgt gctcagtccc


 


tggcatctct aggggtgagg agcgtctgcc tagcaggttc ccaccaggaa gctggatttg


 


agtggatggg gcgctggaat cgtgaggggc agaagcaggc aaagggtcgg ggcgaacctc


 


actaacgtgc cagttccaag cacactgtgg gcagccctgg ccctgactca agcctcttgc


 


cttccagttc cggaactgca tgctcaccac catctgctgc ggcaagaacc cactgggtga


 


cgatgaggcc tctgctaccg tgtccaagac ggagacgagc caggtggccc cggcctaaga


 


cctgcctagg actctgtggc cgactatagg cgtctcccat cccctacacc ttcccccagc


 


cacagccatc ccaccaggag cagcgcctgt gcagaatgaa cgaagtcaca taggctcctt conserved region F


 


aatttttttt ttttttttaa gaaataatta atgaggctcc tcactcacct gggacagcct


 


gagaagggac B atccaccaag acctactgat ctggagtccc acgttcccca aggccagcgg


 


gatgtgtgcc cctcctcctc ccaactcatc tttcaggaac acgaggattc ttgctttctg


 


gaaaagtgtc ccagcttagg gataagtgtc tagcacagaa tggggcacac agtaggtgct conserved region G


 


taataaatgc tggatggatg caggaaggaa tggaggaatg aatgggaagg gagaacatat


 


ctatcctctc agaccctcgc agcagcagca actcatactt ggctaatgat atggagcagt


 


tgtttttccc tccctgggcc tcactttctt ctcctataaa atggaaatcc cagatccctg


 


gtcctgccga cacgcagcta ctgagaagac caaaagaggt gtgtgtgtgt ctatgtgtgt


 


gtttcagcac tttgtaaata gcaagaagct gtacagattc tagttaatgt tgtgaataac


 


atcaattaat gtaactagtt aattactatg attatcacct cctgatagtg aacattttga


 


gattgggcat tcagatgatg gggtttcacc caaccttggg gcaggttttt aaaaattagc


 


taggcatcaa ggccagacca gggctggggg ttgggctgta ggcagggaca gtcacaggaa


 


tgcaggatgc agtcatcaga cctgaaaaaa caacactggg ggagggggac ggtgaaggcc


 


aagttcccaa tgagggtgag attgggcctg gggtctcacc cctagtgtgg ggccccaggt


 


cccgtgcctc cccttcccaa tgtggcctat ggagagacag gcctttctct cagcctctgg


 


aagccacctg ctcttttgct ctagcacctg ggtcccagca tctagagcat ggagcctcta


 


gaagccatgc tcacccgccc acatttaatt aacagctgag tccctgatgt catccttact conserved region H


 


cgaagagctt agaaacaaag agtgggaaat tccactgggc ctaccttcct tggggatgtt


 


catgggcccc agtttccagt ttcccttgcc agacaagccc atcttcagca gttgctagtc


 


cattctccat tctggagaat ctgctccaaa aagctggcca catctctgag gtgtcagaat


 


taagctgcct cagtaactgc tcccccttct ccatataagc aaagccagaa gctctagctt


 


tacccagctc tgcctggaga ctaaggcaaa ttgggccatt aaaagctcag ctcctatgtt


 


ggtattaacg gtggtgggtt ttgttgcttt cacactctat ccacaggata gattgaaact conserved region I


 


gccagcttcc acctgatccc tgaccctggg atggctggat tgagcaatga gcagagccaa


 


gcagcacaga gtcccctggg gctagaggtg gaggaggcag tcctgggaat gggaaaaacc


 


ccaactttgg ggtcatagag gcacaggtaa cccataaaac tgcaaacaag ctt





Sequence 1: Mouse rhodopsin promoter sequence (upper case) ending at the Xho I site (highlighted in bold print), followed by the human rhodopsin 5′UTR, human rhodopsin exons and introns and human rhodopsin 3′ region sequence (lower case). Conserved regions A-I are highlighted in bold print. (SEQ ID NO: 90).






Conserved regions A through I and some sequence flanking the regions (5′ and 3′, were combined (Table 11, SEQ ID NO: 92 through SEQ ID NO: 99, Sequence 2). This sequence was analyzed using MatInspector Release Professional 7.4.1 to identify other regions that may be involved in transcriptional and/or translational control of rhodopsin gene expression. (A small portion of the Matinspector results are presented in Table 12). This table illustrates some sequences within conserved regions A through I that are thought to be involved in the transcription and/or translation and/or stability of rhodopsin. Some of these sequences, such as the CRX binding element in conserved region D and the TATA box in region G are known in the art. Others, such as the CRX binding region in E, are not. The complete set of results from MatInspector are presented in Table 13. 302 putative transcription binding sites and/or regulatory sequences were identified and some are highlighted in bold. On the basis of the conserved nature of regions A though I and the important transcription factor binding sites thought to be located within these regions, the constructs in FIG. 9 were generated. Construct BB16 contains conserved regions A, B, C, D, F and G. In addition an artificial CRX-NRL element (below) was inserted between conserved regions A and B. The components of the artificial CRX-NRL enhancer element include the CRX motif from conserved region D, the CRX motif from conserved region E and NRL binding sites are underlined.







(SEQ ID NO: 91)







TTTCTGCAGCGGGGATTAATATGATTATGAACACCCCCAATCTCCCAGA


 


TGCTGATTCAGCCAGGAGGTACC






All these constructs contain transcription binding sites identified within conserved regions A though I.

Sequence 2: Conserved regions A through I in the rhodopsin gene are highlighted in bold below. The nucleotides of these sequences and a small section of 5′ and 3′ sequence surrounding conserved regions have been numbered 1-1600. This sequence was analysed with MatInspector and the nucleotide numbering system of sequence 2 (1-1600) relates to the nucleotide numbering system in Table 13.








TABLE 11







Conserved region A 1-210 (SEQ ID NO: 92)



CACAACTCCT CCGTCGAGTG TCTAATTGCT TATGATCATG


 


CATGCTCTCT CTCCCACTAA ACATTTATTA ATGTGTTAGG


 


ATTTCCATTA GCGCGTGCCT TGAACTGAAA TCATTTGCAT


 


ATGGCTGGGA AAAAGTGGGG TGAGGGAGGA AACAGTGCCA


 


GCTCCCCAAC AGGCGTCAAT CACAGTGACA GATCAGATGG


 


TTTCTGGCTG 210


 


Conserved region B 210-310 (SEQ ID NO: 93)


AAGGGGGGGG GGGGTCTGCT GACCCAGCAA CACTCTTTCC


 


TTCTGAGGCT TAAGAGCTAT TAGCGTAGGT GACTCAGTCC


 


CTAATCCTCC ATTCAATGCC 310


 


Conserved region C 310-410 (SEQ ID NO: 94)


GGGGCTGACC ACCAGGGTCA GAATCAGAAC CTCCACCTTG


 


ACCTCATTAA CGCTGGTCTT AATCACCAAG CCAAGCTCCT


 


TAAACTGCTA GTGGCCAACT 410


 


Conserved region D 410-690 (SEQ ID NO: 95)


aggcttcctc ctagtgtcac cttggcccct cttagaagcc aattaggccc tcagtttctg cagcggggat


 


taatatgatt atgaacaccc ccaatctccc agatgctgat tcagccagga gcttaggagg gggaggtcac


 


tttataaggg tctggggggg tcagaaccca gagtcatcca gctggagccc tgagtggctg agctcaggcc


 


ttcgcagcat tcttgggtgg gagcagccac gggtcagcca caagggccac agccatgaat ggcacagaag


690


 


Conserved region E 690-850 (SEQ ID NO: 96)


tcctgagccc catgtcaaac agaatccaag acatcccaac ccttcacctt ggctgtgccc ctaatcctca


 


actaagctag gcgcaaattc caatcctctt tggtctagta ccccgggggc agccccctct aaccttgggc


 


ctcagcagca ggggaggcca 850


 


Conserved regions F and G 850-1220 (SEQ ID NO: 97)


cccctacacc ttcccccagc cacagccatc ccaccaggag cagcgcctgt gcagaatgaa cgaagtcaca


 


taggctcctt aatttttttt ttttttttaa gaaataatta atgaggctcc tcactcacct gggacagcct gagaagggac


 


atccaccaag acctactgat ctggagtccc acgttcccca aggccagcgg gatgtgtgcc cctcctcctc


 


ccaactcatc tttcaggaac acgaggattc ttgctttctg gaaaagtgtc ccagcttagg gataagtgtc


 


tagcacagaa tggggcacac agtaggtgct taataaatgc tggatggatg caggaaggaa tggaggaatg


 


aatgggaagg 1220


 


Conserved region H 1220 1230-1316 1330 (SEQ ID NO: 98)


tctagagcat ggagcctcta gaagccatgc tcacccgccc acatttaatt aacagctgag tccctgatgt


 


catccttact cgaagagctt agaaacaaag agtgggaaat 1330


 


Conserved region I 1330 1342-1425 1600 (SEQ ID NO: 99)


gctctagctt tacccagctc tgcctggaga ctaaggcaaa ttgggccatt aaaagctcag ctcctatgtt


 


ggtattaacg gtggtgggtt ttgttgcttt cacactctat ccacaggata gattgaaact gccagcttcc


 


acctgatccc tgaccctggg atggctggat tgagcaatga gcagagccaa gcagcacaga gtcccctggg


 


gctagaggtg gaggaggcag tcctgggaat gggaaaaacc ccaactttgg ggtcatagag 1600





(Conserved regions are in bold)













TABLE 12







Conserved sequence motifs in Rhodopsin











Conserved





region
Position
Name






B
288-304
CRX



C
366-382
CRX



D
470-486
CRX



E
784-764
CRX



G
1172-1177
TATA box



D
500-520
Neuron-restrictive silencer factor



E
794-814
Neuron-restrictive silencer factor



E
831-851
Neuron-restrictive silencer factor
















TABLE 13







Putative Rhodopsin Transcription Regulatory Factors























Sequence











(red: ci-value









>60
SEQ



Further



Core
Matrix
capitals: core
ID


Family/matrix
Information
Opt.
Position
Str.
sim.
sim.
sequence)
NO





V$PDX1/ISL1.01
Pancreatic and
0.82
14-34
(+)
1.000
0.860
tcgagtgtcTAA
100




intestinal lim-





Ttgcttatg



homeodomain



factor


 


V$HOMF/MSX.01
Homeodomain
0.97
18-30
(+)
1.000
0.995
gtgtcTAATtgct
101



proteins MSX-1



and MSX-2


 


V$HOXF/GSH2.01
Homeodomain
0.95
19-35
(+)
1.000
0.975
tgtcTAATtgctt
102



transcription





atga



factor Gsh-2


 


V$GABF/GAGA.01
GAGA-Box
0.78
33-57
(−)
1.000
0.825
gtgggAGAGag
103









agcatgcatgatca


 


