WO2024110625A1

WO2024110625A1 - Cralbp based therapeutics for retinal disorders

Info

Publication number: WO2024110625A1
Application number: PCT/EP2023/082964
Authority: WO
Inventors: Achim Stocker; Walter AESCHIMANN
Original assignee: Universitaet Bern
Current assignee: Universitaet Bern
Priority date: 2022-11-24
Filing date: 2023-11-24
Publication date: 2024-05-30
Anticipated expiration: 2025-05-24

Abstract

The present invention relates to compositions for use in methods of preventing or treating retinal diseases or disorders of an animal or a human, wherein said compositions comprise complexes comprising (a) a CRALBP protein, and (b) a cognate ligand of CRALBP.

Description

CRALBP BASED THERAPEUTICS FOR RETINAL DISORDERS

The present invention relates to compositions for use in methods of preventing or treating diseases or disorders caused by an endogenous retinoid deficiency of an animal or a human, wherein said compositions comprise complexes comprising (a) a CRALBP protein, and (b) a cognate ligand of CRALBP. In particular, the present invention relates to the prevention and treatment of retinal diseases and disorders such as Retinitis Pigmentosa and related diseases and disorders by administration, preferably via intravitreal application, of the described CRALBP based compositions and complexes effecting the recovery of light-sensitive photoreceptors within the eye’s retina.

RELATED ART

The retinoid or visual cycle is described as a cyclic enzymatic pathway that continuously generates 11-cz -retinal, the chromophore of visual pigments in rod and cone photoreceptor cells needed for vision (Maeda, T., et al., 2009, Hum. Molec. Genet. 18: 2277-2287; Kiser, P.D. et al., 2014, Chem. Rev. 114: 194-232; Kiser, P.D., et al., 2021, Journal of Biological Chemistry 296: 100072). Hereby, l l -cz.s retinal is the photon-absorbing partner of opsins in cone and rod photoreceptor cells of the vertebrate eye. Absorption of a photon is coupled to cis-\o-trans photoisomerization in the retinylidene moiety, deactivating the photopigment. Regenerative trans- o-cis recycling does not occur in the rod and cone photoreceptor cells, but instead in adjacent retinal pigment epithelium (RPE) and Muller cells. For this, all-/zz/z/.s-retinol has to be transported into the RPE by interphotoreceptor retinol binding protein (IRBP) when released to the cytosol of rod outer segment. In RPE, all-trans-retinol is acylated by lecithin retinol acyltransferase (LRAT) yielding al 1 -// zzz.s-reti ny 1 ester. This ester is concomitantly hydrolyzed and isomerized by the enzyme (RPE65) to form 11 -cis-retinol. The highly light sensitive l l -cz.s retinol is then taken up by CRALBP chaperoning its oxidation to 1 1 -cz.s retinal by cis-specific retinol dehydrogenase (RDH5) and the reloding process of the rod and cone photoreceptor cells with the freshly regenerated chromophore.

Advances in molecular genetics have not only led to the identification of the genes encoding for enzymes and retinoid-binding proteins involved in the visual cycle and the 1 1 -cz.s retinal regeneration process, but furthermore to the identification of genetic mutations of said genes involved in the visual cycle that impair or even abolish the reloading of photoreceptor cells with fresh 1 1 -cz.s retinal. Thus, mutations in the genes encoding the enzymes and proteins utilized in the visual cycle have been identified that lead to endogenous retinoid deficiencies resulting in visual disorders due to the shortage or depletion of 1 l-czs-retinal.

