*4.1. Pathophysiology of 11-cis-Deficient Retinal Diseases in Mouse Models of Retinal Degeneration*

The progressive loss of rod photoreceptors and shortening of rod outer segments with age has been reported in mice lacking functional *Lrat* or *Rpe65* [45,52]. Additionally both mouse models show rapid degeneration of cone photoreceptors, with complete degeneration of M/L and S opsin occurring at P28 and P42 respectively [73,74]. Both knockout mice display mislocalization of cone opsin at P28 and upon repeated administration of 11-*cis*-retinal opsin trafficking can be partially corrected in young *Lrat<sup>í</sup>/<sup>í</sup>* and *Rpe65<sup>í</sup>/<sup>í</sup>* mice, emphasizing the importance of 11-*cis*-retinal for proper cone opsin conformation and trafficking [54,74,75]. Recently, mounting evidence supports the hypothesis that mislocalization of cone opsin results in increased endoplasmic reticulum stress and induces the early cone cell death seen in LCA mouse models. Pharmacological studies in *Lrat<sup>í</sup>/<sup>í</sup>* and *Rpe65<sup>í</sup>/<sup>í</sup>* mice have demonstrated that cone cell death can be ameliorated by both the ER chemical chaperone tauroursodeoxycholic acid, and proteasome inhibitor MG-132 respectively, suggesting a central role for ER in opsin protein degradation [76,77].

Rod opsin on the other hand is observed to traffic normally in the absence of 11-*cis*-retinal, both in *Lrat* and *Rpe65* knockout mice, suggesting that rod pigment does not necessarily need its chromophore for proper photoreceptor localization [74], whereas 11-*cis*-retinal deficiency can induce abnormality of length and morphological structures of rod outer segments in LCA mouse models [74,78]. Experiments in P23H mutant mice have shown that the 11-*cis*-retinal or 9-*cis*-retinal chromophore is important for increasing protein stability and intracellular transportation of mutant rod opsin, providing evidence that specific protein sequences may also be important for proper functioning and transport of rod opsin [79–81].

The cyclic processing of chromophore can be blocked by removal or mutation in any one of the enzymes required for regeneration. Furthermore cycle interruption may result not only in cessation of 11-*cis*-retinal chromophore production but also in the accumulation of products from the previous steps, leading to cell disruption and death. Mutations in *Lrat* and *Rpe65* disrupt the visual cycle at distinct steps in regeneration and therefore knockout animals present differences in retinoid composition in the eye. The loss of functional RPE65 prevents the conversion of stored retinyl esters to 11-*cis*-retinal and causes the unrestrained accumulation of retinyl esters in the RPE, leading to a nonfunctioning visual cycle [52]. Excessive ester accumulation, appearing as retinosomes, can be clearly seen in young *Rpe65<sup>í</sup>/<sup>í</sup>* mice, while such structures are rare in either *Lrat<sup>í</sup>/<sup>í</sup>* or wild-type mice (Figure 3). In addition noninvasive two-photon imaging techniques have revealed analogous fluorescent structures in RPE of 3 months old *Rpe65<sup>í</sup>/<sup>í</sup>* mice, while these structures were completely absent from *Lrat<sup>í</sup>/<sup>í</sup>*, and minimal in wild-type mice [44].

Whereas *Rpe65<sup>í</sup>/<sup>í</sup>* mice exhibit extremely high concentrations of retinyl ester, mice lacking the enzyme LRAT possess only trace amounts of retinyl esters in the RPE, but likewise have a no detectable 11-*cis*-retinal in the retina, indicating that LRAT is essential for the esterification step of the visual cycle [74]. Furthermore, LRAT activity is required for storage of retinyl esters in other tissues, such as the liver and lungs, thus in addition to the retinal degenerative phenotype *Lrat<sup>í</sup>/<sup>í</sup>* mice are highly susceptible to vitamin A deficiency [30,82]. In conclusion, because of the pathological similarities shared between select human retinal degenerative diseases and both *Rpe65<sup>í</sup>/<sup>í</sup>* and *Lrat<sup>í</sup>/<sup>í</sup>* mice these models provide practical avenues for comprehensively studying aspects of human disease progression and potential treatments.