V$FKHD/FREAC2.01
Fork head related
0.84
52-68
(+)
1.000
0.884
tcccacTAAAc
104



activator-2





atttat



(FOXF2)


 


V$HOXF/HOXC13.01
Homeodomain
0.91
58-74
(−)
1.000
0.914
acattaaTAAAt
105



transcription





gttta



factor HOXC13


 


V$NKXH/HMX2.02
Hmx2/Nkx5-2
0.82
58-72
(−)
0.750
0.835
attaatAAATgtt
106



homeodomain





ta



transcription



factor


 


V$SATB/SATB1.01
Special AT-rich
0.94
58-72
(−)
1.000
0.956
attAATAaatgtt
107



sequence-binding





ta



protein 1,



predominantly



expressed in



thymocytes,



binds to matrix



attachment



regions (MARs)


 


V$BRNF/BRN3.02
Brn-3, POU-IV
0.89
59-77
(−)
1.000
0.892
aacacatTAATa
108



protein class





aatgttt


 


V$PDX1/PDX1.01
Pdx1
0.74
59-79
(−)
1.000
0.744
ctaacacatTAA
109



(IDX1/IPF1)





Taaatgttt



pancreatic and



intestinal



homeodomain TF


 


V$PIT1/PIT1.01
Pit1, GHF-1
0.84
61-73
(+)
1.000
0.857
acatTTATtaatg
110



pituitary specific



pou domain



transcription



factor


 


V$BRNF/BRN3.02
Brn-3, POU-IV
0.89
62-80
(+)
1.000
0.893
catttatTAATgt
111



protein class





gttagg


 


V$LHXF/LMX1B.01
LIM-
0.91
62-76
(−)
1.000
0.946
acacatTAATaa
112



homeodomain





atg



transcription



factor


 


V$HOXH/MEIS1B_HOXA9.01
Meis1b and
0.78
64-78
(−)
0.750
0.823
TAACacattaat
113



Hoxa9 form





aaa



heterodimeric



binding



complexes on



target DNA


 


V$HOXF/HOX1-
Hox-1.3,
0.82
65-81
(+)
1.000
0.826
ttatTAATgtgtt
114


3.01
vertebrate





agga



homeobox



protein


 


V$OCT1/OCT1.04
Octamer-binding
0.80
77-91
(−)
0.846
0.866
ctAATGgaaatc
115



factor 1





cta


 


V$HOXF/PHOX2.01
Phox2a (ARIX)
0.87
78-94
(−)
1.000
0.969
gcgcTAATgga
116



and Phox2b





aatcct


 


V$AHRR/AHRARNT.01
Aryl hydrocarbon
0.92
83-107
(+)
1.000
0.932
ttccattagcgCG
117



receptor/Arnt





TGccttgaactg



heterodimers


 


V$MOKF/MOK2.02
Ribonucleoprotein
0.98
 85-105
(+)
1.000
0.988
ccattagcgcgtg
118



associated zinc





CCTTgaac



finger protein



MOK-2 (human)


 


V$EBOX/MYCMAX.03
MYC-MAX
0.91
 87-101
(−)
1.000
0.918
aaggcaCGCGc
119



binding sites





taat


 


V$HESF/HELT.01
Hey-like bHLH-
0.91
 87-101
(−)
1.000
0.947
aaggCACGcgc
120



transcriptional





taat



repressor


 


V$HOMF/EN1.01
Homeobox
0.77
 97-109
(+)
0.782
0.776
gccTTGAactg
121



protein engrailed





aa



(en-1)


 


V$OCT1/OCT1.02
Octamer-binding
0.85
109-123
(−)
1.000
0.992
catATGCaaatg
122



factor 1





att


 


V$OCTP/OCT1P.01
Octamer-binding
0.86
113-125
(−)
1.000
0.910
gccATATgcaa
123



factor 1, POU-





at



specific domain


 


V$AIRE/AIRE.01
Autoimmune
0.86
119-145
(+)
0.916
0.862
atatggctgggaaa
124



regulator





aagTGGGgtga









gg


 


V$RBPF/RBPJK.02
Mammalian
0.94
122-136
(+)
1.000
0.941
tggcTGGGaaa
125



transcriptional





aagt



repressor RBP-



Jkappa/CBF1


 


V$RXRF/VDR_RXR.06
Bipartite binding
0.75
123-147
(+)
0.812
0.760
ggctgggaaaaag
126



site of





tgGGGTgaggga



VDR/RXR



heterodimers: 4



spacer



nucleotides



between the two



directly repeated



motifs


 


V$NKXH/HMX3.01
H6
0.89
127-141
(+)
1.000
0.910
gggaaaAAGTg
127



homeodomain





gggt



HMX3/Nkx5.1



transcription



factor


 


V$CIZF/NMP4.01
NMP4 (nuclear
0.97
128-138
(+)
1.000
0.998
ggAAAAagtgg
128



matrix protein 4)/



CIZ (Cas-



interacting zinc



finger protein)


 


V$EBOX/SREBP.01
Sterol regulatory
0.90
132-146
(−)
1.000
0.960
cccTCACccca
129



element binding





cttt



protein 1 and 2


 


V$RXRF/VDR_RXR.02
VDR/RXR
0.86
134-158
(+)
1.000
0.878
agtggggtgagg
130



Vitamin D





GAGGaaacagt



receptor RXR





gc



heterodimer site


 


V$ETSF/PU1.01
Pu.1 (Pu120)Ets-
0.89
141-157
(+)
1.000
0.895
tgagggaGGAA
131



like transcription





acagtg



factor identified



in lymphoid B-



cells


 


V$NFAT/NFAT.01
Nuclear factor of
0.95
145-155
(+)
1.000
0.989
ggaGGAAacag
132



activated T-cells


 


V$AREB/AREB6.04
AREB6 (Atp1a1
0.98
146-158
(−)
1.000
0.991
gcactGTTTcctc
133



regulatory



element binding



factor 6)


 


V$COMP/COMP1.01
COMP1,
0.77
163-185
(−)
1.000
0.811
ctgtgATTGacg
134



cooperates with





cctgttgggga



myogenic



proteins in



multicomponent



complex


 


V$PAX6/PAX6.01
Pax-6 paired
0.77
163-181
(−)
0.808
0.781
gaTTGAcgcct
135



domain binding





gttgggga



site


 


V$MYBL/CMYB.01
c-Myb, important
0.90
165-177
(+)
1.000
0.945
ccCAACaggcg
136



in hematopoesis,





tc



cellular



equivalent to



avian



myoblastosis



virus oncogene



v-myb


 


V$CREB/CREB.02
cAMP-
0.89
167-187
(−)
1.000
0.902
cactgtgatTGA
137



responsive





Cgcctgttg



element binding



protein


 


V$WHZF/WHN.01
Winged helix
0.95
169-179
(−)
1.000
0.955
ttgACGCctgt
138



protein, involved



in hair



keratinization



and thymus



epithelium



differentiation


 


V$HOXC/PBX1.01
Homeo domain
0.78
170-186
(−)
1.000
0.840
actgtGATTgac
139



factor Pbx-1





gcctg


 


V$PBXC/PBX1_MEIS1.02
Binding site for a
0.77
170-186
(−)
1.000
0.875
actgTGATtgac
140



Pbx1/Meis1





gcctg



heterodimer


 


V$AP1R/TCF11MAFG.01
TCF11/MafG
0.81
177-201
(+)
1.000
0.838
caatcacagTGA
141



heterodimers,





Cagatcagatggt



binding to



subclass of AP1



sites


 


V$TALE/MEIS1.01
Binding site for
0.95
183-193
(−)
1.000
0.971
atcTGTCactg
142



monomeric



Meis1



homeodomain



protein


 


V$HOXH/MEIS1A_HOXA9.01
Meis1a and
0.77
186-200
(+)
1.000
0.770
TGACagatcag
143



Hoxa9 form





atgg



heterodimeric



binding



complexes on



target DNA


 


V$GATA/GATA3.02
GATA-binding
0.91
187-199
(+)
1.000
0.950
gacAGATcaga
144



factor 3





tg


 


V$AP4R/TAL1BETAE47.01
Tal-1beta/E47
0.87
189-205
(+)
1.000
0.955
cagatCAGAtg
145



heterodimer





gtttct


 


V$NEUR/NEUROG.01
Neurogenin 1
0.92
191-203
(−)
1.000
0.925
aaaCCATctgatc
146



and 3 (ngn1/3)



binding sites


 


V$ZBPF/ZBP89.01
Zinc finger
0.93
205-227
(−)
1.000
0.966
agacccccccCC
147



transcription





CCcttcagcca



factor ZBP-89


 


V$ZBPF/ZNF219.01
Kruppel-like zinc
0.91
207-229
(−)
1.000
0.997
gcagaccCCCC
148



finger protein





ccccccttcagc



219


 


V$INSM/INSM1.01
Zinc finger
0.90
209-221nc
(+)
1.000
0.914
tgaagGGGGgg
149



protein





gg



insulinoma-



associated 1 (IA-



1) functions as a



transcriptional



repressor


 


V$EKLF/KKLF.01
Kidney-enriched
0.91
210-226nc
(+)
1.000
0.934
gaagggGGGG
150



kruppel-like





gggggtc



factor, KLF15


 


V$EGRF/WT1.01
Wilms Tumor
0.92
211-227nc
(+)
0.837
0.945
aagggGGGGg
151



Suppressor





ggggtct


 


V$SP1F/GC.01
GC box elements
0.88
211-225nc
(+)
0.819
0.897
aagggGGGGg
152









ggggt


 


V$EKLF/KKLF.01
Kidney-enriched
0.91
212-228nc
(+)
1.000
0.949
agggggGGGG
153



kruppel-like





gggtctg



factor, KLF15


 


V$SP1F/GC.01
GC box elements
0.88
213-227nc
(+)
0.819
0.908
gggggGGGGg
154









ggtct


 


V$EGRF/WT1.01
Wilms Tumor
0.92
214-230nc
(+)
0.837
0.932
gggggGGGGg
155



Suppressor





gtctgct


 


V$GLIF/ZIC2.01
Zinc finger
0.89
214-228
(−)
1.000
0.967
cagacccCCCC
156



transcription





cccc



factor, Zic family



member 2 (odd-



paired homolog,



Drosophila)


 


V$MAZF/MAZR.01
MYC-associated
0.88
215-227nc
(+)
1.000
0.972
ggggggGGGG
157



zinc finger





tct



protein related



transcription



factor


 


V$AP1R/BACH2.01
Bach2 bound
0.89
221-245
(−)
0.813
0.897
gagtgttgcTGG
158



TRE





Gtcagcagacccc


 


V$AP1R/VMAF.01
v-Maf
0.82
221-245
(+)
1.000
0.957
ggggtctgcTGA
159









Cccagcaacactc


 