Most prominent diseases or disorders caused by such genetic mutations are retinal diseases or dystrophies, in particular, inherited retinal diseases or dystrophies including retinitis pigmentosa (RP) (Shintani K., et al., 2009, Optometry 80:384-401; Maeda, T., et al., 2009, Hum. Molec. Genet. 18: 2277-2287; O'Neal, T.B., et al., 2022, In: StatPearls. Treasure Island (FL): StatPearls Publishing), Leber congenital amaurosis (LCA) (Kumaran N., et al., 2017, Br. J. Ophthalmol. 101 : 1147-1154; Daich Varela M., et al., 2022, Br. J. Ophthalmol. 106:445-451; Anneke I., et al., 2008, Progress in Retinal and Eye Research 27:391-419), age-related macular degeneration (AMD) (Petrukhin, K., et al., 2013, Drug Discovery Today:Therapeutic Strategies 10: l,el l-e20), Stargardt disease (STGD) (Stargardt, K., 1909, Albrecht von Graefes Arch. Klin. Exp. Ophthal. 71 :534-549), cone dystrophy (COD) (Holt, R., et al., 2015, Exp Eye Res. 132: 161-173), cone-rod dystrophy (CRD) (Berger, W., et al., 2010, Prog Retina Eye Res 29, 335-375), retinitis punctata albesciens (RPA) (Littink et al., 2012, Ophthalmology, 119: 1899- 906), and fundus albipunctatus and cone stationary night blindness (CSNB) (Kiser, P.D., et al., 2021, Journal of Biological Chemistry 296: 100072). These retinal diseases or dystrophies are typically associated with a breakdown of light-detecting rod and cone photoreceptor cells over time, destroying vision (Moiseyev, G., et al., 2005, Proc. Nat. Acad. Sci. 102: 12413-12418). There is currently no cure for any of these diseases or dystrophies. Common symptoms include impaired night vision and a loss of peripheral fields leading to tunnel vision, and eventual blindness.

Retinitis pigmentosa (RP) is the most common group of inherited retinal diseases worldwide. It is characterized by progressive photoreceptor degeneration with subsequent degeneration of the retinal pigment epithelium (RPE) (Hartong, D.T. et al., 2006, Lancet, 368: 1795-1809; Shintani K., et al., 2009, Optometry 80:384-401). There are a variety of forms of RP all of which show various limitations of visual performance over time and the course and progression of the disease show considerable variability between individuals. RP is typically characterized by initial symptoms of night blindness, with onset in adolescence or early adulthood, loss of peripheral vision and, as the disease progresses, loss of central vision that can lead to blindness or severe visual impairment. The age-at-onset of symptoms is highly variable and ranges from childhood to mid-adulthood. RP disease classification can be by made by age of onset, for example, congenital RP (sometimes referred to as LCA), juvenile onset RP, teenage onset RP, adult onset RP, and late onset RP. Electroretinogram (ERG) responses are an early indicator of loss of rod and cone function in RP and diminution of ERG responses can be evident within the first few years of life, even though symptoms appear much later. Typical RP presents as primary degeneration of rods, with secondary degeneration of cones, and is consequently described as a rod-cone dystrophy, with rods being more affected than cones. This sequence of photoreceptor involvement explains why some RP subjects initially present with night blindness, and only in later life become visually impaired in all light conditions.

RP can be caused by defects in many different genes and their related disease pathways. At present, more than 200 causative RP mutations have been detected in more than 100 different genes. RP genotypes are heterogeneous, and RP subjects with the same mutation can exhibit different phenotypes. RP may be classified by inheritance type, for example, autosomal dominant (ad) RP, autosomal recessive (ar) RP, X-linked (XL) or sex-linked recessive RP, sporadic RP (simplex RP; most are recessive), or digenic RP. RP is currently estimated to affect at least 300,000 individuals worldwide, of which approximately 20%-30% are autosomal recessive (arRP). In particular, mutations in the LRAT and RPE65 genes have been discovered in RP subjects with arRP or adRP. RPE65 mutations are predominantly associated with early- onset severe retinal dystrophy, with rod-cone degeneration, nystagmus and severe visual loss within the first few years of life. The severity of the disease resulting from mutations in RPE65 appears to be largely independent of the mutation types present in the RP subjects. To date there is no effective treatment that can prevent or reverse the devastating vision loss caused by RP. Some approaches to RP treatment have been investigated, including nutritional retinoid supplementation (Maeda, T., et al., 2009, Invest Ophthalmol Vis Sci. 50(9):4368-78), light reduction (Paskowitz, D.M., et al., 2006, Br J Ophthalmol. 90(8): 1060-6), stimulation of lightsensing cells (Tong, W., et al., 2020, Frontiers in Neuroscience https://www.frontiersin.org/articles/10.3389/fnins.2020.00262), intravitreal use of bone marrow-derived stem cells (Siqueira, R.C., et al., 2011, Retina. 31 : 1207-14) and gene therapy (Piri, N., 2021, et al., Taiwan J. Ophthalmol. 19; 11(4):348-351).