#### *4.2. 11-cis-Retinal Deficiency and Leber Congenital Amaurosis*

Defects in 11-*cis*-retinal regeneration are seen in a number of human inherited degenerative retinopathies including early childhood onset Leber congenital amaurosis (LCA). Diagnosis of LCA is usually confirmed early in life by irregular electroretinographic and papillary responses, and vision commonly declines with age until complete blindness is observed by the third or fourth decade of life [83,84]. Numerous gene mutations have been reported to cause LCA in humans including RPE65, LRAT, CRX (Homeodomain transcription factor), CRB1 (Crumbs like protein 1), TULP1 (Tubby-like protein), AIPL1 (aryl hydrocarbon interacting protein), and various other genes [84]. LCA typically is an autosomal recessive inherited disease, though autosomal dominant patterns have been reported [85]. The early-onset rod-cone dystrophy phenotype is observed in human patients diagnosed with LCA, but also in *Rpe65<sup>í</sup>/<sup>í</sup>* and *Lrat<sup>í</sup>/<sup>í</sup>* mice, as discussed above [45,52,86]. Early in life patients with LCA exhibit visual impairment with attenuated rod and cone function, macular atrophy, severely delayed or minimal ERG responses, nystagmus, and retinal cell degeneration [83,84,87,88]. LCA is currently considered an incurable disease but several promising therapies are presently being investigated, including gene therapy and chromophore replacement therapy [89–91].

**Figure 3.** (**A**) Horizontal EM images of RPE in 3 month old *Rpe65<sup>í</sup>/<sup>í</sup>*, *Lrat<sup>í</sup>/<sup>í</sup>* and wild-type mice. Of particular interest are the large retinosomes present around the perimeter of RPE cells in *Rpe65<sup>í</sup>/<sup>í</sup>* mice (black arrows), these formations are indicative of excessive ester accumulation in the retina. (**B**) Two photon imaging of the RPE in 3 month old *Rpe65<sup>í</sup>/<sup>í</sup>*, *Lrat<sup>í</sup>/<sup>í</sup>* and wild-type mice. Large autofluorescent spots are observed in the RPE of *Rpe65<sup>í</sup>/<sup>í</sup>* mice (white arrows), while such spots are absent in *Lrat<sup>í</sup>/<sup>í</sup>* mice, and are minimally observed in wild type mice. Scale bar 5.0 m.

**5. Artificial Visual Chromophore Therapeutics and Further Applications** 

Pharmacological replacement of 11-*cis*-retinal has been shown to be highly effective at reconstituting functional visual pigment, increasing ERG responses and reducing the rate of retinal degeneration in animals with *Rpe65* or *Lrat* mutations [89,91]. 9-*cis* isomers have proven to be the most successful isomer in replacing the native 11-*cis* chromophore *in vivo*. Moreover 9-*cis*-retinal is preferred over 11-*cis*-retinal for chromophore replacement therapy because of its increased stability, ease of synthesis and ability to form light sensitive isorhodopsin *in vivo* [91]. Once incorporated into the rod outer segment 9-*cis*-retinal forms a schiff base with residue K296 of opsin producing a chromophore molecule analogous to 11-*cis*-retinal bound chromophore, and reducing the amount of endogenous opsin apoprotein [92] (Figure 4).

**Figure 4.** Isomers of the opsin chromophore. Both 11-*cis*-retinal and 9-*cis*-retinal form a Schiff base with residue K296 of the opsin molecule forming the light sensitive chromophore used in vision.

9-*cis-*retinoids taken orally, are converted to pro-drug forms *in vivo*, stored in the liver, transported in the blood, and eventually taken up into retinal tissue similar to dietary vitamin A. The capability to store 9-*cis* retinoids in tissues that naturally sequester vitamin A is especially important in producing a continuous therapeutic effect with retinoid drug administration. To avoid potential negative effects of administering large doses of retinoids future development of targeted delivery systems may lead to lower toxicity and improved effectiveness [93,94].

### *5.1. Prevention of the LCA Phenotype with Administration of 9-cis-Retinoids in Animal Models*

The *Lrat<sup>í</sup>/<sup>í</sup>* and *Rpe65<sup>í</sup>/<sup>í</sup>* mouse provide useful disease models for studying the efficacy and toxicity of chromophore replacement therapy, since these mice lack visual chromophore and develop retinopathy that closely resembles LCA in humans. In *Rpe65<sup>í</sup>/<sup>í</sup>* mice oral gavage of 9-*cis*-retinal, the biologically active form, which binds with opsin, has been shown to produce functional isorhodopsin, restore rod function, increase light sensitivity, and reduce RPE ester concentration *in vivo* [89,91]. Likewise, intravitreal injections of 9-*cis*-retinal increased ERG responses and improved obstacle avoidance in a RPE65 deficient canine model of LCA [95]. 9-*cis*-retinal treatment in both *Lrat<sup>í</sup>/<sup>í</sup>* and *Rpe65<sup>í</sup>/<sup>í</sup>* mice demonstrate the ability of this retinal isomer to bypass essential steps in retinoid regeneration, fully integrate into photoreceptor outer segments and form isorhodopsin capable of sensing light (Figure 5).