V$XBBF/MIF1.01
MIBP-1/RFX1
0.76
225-243
(−)
0.800
0.778
gtgttgctggGTC
160



complex





Agcaga


 


V$XBBF/RFX1.01
X-box binding
0.89
227-245
(+)
1.000
0.907
tgctgacccaGC
161



protein RFX1





AAcactc


 


V$NFAT/NFAT.01
Nuclear factor of
0.95
243-253
(−)
1.000
0.971
gaaGGAAagag
162



activated T-cells


 


V$NKXH/HMX2.01
Hmx2/Nkx5-2
0.83
253-267
(−)
1.000
0.911
gctCTTAagcct
163



homeodomain





cag



transcription



factor


 


V$PAX8/PAX8.01
PAX 2/5/8
0.88
254-266
(−)
0.800
0.901
ctcTTAAgcctca
164



binding site


 


V$NKXH/HMX2.01
Hmx2/Nkx5-2
0.83
256-270
(+)
1.000
0.931
aggCTTAagag
165



homeodomain



transcription



factor


 


V$HOXF/PHOX2.01
Phox2a (ARIX)
0.87
260-276
(−)
1.000
0.898
acgcTAATagc
166



and Phox2b





tcttaa


 


V$CLOX/CDPCR3.01
Cut-like
0.73
266-284
(−)
0.880
0.770
agtcacctacgcta
167



homeodomain





ATAGc



protein


 


V$EGRF/NGFIC.01
Nerve growth
0.80
269-285
(+)
1.000
0.855
attaGCGTaggt
168



factor-induced





gactc



protein C


 


V$AP1R/BACH2.01
Bach2 bound
0.89
271-295
(−)
1.000
0.957
attagggacTGA
169



TRE





Gtcacctacgcta


 


V$CREB/TAXCREB.02
Tax/CREB
0.71
274-294
(+)
1.000
0.744
cgtaggTGACtc
170



complex





agtccctaa


 


V$AP1F/AP1.01
Activator protein 1
0.94
278-288
(+)
0.904
0.967
ggtgACTCagt
171


 


V$AP1F/AP1.03
Activator protein 1
0.94
278-288
(−)
1.000
0.976
acTGAGtcacc
172


 


V$HOXF/CRX.01
Cone-rod B
0.94
288-304
(+)
1.000
0.972
tcccTAATcctc
173



homeobox-





cattc



containing



transcription



factor/otx-like



homeobox gene


 


V$SORY/HBP1.01
HMG box-
0.86
298-310
(−)
1.000
0.905
ggcattgAATG
174



containing





ga



protein 1


 


V$IRFF/IRF7.01
Interferon
0.86
329-347
(+)
0.936
0.865
caGAATcagaa
175



regulatory factor





cctccacc



7 (IRF-7)


 


V$RORA/RORA1.01
RAR-related
0.93
342-360
(−)
1.000
0.953
ttaatgaGGTCa
176



orphan receptor





aggtgga



alpha1


 


V$CSEN/DREAM.01
Downstream
0.95
344-354
(−)
1.000
0.960
agGTCAaggtg
177



regulatory



element-



antagonist



modulator,



Ca2+-binding



protein of the



neuronal



calcium sensors



family that



binds DRE



(downstream



regulatory



element) sites as



a tetramer


 


V$E4FF/E4F.01
GLI-Krueppel-
0.82
345-357
(−)
0.789
0.824
atgAGGTcaag
178



related





gt



transcription



factor, regulator



of adenovirus E4



promoter


 


V$HOXF/BARX2.01
Barx2,
0.95
347-363
(−)
1.000
0.980
gcgtTAATgag
179



homeobox





gtcaag



transcription



factor that



preferentially



binds to paired



TAAT motifs


 


V$MYBL/VMYB.04
v-Myb, AMV v-
0.85
356-368
(+)
1.000
0.881
attAACGctggtc
180



myb


 


V$HOXF/CRX.01
Cone-rod (C)
0.94
366-382
(+)
1.000
0.962
gtctTAATcacc
181



homeobox-





aagcc



containing



transcription



factor/otx-like



homeobox gene


 


V$RCAT/CLTR_CAAT.01
Mammalian C-
0.71
375-399
(+)
1.000
0.718
aCCAAgccaag
182



type LTR





ctccttaaactgct



CCAAT box


 


V$ETSF/ETS1.01
c-Ets-1 binding
0.92
409-425
(−)
1.000
0.921
actaggaGGAA
183



site





gcctag


 


V$SF1F/FTF.01
Alpha (1)-
0.94
426-438
(−)
1.000
0.940
gggcCAAGgtg
184



fetoprotein





ac



transcription



factor (FTF),



liver receptor



homologue-1



(LRH-1)


 


V$BCL6/BCL6.02
POZ/zinc finger
0.77
436-452
(+)
1.000
0.785
cccctctTAGAa
185



protein,





gccaa



transcriptional



repressor,



translocations



observed in



diffuse large cell



lymphoma


 


V$HOXF/GSH2.01
Homeodomain
0.95
443-459
(−)
1.000
1.000
ggccTAATtgg
186



transcription





cttcta



factor Gsh-2


 


V$CAAT/CAAT.01
Cellular and viral
0.90
445-459
(+)
1.000
0.949
gaagCCAAtta
187



CCAAT box





ggcc


 


V$NKXH/NKX25.02
Homeo domain
0.88
446-460
(−)
1.000
0.938
gggccTAATtg
188



factor Nkx-





gctt



2.5/Csx, tinman



homolog low



affinity sites


 


V$HOMF/S8.01
Binding site for
0.97
448-460
(−)
1.000
0.999
gggccTAATtg
189



S8 type





gc



homeodomains


 


V$HOXF/CRX.01
Cone-rod (D)
0.94
470-486
(−)
1.000
0.985
atatTAATcccc
190



homeobox-





gctgc



containing



transcription



factor/otx-like



homeobox gene


 


V$MZF1/MZF1.01
Myeloid zinc
0.99
473-481
(+)
1.000
0.991
gcGGGGatt
191



finger protein



MZF1


 


V$OCTB/TST1.01
POU-factor Tst-
0.90
475-487
(+)
1.000
0.947
ggggATTAatatg
192



1/Oct-6


 


V$CART/CART1.01
Cart-1 (cartilage
0.86
477-493
(−)
1.000
0.926
caTAATcatatt
193



homeoprotein 1)





aatcc


 


V$CART/CART1.01
Cart-1 (cartilage
0.86
479-495
(+)
1.000
0.914
atTAATatgatta
194



homeoprotein 1)





tgaa


 


V$SATB/SATB1.01
Special AT-rich
0.94
479-493
(+)
1.000
0.957
attAATAtgatta
195



sequence-binding





tg



protein 1,



predominantly



expressed in



thymocytes,



binds to matrix



attachment



regions (MARs)


 


V$PDX1/PDX1.01
Pdx1
0.74
480-500
(+)
0.826
0.775
ttaatatgaTTAT
196



(IDX1/IPF1)





gaacaccc



pancreatic and



intestinal



homeodomain TF


 


V$GLIF/ZIC2.01
Zinc finger
0.89
491-505
(+)
1.000
0.932
atgaacaCCCCc
197



transcription





aat



factor, Zic family



member 2 (odd-



paired homolog,



Drosophila)


 


V$CAAT/ACAAT.01
Avian C-type
0.83
497-511
(+)
1.000
0.905
acccCCAAtctc
198



LTR CCAAT





cca



box


 


V$RREB/RREB1.01
Ras-responsive
0.80
499-513
(+)
1.000
0.841
cCCCAatctccc
199



element binding





aga



protein 1


 


V$NRSF/NRSF.01
Neuron-
0.69
500-520
(−)
1.000
0.696
atcAGCAtctgg
200



restrictive





gagattggg



silencer factor


 


V$IKRS/LYF1.01
LyF-1 (Ikaros 1),
0.98
502-514
(−)
1.000
1.000
atcTGGGagattg
201



enriched in B and



T lymphocytes


 


V$AP4R/TAL1ALPHAE47.01
Tal-1alpha/E47
0.87
505-521
(+)
1.000
0.905
tctccCAGAtgc
202



heterodimer





tgatt


 


V$RP58/RP58.01
Zinc finger
0.84
507-519
(−)
1.000
0.865
tcagCATCtggga
203



protein RP58



(ZNF238),



associated



preferentially



with



heterochromatin


 


V$AP1R/NFE2.01
NF-E2 p45
0.85
508-532
(+)
1.000
0.904
cccagatgCTG
204









Attcagccaggagc


 


V$AP1R/NFE2.01
NF-E2 p45
0.85
508-532
(−)
1.000
0.882
gctcctggCTGA
205









atcagcatctggg


 


V$BEL1/BEL1.01
Bel-1 similar
0.81
510-532
(−)
1.000
0.818
gctcctggctgaaT
206



region (defined





CAGcatctg



in Lentivirus



LTRs)


 


V$NRLF/NRL.01
Neural retinal
0.85
511-529
(−)
1.000
0.991
cctggCTGAatc
207



basic leucine





agcatct



zipper factor



(bZIP)


 


V$AP1F/AP1.03
Activator protein 1
0.94
515-525
(+)
0.885
0.970
gcTGATtcagc
208


 


V$AP1F/AP1.03
Activator protein 1
0.94
515-525
(−)
0.857
0.963
gcTGAAtcagc
209


 


V$HOXF/PTX1.01
Pituitary
0.94
523-539
(−)
1.000
0.944
ctcCTAAgctcc
210



Homeobox 1





tggct



(Ptx1, Pitx-1)


 


V$ZBPF/ZNF219.01
Kruppel-like zinc
0.91
528-550
(−)
1.000
0.926
gtgacctCCCCc
211



finger protein





tcctaagctcc



219


 


V$RXRF/VDR_RXR.01
VDR/RXR
0.85
531-555
(+)
1.000
0.889
gcttaggaggggG
212



Vitamin D





AGGtcactttat



receptor RXR



heterodimer site


 


V$ZBPF/ZBP89.01
Zinc finger
0.93
531-553
(−)
1.000
0.958
aaagtgacctCC
213



transcription





CCctcctaagc



factor ZBP-89


 


V$EKLF/KKLF.01
Kidney-enriched
0.91
534-550
(+)
1.000
0.913
taggagGGGGa
214



kruppel-like





ggtcac



factor, KLF15


 


V$GLIF/ZIC2.01
Zinc finger
0.89
536-550
(−)
1.000
0.945
gtgacctCCCCc
215



transcription





tcc



factor, Zic family



member 2 (odd-



paired homolog,



Drosophila)


 


V$RORA/TR2.01
Nuclear hormone
0.92
538-556
(+)
1.000
0.950
agggggaGGTC
216



receptor TR2,





actttata



half site


 


