(AVMD) with less severe mutations, whereas a lack of functional WT BEST-1 protein is the likely cause of recessive bestrinopathy [44, 45, 156, 157].

Similarly, different RS1 mutations cause a wide range of phenotypic variability in males with JXRS and the female carriers [47, 158, 159]. Mutations in RS1 can promote protein misfolding or failure of secretion, failure of oligomerization, or failure of surface binding or cell: cell interactions due to mutations in the conserved discoidin domain [185, 186, 197, 198]. On the other hand, there does not appear to be a strong correlation between the genotype and phenotype in JXRS patients with RS1 mutations and there is a fairly uniform clinical presentation although the age of onset and severity does vary [47, 158].

As cellular function is manifest in the cooperative performance of many biochemical and physiological pathways, it is not surprising that mutations in multiple genes can promote the same clinical syndromes. For example, mutations in ABCR and ELOVL4 both promote the disease known clinically as Stargardt’s macular degeneration; yet, the functional roles of the protein are quite distinct. Overall, however, both the proteins serve to generate and maintain the rod and cone outer segment structure and function in support of phototransduction. In a related way, mutations in a single gene are able to promote diverse clinical anatomic disease patterns. For example, mutations in BEST-1 promote both BMD and AVMD, whereas peripherin/RDS mutations can also cause AVMD. Mutations in ABCR cause STGMD, FF, CORD, RP, and increase the risk for AMD [36, 151, 153]. In a more extreme example, mutations in the peripherin gene (RDS) (alias PRPH2), which is expressed in both the rod and cone photoreceptors, cause a variety of autosomal dominant disease patterns including macular degeneration, AVMD, macular pattern dystrophy, retinitis punctata albescens, central areolar pigment epithelial dystrophy, and RP [160–164, 189, 190].

11.5.1  Genetic Modifiers

and Digenic Inheritance

undiagnosed. As a multitude of genes, for example, specify the development, maintenance, and functional performance of the rod and cone photoreceptors, random mutations in other genes contributing the system structure and performance could affect the penetrance, the time of emergence, and the rate of degeneration due to a mutational hit in a photoreceptor-specific disease gene. Disease emerges in a biological system of coordinately interacting components. Such genes are known as modifiers as they can modify the phenotype of an inherited disease [165]. RPE65, the retinoid isomerase of the RPE, is known to be a genetic modifier in retinal degenerations [166]. This is not surprising given the necessary and essential role that RPE65 plays in retinoid metabolism, which is supportive for the phototransduction apparatus of the photoreceptors. ABCR mutations may be playing a modifier gene role in patients who later develop AMD. A report suggests such modifier associations for JXRS [167]. One might expect the interaction of modifier genes with disease genes underlying juvenile macular degeneration because of the wide spectrum of phenotypic variability that is generally present, even though these degenerations are early onset by definition.

Digenic inheritance means that mutations in two unlinked disease genes are needed to promote a deleterious phenotype. The initial and now classic example is digenic RP which is caused by simultaneous mutations in both peripherin and ROM1 [168]. Peripherin and ROM-1 are proteins expressed to the outer segment of both the rod and cone photoreceptors and form stabilizing protein: protein interactions at the lateral edges of the disk structures [169]. Because of this structural and functional protein: protein association, mutational knockdown of the WT levels of both the proteins leads to the suppression of complex formation and destabilization of the outer segment structures which promotes photoreceptor degeneration. To our knowledge, digenic inheritance has not been reported in association with the disease genes underlying juvenile macular degenerations presented here.

The genotype of the individual can affect the outcome of emergence of a mutation in a separate known disease gene. Any random individual harbors many random mutations or variations. Such random variations may be deleterious and become established and transmitted within the bloodline of a family. Likely, most remain

11.6  Potential Therapeutics for Juvenile Macular Degenerations

The nature of the therapeutic used will strongly depend upon the nature of the cellular problems that emerge from the mutant genes and altered or missing proteins.

There are three different types of mutations at the level of the protein phenotype. Lack of function mutations. Some mutations create a deficiency of WT protein, such as, due to premature stop codon mutations in the mRNA, or missense mutations that promote rapid degradation of the mutant protein to create an effective deficiency. These are also called null mutations, as they result in a lack of WT protein. Such mutations are commonly found in recessive conditions (e.g., STGD1,

JXRS) [191]. Gain-of-function mutations. Most missense mutations are gain-of-function mutations. This means that the mutant protein has properties that are not present in the WT and these new and active properties are deleterious to the cell in which the mutant gene is expressed. This is the common type of mutation found in autosomal dominant conditions (e.g., STGD3, BEST1). There are many potential ways in which a mutant protein can exert toxicity for the cell in which it is expressed. The protein may fold correctly, but have abnormal signal generating capacity, or abnormal enzymatic capacity, or be focally abnormal and unable to build a multiprotein complex or cellular structure. Mutant proteins may also fold abnormally. If such mutant proteins cannot be rescued by cellular chaperones, then they may be earmarked for degradation in the lysosome or proteosome. Or, such mutant proteins can accumulate inside the cells and exert toxic effects on cellular metabolism. For example, many mutant proteins that fail to fold properly may become trapped inside the endoplasmic reticulum and initiate the unfolded protein cellular response that may result in apoptosis. Or, the cell may deposit mutant proteins in intracellular bodies called aggresomes. The build up of mutant proteins in such microenvironments may itself induce toxic effects after a threshold amount is accumulated. Dominant negative mutations. In some cases of missense mutations, the altered protein is able to affect the trafficking or degradation of the WT protein. This event may occur because the WT protein of interest naturally forms dimers, and the mutant protein, if sufficiently structured, is able to trap the WT protein through binding and affect its cellular localization or stability. Dominant negative mutant proteins may also affect the posttranslational processing or trafficking of the WT protein and hence affect the WT levels­ of the protein that are attained.