Various analogs of 9-*cis*-retinal have been investigated that theoretically provide better stability in gastric acidity when ingested and are further metabolized in the liver to produce storage forms of 9-*cis-*retinyl esters, such as 9-*cis-*retinyl palmitate [93,96]. Prolonged improvements in ERG responses in 9-*cis* retinyl acetate treated *Rpe65<sup>í</sup>/<sup>í</sup>* mice implies that 9-*cis*-retinoids are stored in the liver, mobilized, taken up by RPE cells through the circulation as 9-*cis*-retinol and incorporated into the visual cycle similar to the all-*trans* isomer [97]. Uptake of retinol from the circulation into the RPE of *Rpe65<sup>í</sup>/<sup>í</sup>* mice remains functional despite the presence of an abnormally large quantity of retinyl esters, implying that circulating 9-*cis*-retinoids can be absorbed by the RPE even though a functional visual cycle does not exist [98]. Prodrugs, which can be stored by the body, provide a practical approach to 9-*cis*-retinal delivery in humans by increasing drug bioavailability and decreasing the need for frequent dosing.

**Figure 5.** Normal phase HPLC analysis of retinoids in dark adapted mouse models of retinal degeneration. Concentrations of *cis*-retinoids and retinyl esters in the retina differ before and after 9-*cis*-retinoid treatment. Control *Rpe65* knockout mice exhibit significantly increased concentrations of retinyl esters and are devoid of 11-*cis*-retinal. *Rpe65<sup>í</sup>/<sup>í</sup>* mice treated with 9-*cis*-retinoids show increase 9-*cis*-retinal bound to rhodopsin. Likewise, untreated *Lrat* knockout mice lack both 11-*cis*-retinal and retinyl esters, while treated *Lrat* knockout mice show an increase 9-*cis*-retinal bound to rhodopsin. WT mice show normal concentrations of 11-*cis-*retinal, as well as small amounts of retinyl esters.

9-*cis*-retinyl acetate is one such prodrug, since it must be metabolized to the active 9-*cis-*retinal by the liver and then delivered to the eye via the bloodstream. Analogous to 9-*cis*-retinal therapy 9-*cis*-retinyl acetate treatment in *Lrat<sup>í</sup>/<sup>í</sup>* and *Rpe65<sup>í</sup>/<sup>í</sup>* mice generates functional isorhodopsin, maintains retinal thickness, and attenuates the decrease in ERG scotopic and photopic responses consistently seen with increasing age [71,99]. Similar to rod photoreceptors, the rate of age related cone photoreceptor cell death was slowed in 9-*cis*-retinyl acetate treated *Rpe65<sup>í</sup>/<sup>í</sup>* and *Lrat<sup>í</sup>/<sup>í</sup>* mice compared to untreated counterparts [100,101]. Furthermore significantly improved pure cone cell ERG responses were recorded in 9-*cis*-retinyl acetate treated *Rpe65<sup>í</sup>/<sup>í</sup>* and *Lrat<sup>í</sup>/<sup>í</sup>* mice lacking the functional transducin protein required for continued phototransduction in only rod cells. These data suggest that chromophore replacement therapy may be beneficial for also rescuing cone photoreceptor cells in LCA patients, which tend to be lost earlier in disease progression than rod photoreceptor cells [100]. Pharmacokinetic and systemic retinal toxicity studies demonstrated that WT, *Rpe65<sup>í</sup>/<sup>í</sup>*, and *Lrat<sup>í</sup>/<sup>í</sup>* mice administered various dosing regimens of 9-*cis-*retinyl acetate displayed no toxicity at therapeutic dosing [97,99]. A recent study demonstrated that retinas of *Rpe65<sup>í</sup>/<sup>í</sup>* and *Lrat<sup>í</sup>/<sup>í</sup>* mice were well tolerated to continuous exposure of high levels of QLT091001, a 9-*cis*-retinyl acetate drug, without the accumulation of toxic retinoid byproducts, such as A2E, and obvious pathological changes in neural retina and RPE [99]. Importantly QLT091001, developed by QLT, Inc., has been tested in preliminary human clinical trials (ClinicalTrials.gov number, NCT01014052).