V$TBPF/TATA.01
Cellular and viral
0.90
543-559
(−)
1.000
0.915
ccttaTAAAgtg
217



TATA box





acctc



elements


 


V$SRFF/SRF.01
Serum response
0.66
545-563
(−)
1.000
0.722
agaccctTATAa
218



factor





agtgacc


 


V$SRFF/SRF.01
Serum response
0.66
546-564
(+)
1.000
0.712
gtcacttTATAa
219



factor





gggtctg


 


V$TBPF/LTATA.01
Lentivirus LTR
0.82
550-566
(+)
1.000
0.829
cttTATAagggt
220



TATA box





ctggg


 


V$MOKF/MOK2.01
Ribonucleoprotein
0.74
552-572
(−)
1.000
0.772
gacccccccagac
221



associated zinc





CCTTataa



finger protein



MOK-2 (mouse)


 


V$ZBPF/ZNF219.01
Kruppel-like zinc
0.91
553-575
(−)
1.000
0.948
tctgaccCCCCc
222



finger protein





agacccttata



219


 


V$GLIF/ZIC2.01
Zinc finger
0.89
560-574
(−)
1.000
0.967
ctgacccCCCCa
223



transcription





gac



factor, Zic family



member 2 (odd-



paired homolog,



Drosophila)


 


V$MAZF/MAZR.01
MYC-associated
0.88
561-573
(+)
1.000
0.919
tctgggGGGGtca
224



zinc finger



protein related



transcription



factor


 


V$ZNFP/SZF1.01
SZF1,
0.82
579-603
(−)
0.801
0.829
tcaGGGCtccag
225



hematopoietic





ctggatgactctg



progenitor-



restricted KRAB-



zinc finger



protein


 


V$AP4R/AP4.01
Activator protein 4
0.85
584-600UTR
(+)
1.000
0.916
tcatcCAGCtgg
226









agccc


 


V$EBOX/ATF6.01
Member of b-zip
0.93
596-610
(−)
1.000
0.970
cagCCACtcag
227



family, induced





ggct



by ER



damage/stress,



binds to the



ERSE in



association with



NF-Y


 


V$CAAT/CAAT.01
Cellular and viral
0.90
597-611
(−)
0.826
0.937
tcagCCACtcag
228



CCAAT box





ggc


 


V$HEAT/HSF1.01
Heat shock factor 1
0.84
621-645
(−)
1.000
0.857
tgctcccacccaA
229









GAAtgctgcgaa


 


V$OAZF/ROAZ.01
Rat C2H2 Zn
0.73
625-641
(+)
0.750
0.779
caGCATtcttgg
230



finger protein





gtggg



involved in



olfactory



neuronal



differentiation


 


V$OAZF/ROAZ.01
Rat C2H2 Zn
0.73
626-642nc
(−)
0.750
0.744
tcCCACccaag
231



finger protein





aatgct



involved in



olfactory



neuronal



differentiation


 


V$EGRF/EGR2.01
Egr-2/Krox-20
0.79
631-647UTR
(+)
0.766
0.828
tcttGGGTggga
232



early growth





gcagc



response gene



product


 


V$EGRF/WT1.01
Wilms Tumor
0.92
633-649
(+)
1.000
0.930
ttgggTGGGag
233



Suppressor





cagcca


 


V$RBPF/RBPJK.01
Mammalian
0.84
634-648UTR
(+)
1.000
0.847
tgggTGGGagc
234



transcriptional





agcc



repressor RBP-



Jkappa/CBF1


 


V$OAZF/ROAZ.01
Rat C2H2 Zn
0.73
641-657
(+)
0.750
0.818
gaGCAGccacg
235



finger protein





ggtcag



involved in



olfactory



neuronal



differentiation


 


V$EBOX/USF.03
Upstream
0.89
643-657
(−)
1.000
0.904
ctgaccCGTGg
236



stimulating factor





ctgc


 


V$EBOX/MYCMAX.03
MYC-MAX
0.91
644-658
(+)
0.842
0.919
cagccaCGGGt
237



binding sites





cagc


 


V$PAX5/PAX5.03
PAX5 paired
0.80
659-687gen
(+)
0.894
0.833
cacaagggCCA
238



domain protein





Cagccatgaatgg









cacag


 


V$CLOX/CDPCR3.01
Cut-like
0.73
665-683gen
(+)
1.000
0.735
ggccacagccatg
239



homeodomain





aATGGc



protein


 


V$PAX5/PAX5.03
PAX5 paired
0.80
674-702gen
(+)
1.000
0.800
catgaatgGCAC
240



domain protein





agaagtcctgagcc









cca


 


V$ZNFP/ZBRK1.01
Transcription
0.77
680-704
(−)
0.813
0.847
catggggcTCA
241



factor with 8





Ggacttctgtgcca



central zinc



fingers and an N-



terminal KRAB



domain


 


V$PBXC/PBX1_MEIS1.02
Binding site for a
0.77
699-715
(−)
0.750
0.860
attcTGTTtgaca
242



Pbx1/Meis1





tggg



heterodimer


 


V$TALE/TGIF.01
TG-interacting
1.00
700-710nc
(+)
1.000
1.000
ccatGTCAaac
243



factor belonging



to TALE class of



homeodomain



factors


 


V$SNAP/PSE.02
Proximal
0.73
745-763
(+)
1.000
0.734
gtgccCCTAatc
244



sequence element





ctcaact



(PSE) of RNA



polymerase III-



transcribed genes


 


V$HOXF/CRX.01
Cone-rod (E)
0.94
748-764
(+)
1.000
0.965
ccccTAATcctc
245



homeobox-





aacta



containing



transcription



factor/otx-like



homeobox gene


 


V$FAST/FAST1.01
FAST-1 SMAD
0.81
749-763
(−)
0.983
0.829
agttgagGATTa
246



interacting





ggg



protein


 


V$NR2F/HPF1.01
HepG2-specific
0.78
767-787
(+)
0.750
0.801
ctaggcgcAAA
247



P450 2C factor-1





Ttccaatcct


 


V$SORY/HMGIY.01
HMGI(Y) high-
0.92
770-782
(−)
1.000
0.938
tggAATTtgcgcc
248



mobility-group



protein I (Y),



architectural



transcription



factor organizing



the framework of



a nuclear protein-



DNA



transcriptional



complex


 


V$HMTB/MTBF.01
Muscle-specific
0.90
774-782
(−)
1.000
0.953
tggaATTTg
249



Mt binding site


 


V$LEFF/LEF1.01
TCF/LEF-1,
0.86
783-799
(−)
1.000
0.889
actagacCAAA
250



involved in the





gaggat



Wnt signal



transduction



pathway


 


V$NRSE/NRSF.01
Neural-
0.67
794-814nc
(+)
0.782
0.762
tctagtacccCGG
251



restrictive-





Gggcagcc



silencer-element


 


V$ZBPF/ZF9.01
Core promoter-
0.87
803-825nc
(+)
0.769
0.878
ccgggggCAG
252



binding protein





Ccccctctaacct



(CPBP) with 3



Krueppel-type



zinc fingers


 


V$HICF/HIC1.01
Hypermethylated
0.93
804-816
(−)
1.000
0.970
ggggcTGCCcc
253



in cancer 1,





cg



transcriptional



repressor



containing five



Kruppel-like



C2H2 zinc



fingers, for



optimal binding



multiple binding



sites are required.


 


V$NFKB/NFKAPPAB50.01
NF-kappaB (p50)
0.83
806-818
(−)
0.750
0.865
aggGGGCtgcc
254









cc


 


V$STAF/ZNF76_143.01
ZNF143 is the
0.76
810-832
(+)
0.809
0.761
cagcCCCCtcta
255



human ortholog





accttgggcct



of Xenopus Staf,



ZNF76 is a DNA



binding protein



related to



ZNF143 and Staf


 


V$SF1F/SF1.01
SF1
0.95
819-831
(−)
1.000
0.966
ggccCAAGgtt
256



steroidogenic





ag



factor 1


 


V$RXRF/VDR_RXR.06
Bipartite binding
0.75
825-849nc
(+)
0.812
0.787
ttgggcctcagcag
257



site of





cAGGGgaggcc



VDR/RXR



heterodimers: 4



spacer



nucleotides



between the two



directly repeated



motifs


 


V$MYOD/MYF5.01
Myf5 myogenic
0.90
831-847nc
(+)
1.000
0.903
ctcagCAGCag
258



bHLH protein





gggagg


 


V$NRSF/NRSF.01
Neuron-
0.69
831-851nc?
(+)
1.000
0.705
ctcAGCAgcag
259



restrictive





gggaggccac



silencer factor


 


V$ZBPF/ZF9.01
Core promoter-
0.87
832-854
(−)
0.820
0.890
ggggtggCCTC
260



binding protein





ccctgctgctga



(CPBP) with 3



Krueppel-type



zinc fingers


 


V$ZBPF/ZF9.01
Core promoter-
0.87
841-863nc
(+)
0.923
0.937
ggggaggCCA
261



binding protein





Cccctacaccttc



(CPBP) with 3



Krueppel-type



zinc fingers


 


V$PLAG/PLAG1.01
Pleomorphic
0.88
847-867
(−)
0.958
0.929
GGGGgaaggtg
262



adenoma gene





taggggtggc



(PLAG) 1, a



developmentally



regulated C2H2



zinc finger



protein


 


V$ZBPF/ZNF202.01
Transcriptional
0.73
859-881
(+)
1.000
0.776
ccttccCCCAgc
263



repressor, binds





cacagccatcc



to elements



found



predominantly in



genes that



participate in



lipid metabolism


 


V$INSM/INSM1.01
Zinc finger
0.90
860-872
(−)
1.000
0.965
tggctGGGGga
264



protein





ag



insulinoma-



associated 1 (IA-



1) functions as a



transcriptional



repressor


 


V$MZF1/MZF1.02
Myeloid zinc
0.99
860-868
(−)
1.000
0.994
tgGGGGaag
265



finger protein



MZF1


 


V$HAML/AML3.01
Runt-related
0.84
863-877
(−)
1.000
0.845
ggctGTGGctg
266



transcription





gggg



factor 2/CBFA1



(core-binding



factor, runt



domain, alpha



subunit 1)


 


V$NRF1/NRF1.01
Nuclear
0.78
889-905nc
(−)
0.750
0.828
tctGCACaggc
267



respiratory factor





gctgct



1 (NRF1), bZIP



transcription



factor that acts on



nuclear genes



encoding



mitochondrial



proteins


 


V$NRF1/NRF1.01
Nuclear
0.78
890-906nc
(+)
1.000
0.801
gcaGCGCctgt
268



respiratory factor





gcagaa



1 (NRF1), bZIP



transcription



factor that acts on



nuclear genes



encoding



mitochondrial



proteins


 


V$SORY/HBP1.01
HMG box-
0.86
898-910nc
(+)
1.000
0.862
tgtgcagAATG
269



containing





aa



protein 1


 