Leber congenital amaurosis (LCA) is the most severe retinal dystrophy causing blindness or severe visual impairment before the age of 1 year. Patients with LCA lack the ability to generate l l -cv.s-retinal in adequate quantities and therefore suffer from severe vision loss at birth, nystagmus, poor pupillary responses and severely diminished electroretinograms (ERGs). LCA is so far known to be caused by mutations in at least 14 different genes. One of the most explored and widely publicized forms of LCA is due to a mutation in the gene encoding RPE65 (Rafael, C., et al., 2010, Invest. Ophthalmol. Vis. Sci. 51(10):5304-5313.). Treatment of this form of LCA has recently been shown to be safe and efficacious in independent early-phase clinical trials of subretinal injection of a gene therapeutic named Luxturna® (INN: voretigene neparvovec) (Cross, N., et al., 2022, Clin Ophthalmol. 16:2909-2921). Studies of canine and murine animal models with RPE65 deficiency have had a major positive impact on the relatively rapid translation to human clinical trials of this gene therapy (den Hollander, et al., 2008, Prog Retin Eye Res. 27:391- 419.; Cideciyan A. V., et al., 2010, Prog Retin Eye Res. 29: 398 - 427).

Retinitis Punctata Albesciens (RPA) is a form of RP that exhibits a shortage of l l -cz.s- retinal in the rods. Recently, homozygous frameshift mutations in LRAT were identified as a cause of RPA in certain subjects and it has been reported that LRAT is the fourth gene involved in the visual cycle that may cause a white-dot retinopathy (Littink et al., Ophthalmology, 119: 1899-906 (2012)).

Congenital Stationary Night Blindness (CSNB) and Fundus Albipunctatus are a group of diseases that are manifested as night blindness, but there is not a progressive loss of vision as in RP. Some forms of CSNB are due to a delay in the recycling of 1 l-czs-retinal. Fundus Albipunctatus has been shown to be also a progressive disease although much slower than RP. It is caused by gene defects that lead to a delay in the cycling of l l-czs-retinal, including heterozygous mutations in RPE65 (Schatz et al., Ophthalmology, 118:888-94 (2011)).

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world. Clinically, atrophic AMD represents a slowly progressing neurodegenerative disorder in which specialized neurons (rod and cone photoreceptors) die in the central part of the retina called the macula. Histopathological and clinical imaging studies indicate that photoreceptor degeneration in dry AMD may be triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath photoreceptors and provides crucial metabolic support to these lightsensing neuronal cells. There is no FDA-approved treatment for the most prevalent dry (atrophic) form of AMD (Petrukhin, K., et al., 2013, Drug Discovery Today:Therapeutic Strategies 10: l,el l-e20).

The major part of strategies directed for the treatment of retinal diseases including RP are based on targeted therapies such as gene therapy. From the 133 drugs currently in clinical development for RP, around 50% are represented by gene or cell therapies, i.e. targeted approaches, while the remaining ones can be divided into antibody, recombinant protein, peptide and small molecule therapies (Cross, N., et al., 2022, Clin Ophthalmol. 16:2909-2921). Targeted therapies are complicated by multiple gene mutations being linked to the same subgroup of retinal dystrophy. Another approach for the treatment of retinal diseases is a retinoid- based approach and applies artificial visual chromophore therapy for the treatment of retinal diseases caused by defects in the visual cycle. This approach however requires the development of synthetic retinoids since retinoids involved in the vision cycle, in particular, the involved aldehydes are not only very unstable and thus not conducive to pharmaceutical development (Koenekoop, R. K., et al., 2014, Lancet 384: 1513-1520; Palczewski K., 2010, Trends Pharmacol Sci, 31 :284-295) but, furthermore, they have been shown to be toxic in numerous studies (Travis, GH., et al., 2007, Annu Rev Pharmacol Toxicol 2007, 47:469-512; Collins MD. and Mao GE., 1999, Annu Rev Pharmacol Toxicol, 39:399-430). As an example for such artificial visual chromophore therapy, an exogenous chromophore, 9-cv.s-retinyl acetate, is used as a prodrug that undergoes hydrolysis and oxidation in vivo to generate the pharmacologically active 9-cv.s-retinal molecule. An open-label phase lb trial with patients aged 6 years or older with LCA and RPE65 or LRAT mutations showed that the treatment was well tolerated and resulted in clinically meaningful improvements in visual function in most patients (Koenekoop, R. K., et al., 2014, Lancet 384: 1513-1520; WO 2013/134867). However, no follow-up studies have ever since been reported.