More recently, subcutaneous implantations of microparticle-hydrogels loaded with 9-*cis*-retinyl acetate have improved ERG responses and maintained retinal morphology in *Lratí/í* mice, suggesting that the use of such implants can reduce the frequency of dosing and therefore decrease the risk of hypervitaminosis A in patients [94,102–107]. The side effects observed from extreme over supplementation of vitamin A derivatives have prompted extensive research and development of chemically modified retinoids, as well as novel delivery systems that decrease toxicity and increase drug effectiveness. Excess natural or synthetic retinoids pose serious teratogenic risks and can possibly lead to craniofacial, cardiac, thymic, and central nervous system malformations in infants [106–108]. In adults chronic hypervitaminosis A, caused by long-term retinoid administration, can result in fibrosis and cirrhosis of the liver, hypercalcemia and bone loss [102–104]. The severe side effects observed from over supplementing vitamin A derivatives have recently prompted extensive research and development of chemically modified retinoids, as well as novel delivery systems that decrease toxicity and increase drug effectiveness. Advancements in slow release therapies may in the future reduce the necessity for frequent dosing allowing patients to visit clinicians less frequently, while providing a consistent dosing of the drug.

#### *5.2. Therapeutics of 9-cis-Carotenoids in the Treatment of LCA*

Proretinoid compounds, such as carotenoids, have also been investigated for their potential use in treating chromophore deficiency in retinal diseases. Of particular interest is the naturally occurring carotenoid isomer 9-*cis*-ȕ-carotene, because if metabolized and cleaved symmetrically by BCMO1, this carotene isomer has the potential to produce 9-*cis*-retinal *in vivo*. In addition substituting carotenes in place of retinoid supplements is particularly appealing since it eliminates the risk of developing hypervitaminosis A, given that ȕ-carotene uptake and catabolic cleavage is negatively regulated by dietary vitamin A intake [17,109].

Recently a clinical study in patients with fundus albipunctatus, a congenital form of night blindness resulting from a genetic mutation in gene RDH5 required for the oxidation of 11-*cis*-retinol to 11-*cis*-retinal, demonstrated that administration of 9-*cis*-carotene rich supplements improved ERG responses and enhanced patients mean visual field score [110,111]. Conversely, a similar experiment was performed in both the *Lrat<sup>í</sup>/<sup>í</sup>* and *Rpe65<sup>í</sup>/<sup>í</sup>* mouse and demonstrated extremely limited delivery of 9-*cis*-retinal to the eye when these animals were administrated 9-*cis*-ȕ-carotene isolated from *D. barawil* extracts [112]. Furthermore it was demonstrated *in vitro* that a second carotenoid cleavage enzyme, ȕ-carotene dioxygenase 2 (BCDO2), exists in the intestine and favorably cleaves 9-*cis*-ȕ-carotene asymmetrically, producing products which are further metabolized into all-*trans*-retinal by BCMO1 [112]. The results from the latter study provide evidence that *cis*-carotenoids are far less effective than 9-*cis*-retinoids for delivery of 9-*cis*-retinal to the eye because of the different catabolic pathways used to produce active retinoid compounds from proretinoids. The above mentioned clinical study included a small sample size of seven patients, and did not contain a control or placebo group; therefore the results observed in this study could be attributed to the increase in the overall intake of dietary vitamin A and not the specific action of the 9-*cis* isomer of ȕ-carotene.

### **6. Conclusions**

To sustain vision vertebrates require the continued cyclic regeneration of the vitamin A derivative 11-*cis*-retinal. Prolonged insufficient dietary supply of vitamin A, select genetic defects in genes required for the production of 11-*cis*-retinal chromophore or discontinuous retinoid cycling may have devastating effects on the overall health of the retina and the quality of vision. Particular human diseases, such as LCA, lack the functional enzymatic reactions to regenerate 11-*cis*-retinal, and therefore patients with these defects exhibit decreased visual responses at an early age, which additionally decline steadily throughout life.

Supplementation with preformed *cis*-retinoid derivatives has been show to bypass defective steps in the visual cycle, and regenerate pigments necessary for vision in animal models of retinal degenerative diseases. The main caveats to retinoid treatment are the myriad of toxic effects seen with administration of pharmacological doses of vitamin A derivatives for prolonged lengths of time. Therefore novel methods for reducing the risk of vitamin A toxicity, improving drug effectiveness, and reducing the frequency of dosing are absolutely necessary for designing a safe retinoid derived drug for treatment of retinal degenerative diseases associated with chromophore deficiency.

#### **Acknowledgments**

The authors would like to kindly thank H. Fujioka and M. Hitomi for electron microscopy images. Additionally the authors would like to thank A. Maeda, K. Palczewski, S. Howell and D. Peck for critically reviewing the manuscript. This work was supported in part by funding from the National Institutes of Health (K08EY019880, P30 EY11373); the Research to Prevent Blindness Foundation; Foundation Fighting Blindness; Midwest Eye Bank and the Ohio Lions Eye Research Foundation.

#### **Conflict of Interest**

The authors declare no conflicts of interest.

#### **References**