V$BRNF/BRN2.03
Brn-2, POU-III
0.92
923-941
(+)
1.000
0.932
ggctccttaATT
270



protein class





Ttttttt


 


V$NKXH/NKX25.02
Homeo domain
0.88
925-939
(+)
1.000
0.956
ctcctTAATtttttt
271



factor Nkx-



2.5/Csx, tinman



homolog low



affinity sites


 


V$CDXF/CDX1.01
Intestine specific
0.94
939-957
(+)
1.000
0.948
tttttttTTTAaga
272



homeodomain





aataa



factor CDX-1


 


V$HOXF/HOXB9.01
Abd-B-like
0.88
940-956
(−)
1.000
0.888
tatttctTAAAaa
273



homeodomain





aaaa



protein Hoxb-9


 


V$CEBP/CEBPB.01
CCAAT/enhancer
0.94
943-957nc
(+)
1.000
0.942
ttttttaaGAAAt
274



binding protein





aa



beta


 


V$HNF1/HNF1.01
Hepatic nuclear
0.80
947-963
(−)
0.790
0.824
cATTAattatttct
275



factor 1





taa


 


V$HOXF/BARX2.01
Barx2,
0.95
948-964
(−)
1.000
0.967
tcatTAATtatttc
276



homeobox





tta



transcription



factor that



preferentially



binds to paired



TAAT motifs


 


V$BRNF/BRN3.01
Brn-3, POU-IV
0.78
949-967
(−)
1.000
0.990
gcctcattaATT
277



protein class





Atttctt


 


V$BRNF/BRN4.01
POU domain
0.89
949-967nc
(+)
1.000
0.894
aagaaataatTA
278



transcription





ATgaggc



factor brain 4


 


V$LHXF/LMX1B.01
LIM-
0.91
949-963nc
(+)
1.000
0.962
aagaaaTAATta
279



homeodomain





atg



transcription



factor


 


V$HOMF/S8.01
Binding site for
0.97
950-962nc
(+)
1.000
0.997
agaaaTAATtaat
280



S8 type



homeodomains


 


V$HOXF/GSH1.01
Homeobox
0.85
952-968nc
(+)
1.000
0.863
aaataatTAATg
281



transcription





aggct



factor Gsh-1


 


V$LHXF/LMX1B.01
LIM-
0.91
952-966
(−)
1.000
0.946
cctcatTAATtat
282



homeodomain





tt



transcription



factor


 


V$RBIT/BRIGHT.01
Bright, B cell
0.92
952-964nc
(+)
1.000
0.961
aaataATTAatga
283



regulator of IgH



transcription


 


V$HOMF/S8.01
Binding site for
0.97
953-965
(−)
1.000
0.992
ctcatTAATtatt
284



S8 type



homeodomains


 


V$LHXF/LHX3.01
Homeodomain
0.81
953-967nc
(+)
1.000
0.851
aataaTTAAtga
285



binding site in





ggc



LIM/Homeodomain



factor LHX3


 


V$SORY/HBP1.01
HMG box-
0.86
953-965nc
(+)
1.000
0.876
aataattAATGag
286



containing



protein 1


 


V$HOXF/BARX2.01
Barx2,
0.95
955-971nc
(+)
1.000
0.987
taatTAATgagg
287



homeobox





ctcct



transcription



factor that



preferentially



binds to paired



TAAT motifs


 


V$RXRF/VDR_RXR.02
VDR/RXR
0.86
960-984
(−)
1.000
0.871
tcccaggtgagtG
288



Vitamin D





AGGagcctcatt



receptor RXR



heterodimer site


 


V$AP1F/AP1.03
Activator protein 1
0.94
969-979
(−)
1.000
0.940
ggTGAGtgagg
289


 


V$AREB/AREB6.01
AREB6 (Atp1a1
0.93
972-984nc
(+)
1.000
0.933
cactcACCTgg
290



regulatory





ga



element binding



factor 6)


 


V$PAX6/PAX6.02
PAX6 paired
0.89
973-991
(−)
1.000
0.893
caggctgtcCCA
291



domain and





Ggtgagt



homeodomain



are required for



binding to this



site


 


V$AP2F/AP2.01
Activator protein 2
0.90
1033-1047
(−)
1.000
0.911
ctgGCCTtggg
292









gaac


 


V$EREF/ERR.01
Estrogen related
0.87
1033-1051nc
(+)
1.000
0.897
gttccccAAGG
293



receptor





ccagcggg


 


V$MZF1/MZF1.02
Myeloid zinc
0.99
1033-1041
(−)
1.000
0.994
ttGGGGaac
294



finger protein



MZF1


 


V$SF1F/SF1.01
SF1
0.95
1035-1047nc
(+)
1.000
0.992
tcccCAAGgcc
295



steroidogenic





ag



factor 1


 


V$TEAF/TEF.01
Thyrotrophic
0.88
1044-1060
(−)
0.968
0.894
ggcacaCATCc
296



embryonic factor





cgctgg


 


V$SP1F/TIEG.01
TGFbeta-
0.83
1046-1060nc
(+)
0.750
0.878
agcGGGAtgtgt
297



inducible early





gcc



gene (TIEG)/



Early growth



response gene



alpha



(EGRalpha)


 


V$MAZF/MAZ.01
Myc associated
0.90
1056-1068
(−)
1.000
0.909
ggagGAGGgg
298



zinc finger





cac



protein (MAZ)


 


V$RXRF/VDR_RXR.03
Bipartite binding
0.74
1056-1080
(−)
0.823
0.750
gatgAGTTggg
299



site of





aggaggaggggc



VDR/RXR





ac



heterodimers



without a spacer



between directly



repeated motifs


 


V$EVI1/MEL1.02
MEL1
0.99
1071-1087
(−)
1.000
0.997
cctgaaaGATG
300



(MDS1/EVI1-





agttgg



like gene 1)



DNA-binding



domain 2


 


V$HEAT/HSF1.01
Heat shock factor 1
0.84
1073-1097
(−)
0.857
0.849
tcctcgtgttccTG
301









AAagatgagtt


 


V$MYT1/MYT1L.01
Myelin
0.92
1073-1085
(−)
0.818
0.927
tgaaAGATgag
302



transcription





tt



factor 1-like,



neuronal C2HC



zinc finger



factor 1


 


V$STAT/STAT1.01
Signal transducer
0.77
1075-1093
(−)
0.767
0.774
cgtgttcctGAA
303



and activator of





Agatgag



transcription 1


 


V$STAT/STAT.01
Signal
0.87
1077-1095
(+)
1.000
0.911
catctttcaGGA
304



transducers and





Acacgag



activators of



transcription


 


V$EBOX/NMYC.01
N-Myc
0.92
1085-1099
(−)
1.000
0.923
aatcctCGTGttc
305









ct


 


V$HEAT/HSF2.02
Heat shock factor 2
0.95
1089-1113
(−)
1.000
0.967
ttccagaaagcaA
306









GAAtcctcgtgt


 


V$HEAT/HSF1.01
Heat shock factor 1
0.84
1097-1121
(−)
1.000
0.874
ggacacttttccA
307









GAAagcaagaat


 


V$STAT/STAT1.01
Signal transducer
0.77
1099-1117
(−)
0.767
0.798
acttttccaGAA
308



and activator of





Agcaaga



transcription 1


 


V$STAT/STAT.01
Signal
0.87
1101-1119
(+)
1.000
0.895
ttgctttctGGAA
309



transducers and





aagtgt



activators of



transcription


 


V$BCL6/BCL6.02
POZ/zinc finger
0.77
1102-1118
(+)
0.800
0.808
tgctttcTGGAa
310



protein,





aagtg



transcriptional



repressor,



translocations



observed in



diffuse large cell



lymphoma


 


V$BNCF/BNC.01
Basonuclin,
0.85
1107-1125
(+)
1.000
0.852
tctggaaaagTG
311



cooperates with





TCccagc



USF1 in rDNA



PolI



transcription)


 


V$GATA/GATA2.01
GATA-binding
0.92
1127-1139
(+)
1.000
0.938
taggGATAagt
312



factor 2





gt


 


V$NKXH/NKX32.01
Homeodomain
0.96
1128-1142
(+)
1.000
0.962
agggataAGTG
313



protein NKX3.2





tcta



(BAPX1,



NKX3B,



Bagpipe



homolog)


 


V$PAX1/PAX1.01
Pax1 paired
0.62
1135-1153
(−)
0.750
0.696
cCATTctgtgct
314



domain protein,





agacact



expressed in the



developing



vertebral column



of mouse



embryos


 


V$SORY/HBP1.01
HMG box-
0.86
1142-1154nc
(+)
1.000
0.860
agcacagAATG
315



containing





gg



protein 1


 


V$NKXH/NKX25.02
Homeo domain
0.88
1166-1180
(+)
1.000
0.898
gtgctTAATaaa
316



factor Nkx-





tgc



2.5/Csx, tinman



homolog low



affinity sites


 


V$HOXF/HOXC13.01
Homeodomain
0.91
1167-1183
(+)
1.000
0.944
tgcttaaTAAAt
317



transcription





gctgg



factor HOXC13


 


V$HOXC/HOX_PBX0.1
HOX/PBX
0.81
1178-1194
(+)
0.944
0.862
tgctGGATggat
318



binding sites





gcagg


 


V$AIRE/AIRE.01
Autoimmune
0.86
1184-1210
(+)
1.000
0.877
atggatgcaggaa
319



regulator





ggaaTGGAgga









atg


 


V$ETSF/ELF2.01
Ets - family
0.90
1186-1202
(+)
1.000
0.933
ggatgcaGGAA
320



member ELF-2





ggaatg



(NERF1a)


 


V$GKLF/GKLF.01
Gut-enriched
0.86
1191-1203
(+)
0.779
0.864
caggaaggaAT
321



Krueppel-like





GG



factor


 


V$SORY/HBP1.01
HMG box-
0.86
1192-1204
(+)
1.000
0.904
aggaaggAATG
322



containing





ga



protein 1


 


V$TEAF/TEF1.01
TEF-1 related
0.84
1192-1208
(−)
1.000
0.859
ttcctcCATTcct
323



muscle factor





tcct


 


V$ETSF/PU1.01
Pu.1 (Pu120) Ets-
0.89
1198-1214
(+)
1.000
0.899
gaatggaGGAA
324



like transcription





tgaatg



factor identified



in lymphoid B-



cells


 


V$SORY/HBPI.01
HMG box-
0.86
1200-1212
(+)
1.000
0.916
atggaggAATG
325



containing





aa



protein 1


 


V$TEAF/TEF1.01
TEF-1 related
0.84
1200-1216
(−)
1.000
0.884
cccattCATTcct
326



muscle factor





ccat


 


V$SORY/HBP1.01
HMG box-
0.86
1204-1216
(+)
1.000
0.949
aggaatgAATG
327



containing





gg



protein 1


 