The strategy of therapy for a lack of function mutation is clear – one must replace the expression of the ineffective genes. It is important to understand that

the amount of expression of the WT protein can dictate the level of function recovered, or can even promote toxicity in overexpression. Hence, any gene therapy must be considered from the point of view of regulation. To achieve sufficient expression to maintain cell vitality, proper expression to optimize cellular function, and controlled expression to prevent excess WT that could be deleterious to the cell, any gene therapy must be carefully regulated in order to obtain maximal therapeutic effect and minimal toxicity. Ideally, titrated expression of the WT gene would occur based upon the quantitative measures of retinal cellular function in vivo, for example, by ERG, or OCT, or other measures.

The strategy for gain-of-function mutations is considerably more complex. In most cases, these patients will be those in pedigrees following an autosomal dominant pattern of inheritance, where both WT and mutant proteins are expressed in the cells that manifest at least the origins of the genetic diseases. The mutant protein with gain-of-function phenotype exerts a toxic effect on the cell with only a 50% level of expression (other 50% is WT protein, assuming equivalent transcription of both the alleles). In order to rescue the cell from the toxicity, it is necessary to suppress the expression of the mutant protein. It may also be necessary to reconstitute the expression of the WT protein to near 100% normal levels. Posttranscriptional gene silencing (PTGS) strategies that target mRNAs have a strong potential for therapy of such conditions [170, 171]. Developed technologies included ribozymes or catalytic RNAs and RNA interference. These technologies design generally small RNAs to seek out larger target mRNAs, bind to them in an accessible region by base pair complementarity (Watson–Crick), and cleave the targettopromoteitsmorerapiddegradation(Fig. 11.16). A lower steady state level of the target mRNA leads to lower levels of the target protein. In some cases, it may be possible to selectively promote the degradation of only the mutant mRNA, but this is an unrealistic strategy because most mutant mRNAs cannot be discretely targeted since the regions of the mutations are not accessible in the target mRNA. A more generalized PTGS strategy is to design the best agent to target the most accessible region of the target mRNA, which would be identical in both the mutant and WT mRNAs, and use such an agent to suppress both the mutant and WT protein expression. WT protein must then be reconstituted through the expression of an mRNA that encodes the WT protein, but is resistant or hardened to

Disease Target mRNA

600

500

1 0 0 0

400

300

200

100

700

1300

Ribozyme RNA

1600

1700

Target

C

A U

A Mg2+ GA

G GU

A

C : G

A : U

G : C

G : C

A G

G U

Ribozyme + Products

Fig. 11.16  Ribozyme attack on a disease target mRNA (upper). The secondary structure of a model disease target mRNA is shown. This is the most stable secondary structure of human rod opsin mRNA as determined by a computational algorithm. Note the dense secondary structure with rare and small regions that are in a single stranded conformational state with the capacity to support ribozyme annealing by Watson–Crick base pairing. The smaller ribozyme is also shown in the schematic (lower). A model hammerhead ribozyme is shown with its core enzyme

that binds Mg2+ and its two antisense flanks that are programmed to anneal to the exposed and accessible target region by Watson– Crick base pairing. Once annealed to form a hybrid, the core enzyme can act to cleave the phosphodiester bond (red arrow) after the cleavage motif GUC . This allows the two products to dissociate from the ribozyme antisense flanks. With the target mRNA broken, the pieces are rapidly degraded in vivo and the mRNA target can no longer be used to translate mutant target protein

cleavage by the ribozyme or RNAi agent. Here, the expression levels of the PTGS agent would need to be regulated in order to modulate the level of knockdown of the target proteins. The expression level of the PTGS-resistant WT allele would also need to be modulated. This might be feasible with the component(s) of the intrinsic WT promoter, if the levels of WT expression are modulated by the feedback to the transcriptional level, or a promoter that is modulated by a small molecule that could be supplied as a drug. Here too, the levels of WT function would need to be quantitatively assessed over real time in the patients receiving such therapies.

The strategy for dominant negative mutations is likely to be as complex as gain-of-function mutations. One could think that simple overexpression of the WT protein would both rescue the dominant negative effect of the mutant protein and mitigate haploinsufficiency. However, WT overexpression may simply feed the dominant negative effect of the mutant protein before the haploinsufficiency can be rescued. Rescue of the dominant negative effect of the mutant protein may require the suppression of the same (e.g., by PTGS strategies), followed by a careful titration of the WT expression.

What is clear at this time is that gene-based therapies for juvenile macular degenerations will take substantial

time to manifest. Gene replacement strategies for null mutations are the simplest and the closest to the clinic at this time. Gene therapeutics for dominant mutations such as ribozymes will require more time to bring to clinic. A potentially more challenging issue is the approach to gene delivery for a juvenile macular degeneration. Current forms of vector gene delivery to the outer retina require retinal detachments by fluid delivery into the implicit subretinal space. Macula-off retinal detachment has notoriously poor clinical outcomes for vision in humans [172–175]. While an argument that short-term and shallow retinal detachments are less likely to be toxic is generally made, these will still involve loss of photoreceptor outer segments, induction of apoptosis, and prolonged recovery times with remaining photoreceptor outer segment outgrowth and realignment to achieve final visual acuity. It is likely that new methods of vectorial gene delivery will be needed to address this issue. One potential approach could be smart nanotechnology vectors which cross the blood retinal barrier to transduce selective retinal subtypes. Early success in such technology development has already appeared in the literature [176–178].

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