V$IRFF/IRF7.01
Interferon
0.86
1208-1226nc
(+)
0.936
0.885
atGAATgggaa
328



regulatory factor





ggtctaga



7 (IRF-7)


 


V$RBPF/RBPJK.02
Mammalian
0.94
1209-1223nc
(+)
1.000
0.942
tgaaTGGGaag
329



transcriptional





gtct



repressor RBP-



Jkappa/CBF1


 


V$IKRS/IK1.01
Ikaros 1,
0.92
1210-1222nc
(+)
1.000
0.925
gaatGGGAagg
330



potential





tc



regulator of



lymphocyte



differentiation


 


V$RORA/NBRE.01
Monomers of the
0.89
1212-1230nc
(+)
1.000
0.947
atgggAAGGtct
331



nur subfamily of





agagcat



nuclear receptors



(nur77, nurr1,



nor-1)


 


V$ZFIA/ZID.01
Zinc finger with
0.85
1225-1237
(−)
1.000
0.916
agGCTCcatgctc
332



interaction



domain


 


V$AIRE/AIRE.01
Autoimmune
0.86
1238-1264
(−)
0.916
0.863
atgtgggcgggtg
333



regulator





agcaTGGCttct









ag


 


V$EGRF/WT1.01
Wilms Tumor
0.92
1246-1262
(−)
0.953
0.930
gtgggCGGGtg
334



Suppressor





agcatg


 


V$SP1F/SP1.01
Stimulating
0.88
1250-1264
(−)
1.000
0.907
atgtGGGCgggt
335



protein 1,





gag



ubiquitous zinc



finger



transcription



factor


 


V$NKXH/HMX3.02
Hmx3/Nkx5-1
0.92
1258-1272
(−)
1.000
0.933
ttaaTTAAatgtg
336



homeo domain





gg



transcription



factor


 


V$CREB/E4BP4.01
E4BP4, bZIP
0.80
1259-1279
(+)
0.758
0.801
ccacatttaaTTA
337



domain,





Acagctga



transcriptional



repressor


 


V$BRNF/BRN3.02
Brn-3, POU-IV
0.89
1260-1278
(−)
1.000
0.940
cagctgtTAATt
338



protein class





aaatgtg


 


V$LHXF/LHX3.01
Homeodomain
0.81
1260-1274
(+)
1.000
0.944
cacatTTAAtta
339



binding site in





aca



LIM/Homeodomain



factor LHX3


 


V$OCT1/OCT1.05
Octamer-binding
0.89
1260-1274
(+)
0.900
0.942
caCATTtaattaa
340



factor 1





ca


 


V$HOMF/S8.01
Binding site for
0.97
1261-1273
(+)
1.000
0.997
acattTAATtaac
341



S8 type



homeodomains


 


V$HOXF/PHOX2.01
Phox2a (ARIX)
0.87
1262-1278
(+)
1.000
0.877
cattTAATtaac
342



and Phox2b





agctg


 


V$NKXH/NKX25.02
Homeo domain
0.88
1262-1276
(−)
1.000
0.898
gctgtTAATtaa
343



factor Nkx-





atg



2.5/Csx, tinman



homolog low



affinity sites


 


V$PBXC/PBX1_MEIS1.02
Binding site for a
0.77
1262-1278
(+)
0.750
0.781
cattTAATtaac
344



Pbx1/Meis1





agctg



heterodimer


 


V$RBIT/BRIGHT.01
Bright, B cell
0.92
1262-1274
(−)
1.000
0.967
tgttaATTAaatg
345



regulator of IgH



transcription


 


V$FAST/FAST1.01
FAST-1 SMAD
0.81
1263-1277
(−)
0.850
0.845
agctgttAATTa
346



interacting





aat



protein


 


V$LHXF/LHX3.01
Homeodomain
0.81
1263-1277
(−)
1.000
0.870
agctgTTAAtta
347



binding site in





aat



LIM/Homeodomain



factor LHX3


 


V$RBIT/BRIGHT.01
Bright, B cell
0.92
1263-1275
(+)
1.000
0.941
atttaATTAacag
348



regulator of IgH



transcription


 


V$ZNFP/SZF1.01
SZF1,
0.82
1263-1287
(−)
0.875
0.866
tcaGGGActca
349



hematopoietic





gctgttaattaaat



progenitor-



restricted KRAB-



zinc finger



protein


 


V$ATBF/ATBF1.01
AT-binding
0.79
1264-1280
(−)
1.000
0.812
ctcagctgttAAT
350



transcription





Taaa



factor 1


 


V$HOMF/S8.01
Binding site for
0.97
1264-1276
(−)
1.000
0.997
gctgtTAATtaaa
351



S8 type



homeodomains


 


V$HEN1/HEN1.02
HEN1
0.81
1265-1285
(−)
1.000
0.845
agggactcaGCT
352









Gttaattaa


 


V$NKXH/HMX3.02
Hmx3/Nkx5-1
0.92
1265-1279
(+)
1.000
0.927
ttaaTTAAcagc
353



homeodomain





tga



transcription



factor


 


V$AP4R/AP4.02
Activator protein 4
0.92
1267-1283
(−)
1.000
0.950
ggactcAGCTgt
354









taatt


 


V$AP1R/NFE2.01
NF-E2 p45
0.85
1268-1292
(+)
1.000
0.865
attaacagCTGA
355









gtccctgatgtca


 


V$BEL1/BEL1.01
Bel-1 similar
0.81
1270-1292-
(−)
1.000
0.842
tgacatcagggac
356



region (defined





TCAGctgtta



in Lentivirus



LTRs)


 


V$CREB/CREBP1.01
cAMP-
0.85
1278-1298
(−)
1.000
0.851
taaggaTGACat
357



responsive





cagggactc



element binding



protein 1


 


V$CREB/ATF2.01
Activating
0.87
1279-1299
(+)
0.814
0.871
agtcccTGATgt
358



transcription





catccttac



factor 2


 


V$E4FF/E4F.01
GLI-Krueppel-
0.82
1284-1296
(+)
0.842
0.824
ctgATGTcatcct
359



related



transcription



factor, regulator



of adenovirus E4



promoter


 


V$HOXF/PTX1.01
Pituitary
0.94
1299-1315
(−)
1.000
0.949
tttCTAAgctctt
360



Homeobox 1





cgag



(Ptx1, Pitx-1)


 


V$TBPF/ATATA.01
Avian C-type
0.78
1302-1318
(−)
1.000
0.781
ttgtttcTAAGct
361



LTR TATA box





cttc


 


V$XBBF/RFX1.01
X-box binding
0.89
1302-1320
(+)
0.881
0.890
gaagagcttaGA
362



protein RFX1





AAcaaag


 


V$LEFF/LEF1.01
TCF/LEF-1,
0.86
1309-1325nc
(+)
1.000
0.884
ttagaaaCAAA
363



involved in the





gagtgg



Wnt signal



transduction



pathway


 


V$RBPF/RBPJK.02
Mammalian
0.94
1319-1333
(+)
1.000
0.977
agagTGGGaaa
364



transcriptional

nc



tgct



repressor RBP-



Jkappa/CBF1


 


V$CP2F/CP2.01
CP2
0.90
1331-1349
(−)
1.000
0.932
agCTGGgtaaa
365









gctagagc


 


V$SRFF/SRF.02
Serum response
0.84
1362-1380
(+)
0.888
0.842
taaggCAAAttg
366



factor





ggccatt


 


V$CART/XVENT2.01
Xenopus
0.82
1366-1382
(+)
0.750
0.882
gcAAATtgggc
367



homeodomain





cattaa



factor Xvent-2;



early BMP



signaling



response


 


V$CART/XVENT2.01
Xenopus
0.82
1367-1383
(−)
1.000
0.835
ttTAATggccca
368



homeodomain





atttg



factor Xvent-2;



early BMP



signaling



response


 


V$PDX1/ISL1.01
Pancreatic and
0.82
1370-1390
(−)
1.000
0.875
ctgagctttTAAT
369



intestinal lim-





ggcccaat



homeodomain



factor


 


V$NKXH/HMX3.02
Hmx3/Nkx5-1
0.92
1372-1386
(−)
1.000
0.946
gcttTTAAtggc
370



homeodomain





cca



transcription



factor


 


V$HOXF/HOXC13.01
Homeodomain
0.91
1373-1389
(+)
1.000
0.932
gggccatTAAA
371



transcription





agctca



factor HOXC13


 


V$NKXH/HMX3.02
Hmx3/Nkx5-1
0.92
1375-1389
(+)
1.000
0.953
gccaTTAAaag
372



homeodomain





ctca



transcription



factor


 


V$MYBL/VMYB.05
v-Myb, variant of
0.90
1404-1416
(+)
1.000
0.990
attAACGgtggtg
373



AMV v-myb


 


V$AHRR/AHRARNT.02
Aryl hydrocarbon/
0.77
1423-1447
(−)
0.750
0.781
cctgtggataGA
374



Arnt





GTgtgaaagcaac



heterodimers,



fixed core


 


V$EVI1/IEVI1.06
Ecotropic viral
0.83
1440-1456
(+)
0.750
0.835
tccacaGGATa
375



integration site 1





gattga



encoded factor,



amino-terminal



zinc finger



domain


 


V$HOXC/HOX_PBX.01
HOX/PBX
0.81
1442-1458
(+)
0.944
0.814
cacaGGATaga
376



binding sites





ttgaaa


 


V$HOXC/PBX1.01
Homeo domain
0.78
1446-1462
(+)
1.000
0.809
ggataGATTga
377



factor Pbx-1





aactgc


 


V$IRFF/ISRE.01
Interferon-
0.81
1447-1465
(+)
1.000
0.829
gatagattGAAA
378



stimulated





ctgccag



response element


 


V$HOXH/MEIS1B_HOXA9.01
Meis1b and
0.78
1450-1464
(−)
0.750
0.781
TGGCagtttcaat
379



Hoxa9 form





ct



heterodimeric



binding



complexes on



target DNA


 


V$NR2F/ARP1.01
Apolipoprotein
0.82
1469-1489
(−)
0.857
0.897
ccagggtcaggG
380



AI regulatory





ATCaggtgg



protein 1, NR2F2


 


V$MEF3/MEF3.01
MEF3 binding
0.89
1474-1486
(−)
1.000
0.943
gggTCAGggat
381



site, present in





ca



skeletal muscle-



specific



transcriptional



enhancers


 


V$RORA/TR4.01
Nuclear hormone
0.84
1474-1492
(−)
1.000
0.841
atcccagGGTC
382



receptor TR4





agggatca



homodimer



binding site


 


V$CSEN/DREAM.01
Downstream
0.95
1476-1486
(−)
1.000
0.974
ggGTCAgggat
383



regulatory



element-



antagonist



modulator,



Ca2+-binding



protein of the



neuronal



calcium sensors



family that



binds DRE



(downstream



regulatory



element) sites as



a tetramer


 


V$CP2F/CP2.01
CP2
0.90
1493-1511
(+)
1.000
0.969
ggCTGGattga
384









gcaatgag


 


V$HOXC/PBX1.01
Homeo domain
0.78
1493-1509
(+)
1.000
0.811
ggctgGATTga
385



factor Pbx-1





gcaatg


 


V$CEBP/CEBPB.01
CCAAT/enhancer
0.94
1496-1510
(+)
1.000
0.984
tggattgaGCAA
386



binding protein





tga



beta


 


V$CAAT/NFY.03
Nuclear factor Y
0.81
1513-1527
(+)
1.000
0.873
agagCCAAgca
387



(Y-box binding





gcac



factor)


 


V$STAF/ZNF76_143.01
ZNF143 is the
0.76
1522-1544
(−)
1.000
0.765
tagcCCCAggg
388



human ortholog





gactctgtgctg



of Xenopus Staf,



ZNF76 is a DNA



binding protein



related to



ZNF143 and Staf


 


V$NOLF/OLF1.01
Olfactory
0.82
1526-1548
(+)
1.000
0.879
acagagTCCCct
389



neuron-specific





ggggctagagg



factor


 


V$AP2F/AP2.02
Activator protein
0.92
1531-1545
(−)
0.905
0.941
ctaGCCCcagg
390



2 alpha





ggac


 


V$ZBPF/ZNF202.01
Transcriptional
0.73
1536-1558
(−)
0.761
0.739
gcctccTCCAcc
391



repressor, binds





tctagccccag



to elements



found



predominantly in



genes that



participate in



lipid metabolism


 


V$IKRS/IK1.01
Ikaros 1,
0.92
1561-1573
(+)
1.000
0.933
tcctGGGAatggg
392



potential



regulator of



lymphocyte



differentiation


 


V$TEAF/TEF1.01
TEF-1 related
0.84
1561-1577
(−)
1.000
0.855
ttttccCATTccc
393



muscle factor





agga


 


V$IRFF/IRF7.01
Interferon
0.86
1565-1583nc
(+)
0.936
0.895
ggGAATggga
394



regulatory factor





aaaacccca



7 (IRF-7)


 


V$LTUP/TAACC.01
Lentiviral TATA
0.71
1565-1587nc
(+)
1.000
0.721
gggaatgggaaa
395



upstream element





AACCccaactt


 


V$RBPF/RBPJK.02
Mammalian
0.94
1566-1580nc
(+)
1.000
0.947
ggaaTGGGaaa
396



transcriptional





aacc



repressor RBP-



Jkappa/CBF1


 


V$IKRS/IK1.01
Ikaros 1,
0.92
1567-1579nc
(+)
1.000
0.927
gaatGGGAaaa
397



potential





ac



regulator of



lymphocyte



differentiation


 


V$NFKB/CREL.01
c-Rel
0.91
1571-1583
(−)
1.000
0.971
tggggtttTTCCc
398


 


V$CIZF/NMP4.01
NMP4 (nuclear
0.97
1572-1582nc
(+)
1.000
0.986
ggAAAAacccc
399



matrix protein 4)/



CIZ (Cas-



interacting zinc



finger protein)


 


V$SRFF/SRF.02
Serum response
0.84
1576-1594
(−)
0.888
0.881
gacccCAAAgt
400



factor





tggggttt


 


V$MYT1/MYT1.02
MyT1 zinc
0.88
1578-1590
(−)
1.000
0.882
ccaAAGTtggg
401



finger





gt



transcription



factor involved



in primary



neurogenesis





Cartharius K, et al. (2005) Bioinformatics 21, 2933-42.






Example 4
Utilising the data from Examples 2 and 3, a suite of constructs are generated containing various shRNA suppressors and/or replacement rhodopsin nucleic acids enhanced with additional promoter sequences, known to be conserved between vertebrate species and various sequences known to enhance expression at RNA and/or protein levels. FIGS. 9 and 16 represents diagrammatically sequences cloned in suppression and/or replacement constructs. Notably, any combination of the elements and conserved regions outlined and indeed other elements that can modulate gene expression could be used in the invention to control expression of suppression and/or replacement components.
Suppression and/or replacement constructs (FIG. 9) were then used to generate recombinant AAV2/5 viruses using the procedures provided in Example 1. AAV2/5 suppression and/or replacement vectors were evaluated in 129 wild type (WT) mice for levels of expression of suppressors and/or replacement nucleic acids at the RNA and protein levels as detailed in Example 1. FIG. 10A illustrates a comparison using an RNAse protection assay of levels of human rhodopsin expression from the RHO-M transgene in RHO-M mice (lane M) versus the rhodopsin expression obtained from the suppression and replacement constructs in rAAV2/5 subretinally injected into wild type 129 mice (lanes B8, B9, B11, B12, B13, B16, B8). FIG. 10A illustrates that AAV-BB8, AAV-BB10, AAV-BB11, AAV-BB12, AAV-BB13 and AAV-BB16 express the human rhodopsin replacement gene in RNA extracted from 129 wild type mice subretinally injected with these suppression and or replacement constructs. AAV-BB8, AAV-BB10 and AAV-BB11 express human rhodopsin at lower levels than AAV-BB12, AAV-BB13 and AAV-BB16.
Further evaluation of suppression and replacement vectors was undertaken. FIG. 11 provides a comparative analysis of human rhodopsin expression from rAAV2/5 suppression and replacement vectors using real time RT-PCR. FIG. 11 illustrates replacement rhodopsin expression levels in RNA extracted from 129 wild type mice subretinally injected with suppression and/or replacement constructs. Expression levels were also determined in Rho-M transgenic mice which express a rhodopsin replacement construct termed rCC and display normal retinal function. Suppression and replacement vectors AAV-BB12, AAV-BB13, AAV-BB16 and AAV-BB18 express approximately in the same order of magnitude as levels of replacement rhodopsin transcript in Rho-M mice, indicating that enhanced replacement constructs with enhancer elements and conserved regions may express sufficient levels of rhodopsin to sustain a functional retina in vivo.
FIG. 12 illustrates retinal histology of adult wild type mouse retinas subretinally injected with 2 ul of 2×1012 particle/ml of different suppression and replacement rhodopsin AAV vectors (see FIG. 9). Two weeks post-injection of AAV vectors transduced eyes were removed, fixed in 4% paraformaldehyde and cryosectioned (12 um). Subsequently, sections were stained with human specific anti-RHO antibody to visualise expression of replacement-RHO using Cy3 label (red) on the secondary antibody; cell nuclei were counterstained with DAPI (blue). A: AAV-BB8, B: AAV-BB13, C: AAV-BB24, D: AAV-Q8, E: AAV-Q26, F: retina from uninjected Rho-M transgenic mouse expressing RHO (positive control). Clear evidence of human rhodopsin expression from AAV suppression and replacement vectors was obtained. Sections indicate different levels of human RHO expression from the AAV suppression and replacement vectors under evaluation. OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.
To explore efficacy of the suppression component of the suppression and replacement approach delivered using AAV, a variety of suppression only vectors were generated with an EGFP reporter gene (see FIG. 9). Adult NHR transgenic mice on a rho−/− background, therefore expressing normal human RHO but not mouse rho, were transduced by subretinal injection of 2 ul of 2×1012 particle/ml of AAV-shQ1-EGFP (A) or AAV-shNT-EGFP (B). Two weeks after injection, eyes were removed, fixed in 4% paraformaldehyde and cryosectioned (FIG. 13). AAV-shQ1-EGFP expresses shRNA-Q1, which targets RHO, while AAV-shNT-EGFP expresses a non-targeting shRNA (see FIG. 9 for constructs). Both constructs express EGFP allowing tracking of the transduced cell populations (green). Sections were counterstained with DAPI (blue) to label the position of the nuclear layers. A significant reduction in the photoreceptor cell number in the transduced part of the outer nuclear layer was apparent in the AAV-shQ1-EGFP injected (A) retinas compared to those of injected with AAV-shNT-EGFP (B) (FIG. 13). IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.
Adult RHO-347 transgenic mice carrying a dominant RHO mutation causing retinal degeneration akin to human RP, were subretinally injected with 2 ul of 2×1012 particle/ml of AAV-shNT (A) or AAV-shQ1 (B) vectors (FIG. 14A). Two weeks post-injection transduced eyes were removed, fixed in 4% paraformaldehyde and cryosectioned (12 um). AAV-shQ1 expresses shRNA-Q1, which targets RHO, while AAV-shNT expresses a non-targeting shRNA. Both constructs express EGFP allowing tracking of the transduced part of the retina (green). Sections were counterstained with DAPI (blue) to indicate positions of the nuclear layers. A significant reduction of the photoreceptor cell numbers in the transduced part of the outer nuclear layer in the AAV-shNT injected or the uninjected (C) retinas was apparent due to the degenerative effects of RHO-347 transgene (FIG. 14A). A significantly preserved outer nuclear layer is detected in the AAV-shQ1 transduced retinas, where shRNA-Q1 effectively suppresses the RHO-347 transcript therefore reducing retinal degeneration (FIG. 14A). Note that the mouse rhodopsin gene (expressed in these retinas) was refractory to suppression by shRNA-Q1 due to the presence of nucleotide changes at the target site for Q1 siRNA-based suppression. Suppression of human rhodopsin and replacement using the degeneracy of the genetic code provided therapeutic benefit at a histological level in RHO-347 mice.
In addition, FIG. 14D provides evidence of an improvement in the electroretinogram (ERG) in RHO-347 eyes treated with AAV-shQ1-EGFP versus eyes treated with AAV-shNT-EGFP. In FIG. 14D a representative maximum ERG response of a RHO-347 mouse, containing a human rhodopsin transgene with a mutation at codon 347, subretinally injected with AAV2/5 constructs is presented. This RHO-347 mouse normally displays a phenotype similar to autosomal dominant RP. The top figure is the response of the right eye, which received an injection of AAV-shQ1-EGFP, a AAV2/5 vector containing suppressor siRNA Q1 driven by an H1 promoter (shQ1) and a CMV-driven EGFP gene. The left eye received an AAV-shNT-EGFP, a AAV2/5 containing a non-targeting (control) siRNA driven by an H1 promoter (shNT) and a CMV-driven EGFP gene. As can be seen above, the maximum response is significantly greater in the treated right eye than in the control left eye, indicating that suppression of the mutant rhodopsin transgene leads to some rescue at the ERG level.
Example 5
Sequences of Various Elements Designed to Enhance Expression of Replacement Constructs
As described, enhancer elements, conserved regions A through I and/or transcription factor binding sites and/or other regulatory elements and/or epigenetic elements may be combined to improve expression of replacement constructs (see FIGS. 9 and 15 and Tables 1, 2, 9-13). These elements can be used in many different combinations to achieve optimum expression, as demonstrated in the Examples provided above. Additional examples include inter alia a construct comprising a human rhodopsin gene expressed from a composite promoter element containing the 484 by mouse rhodopsin promoter together with the CMV enhancer, the rhodopsin promoter enhancer element, the rhodopsin promoter conserved region B and flanked at the 3′ end of the gene by a woodchuck posttranscriptional regulatory element and a minimal poly A sequence. Another example is similar to the one above but instead of the CMV enhancer, it contains multiples of the CRX and/or NRL binding sites.
Example 6
Utilisation of Neuroprotective/Neurotrophic Factors in Conjunction with Suppression and Replacement
As described above, there is evidence from the prior art that neurotrophic/neuroprotective factors can improve cell viability and or cell functioning, the sequences encoding a number of these factors are provided in FIG. 17. FIG. 18 provides suppression and replacement constructs containing genetic elements that are beneficial for neuronal cell survival. In the example, the suppression and replacement construct pAAV-BB18 (FIG. 9) has been combined with the gene encoding the neurotrophic factor GDNF, driven by a small UCOE (chromatin opening element. A Thrasher, Abstract 36, British Society for Gene Therapy 5th Annual Conference 2008) promoter. Notably other neurotrophic factors and or genes encoding neurotrophic factors such as, for example, Neurturin may also be used in combination with any of the suppression and replacement constructs described. In example A (FIG. 18), the additional element, in this case sequence encoding GDNF is co-located with the suppression and replacement construct within the two AAV inverted terminal repeat sequences, ITS1 and ITS2. In the second example, B (FIG. 18), the GDNF gene and its promoter are not co-located with the suppression and replacement elements within ITS1 and ITS2, but are located within the backbone of the plasmid used to generate AAV. Since a small proportion of the backbone is packaged during AAV production, this results in a mixed population of AAVs with the majority containing the suppression and replacement elements and a minority the GDNF elements.
AAV vectors generated to contain suppression, replacement and neurotrophic/neuroprotection components can be subretinally injected into wild type mice and or into mice with inherited retinal degenerations such as the RHO-347 and Pro23His mice described in the Examples above.







TABLE 14





Enhancers
















CMV Enhancer (SEQ ID NO: 402)



CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTG A


 


CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT


 


AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC


 


GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG


 


CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA


 


GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG


 


TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT


 


GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT


 


CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGAC


 


Rhodopsin promoter conserved REGION A (SEQ ID NO: 403)


GAGTGTCTAATTGCTTATGATCATGCATGCTCTCTCTCCCACTAAACATTT


 


ATTAATGTGTTAGGATTTCCATTAGCGCGTGCCTTGAACTGAAATCATTT


 


GCATATGGCTGGGAAAAAGTGGGGTGAGGGAGGAAACAGTGCCAGCTCCC


 


CAACAGGCGTCAATCACAGTGACAGATCAGATGG


 


Rhodopsin Promoter Enhancer Element (contains Crx D(−) & CrxE (+) &


NRL binding sites) (SEQ ID NO: 404


TTTCTGCAGCGGGGATTAATATGATTATG


 


AACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGGTACC


 


Crx D(−) (SEQ ID NO: 405)


GCGGGGATTAATAT


 


CrxE (+)(SEQ ID NO: 406)


TGAACACCCCCAATCTC


 


NRL (SEQ ID NO: 407)


TGCTGATTCAGC


 


Rhodopsin promoter conserved region B (SEQ ID NO: 408)


TCTGCTGACCCAGCAACACTCTTTCCTTCTGAGGCTTAAGAGCTATTAGCGTAGGTG


 


ACTCAGTCCCTAATCCTCC


 


Human rhodopsin polyA region F (SEQ ID NO: 409)


GACCTGCCTAGGACTCTGTGGCCGACTATAGGCGTCTCCCATCCCCTACACCTTCCCC


 


CAGCCACAGCCATCCCACCAGGAGCAGCGCCTGTGCAGAATGAACGAAGT


 


CACATAGGCTCCTTAATTTTTTTTTTTTTTTTAAGAAATAATTAATGAGG


 


CTCCTCACTC


 


Human rhodopsin polyA region G (SEQ ID NO: 410)


ACCTGGGACAGCCTGAGAAGGGACATCCACCAAGACCTAC


 


TGATCTGGAGTCCCACGTTCCCCAAGGCCAGCGGGATGTGTGCCCCTCCT


 


CCTCCCAACTCATCTTTCAGGAACACGAGGATTCTTGCTTTCTGGAAAAG


 


TGTCCCAGCTTAGGGATAAGTGTCTAGCACAGAATGGGGCACACAGTAGG


 


TGCTTAATAAATGCTGGATGGATGCAGGAAGGAATGGAGGAATGAATGGG


 


AAGGGAGAACATAGGATCC


 


SV40 Minimal polyA (SEQ ID NO: 411)


AATAAAGGAAATTTATTTTCATGCAATAGTGTGTTGGTTTTTTGTGTG


 


WPRE from pSK11 (SEQ ID NO: 412)


GGATCC AATCAACCTC


 


TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA CTATGTTGCT


 


CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT ATCATGCTAT


 


TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA TCCTGGTTGC


 


TGTCTCTTTA TGAGGAGTTG TGGCCCGTTG TCAGGCAACG TGGCGTGGTG


 


TGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCA TTGCCACCAC


 


CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT CCCCCTCCCT ATTGCCACGG


 


CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG GGCTCGGCTG


 


TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAGCTGA CGTCCTTTCC


 


ATGGCTGCTG GCCTGTGTTG CCACCTGGAT TCTGCGCGGG ACGTCCTTCT


 


GCTACGTCCC TTCGGCCCTC AATCCAGCGG ACCTTCCTTC CCGCGGCCTG


 


CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT CGCCTTCGCC CTCAGACGAG


 


TCGGATCTCC CTTTGGGCCG CCTCCCC


 


WPRE from pSin11 (SEQ ID NO: 413)


GAGCAT CTTACCGCCA


 


TTTATTCCCA TATTTGTTCT GTTTTTCTTG ATTTGGGTAT ACATTTAAAT


 


GTTAATAAAA CAAAATGGTG GGGCAATCAT TTACATTTTT AGGGATATGT


 


AATTACTAGT TCAGGTGTAT TGCCACAAGA CAAACATGTT AAGAAACTTT


 


CCCGTTATTT ACGCTCTGTT CCTGTTAATC AACCTCTGGA TTACAAAATT


 


TGTGAAAGAT TGACTGATAT TCTTAACTAT GTTGCTCCTT TTACGCTGTG


 


TGGATATGCT GCTTTATAGC CTCTGTATCT AGCTATTGCT TCCCGTACGG


 


CTTTCGTTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTC TCTTTTAGAG


 


GAGTTGTGGC CCGTTGTCCG TCAACGTGGC GTGGTGTGCT CTGTGTTTGC


 


TGACGCAACC CCCACTGGCT GGGGCATTGC CACCACCTGT CAACTCCTTT


 


CTGGGACTTT CGCTTTCCCC CTCCCGATCG CCACGGCAGA ACTCATCGCC


 


GCCTGCCTTG CCCGCTGCTG GACAGGGGCT AGGTTGCTGG GCACTGATAA


 


TTCCGTGGTG TTGTC









INCORPORATION BY REFERENCE
The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby expressly incorporated by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, which are well known in the art.
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

## Claims

1. An enhanced viral expression vector comprising (i) conserved region B from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NO: 93, and optionally (ii) at least one of the conserved regions selected from conserved region C from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NO: 94; conserved region F and G from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NO: 97; and conserved region A from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NO: 92.

2. The vector according to claim 1, wherein the vector additionally comprises:
(i) conserved region D from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NO: 95; and/or
(ii) at least one of conserved regions H and I from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NOs: 98 and 99 respectively.

3. The vector according to claim 1, wherein the vector comprises at least one of each of conserved regions B, C, D, E, F and G, H, I and A, from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NOs: 93, 94, 95, 96, 97, 98, 99 and 92.

4. The vector according to claim 1, wherein the viral vector is an AAV vector.

5. The vector according to claim 1, wherein the vector comprises at least one sequence selected from the group consisting of a stuffer, an insulator, a silencer, an intron sequence, a post translational regulatory element, a transcription factor binding site, and an enhancer.

6. The vector of claim 5, wherein said sequence(s) is:
(i) an enhancer selected from the group consisting of SEQ ID NOs: 87-89 and 91; or
(ii) a sequence selected from the group consisting of SEQ ID NOs: 402-413.

7. The vector according to claim 5, wherein the vector comprises at least one transcription factor binding site sequence selected from the group consisting of SEQ ID NOs: 100-401.

8. The vector according to claim 1, wherein the vector comprises a chromatin opening element and/or a sequence encoding a neurotrophic or neuroprotective factor.

9. The vector according to claim 1, wherein the vector comprises at least one suppression agent and/or at least one replacement nucleic acid.

10. The vector according to claim 1, wherein the replacement nucleic acid encodes a rhodopsin gene.

11. The vector according to claim 1, wherein said vector comprises at least one suppression agent, wherein said suppression agent comprises:
(i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 75, 77, 79, 81, 83, 85, 414, 415, 416, 417, 418, 419, 420 and 421;
(ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33; or
(iii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-67.

12. The vector according to claim 1, wherein said vector comprises at least one replacement nucleic acid, wherein said replacement nucleic acid comprises:
(i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 68; or
(ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76, 78, 80, 82, 84, and 86.

13. The vector according to claim 11, wherein said vector comprises at least one replacement nucleic acid, wherein said replacement nucleic acid comprises:
(i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 68; or
(ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76, 78, 80, 82, 84, and 86.

14. A composition comprising at least one vector according to claim 1.

15. A cell comprising the vector of claim 1.

16. A method of suppressing the expression of a mutant gene and replacing expression of the mutant gene with a replacement nucleic acid, the method comprising the steps of administering to a mammal the composition of claim 13.

17. A kit comprising (i) the vector according to claim 11 and (ii) the vector according to claim 12.

18. The vector according to claim 1, wherein the vector comprises each of conserved regions B and C from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NOs: 93 and 94.

19. The vector according to claim 1, wherein the vector comprises
(i) at least one of each of conserved regions B, C, D, F and G from the rhodopsin gene having the nucleic acid sequence set forth in SEQ ID NOs: 93, 94, 95, and 97;
(ii) the nucleic acid sequence having the nucleic acid sequence set forth in SEQ ID NO: 411;
(iii) the enhancer having the nucleic acid sequence set forth in SEQ ID NO: 91; and
(iv) replacement nucleic acid having the nucleic acid sequence set forth in SEQ ID NO: 76.

20. A kit comprising (i) the vector according to claim 19 and (ii) a vector comprising a suppression agent comprising a nucleotide sequence having the nucleic acid sequence set forth in SEQ ID NO: 75.