Thus, there is at present no cure or effective treatment to slow or stop disease progression for most individuals with retinal diseases, disorders and dystrophies.

SUMMARY OF THE INVENTION

The present invention relates to the use of CRALBP based compositions and complexes for the treatment of multiple groups of retinal diseases and disorders that are, in particular caused by endogenous retinoid deficiency. Thus, we have surprisingly found that treatment using compositions comprising mono-, di- or oligomeric complexes of either wild-type or engineered CRALBP mutant protein with its cognate ligand, preferably with its cognate cisretinoid, can overcome endogenous retinoid deficiency. In detail, we have found that the application of these CRALBP based compositions and complexes to the vitreous of the eye through intravitreal injection, under scotopic conditions, surprisingly revealed rod photoreceptor light sensitivity recovery to near physiologic levels. The collection of in vitro and in vivo data using a chromophore deficient RPE65-/- mouse model yielded consistent data of rod receptor recovery and increased bleaching resistance. The RPE65-/- mouse model was chosen because the enzyme termed RPE65 catalyses the conversion of the light-insensitive all- trans retinyl ester to light-sensitive 11-cis retinol for promoting c/.s-retinoid regeneration within the pigment epithelium of the mouse retina (RPE) (Moiseyev, G., et al., 2005, Proc. Nat. Acad. Sci. 102: 12413-12418). The abrogation of the enzyme’s activity in the RPE65-/- mouse is characterized by the virtual absence of l l -cv.s retinal chromophore regeneration and related absence of light-sensitive rod photoreceptors i.e. a hallmark of impaired vision (Maeda, T., et al., 2009, Invest Ophthalmol Vis Sci. 50(9):4368-78). In vitro and in vivo restoration of lightsensitive rod photoreceptors in such mouse model is a strong indicator for the high biological potency of the treatment. The compositions and complexes used in the present invention and acting as storage devices for an efficient and safe delivery of the retinoids, preferably cisretinoids, to the retinal photoreceptors, are efficiently addressing retinoid deficiencies of the retina.

Thus, in a first aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, preferably of said human, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cA-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein preferably said disease or disorder is a retinal disease or disorder, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cA-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a retinal disease or disorder of an animal, preferably of a human, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cA-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, preferably of said human, wherein said disease or disorder is a retinal disease or disorder, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cA-retinoid. In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is an inherited retinal disease or disorder, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cv.s-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is an inherited retinal disease or disorder associated with a retinoid deficiency of the retina of said animal, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cv.s-retinoid.

In a preferred emdodiment, said disease or disorder is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said disease or disorder is RP.

In a further aspect, the present invention provides a composition for use in a method of maintaining or improving visual function in an animal, preferably in a human, having an endogenous retinoid deficiency, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

In a further aspect, the present invention provides a composition for use in a method of maintaining or improving visual function in an animal, preferably in a human, having a retinoid deficiency of the retina, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid. In another aspect, the present invention provides a composition for use in a method of reducing or preventing chromphore depletion in an animal, preferably in a human, having an endogenous retinoid deficiency, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

In another aspect, the present invention provides a composition for use in a method of reducing or preventing chromphore depletion in an animal, preferably in a human, having a retinoid deficiency of the retina, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

In a preferred emdodiment, said endogenous retinoid deficiency is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), preferably dry AMD, Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said endogenous retinoid deficiency is RP. In a preferred embodiment, said animal is a human.

In a preferred emdodiment, said retinoid deficiency of the retina is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), preferably dry AMD, Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said endogenous retinoid deficiency is RP. In a preferred embodiment, said animal is a human.

In a further preferred embodiment, said CRALBP protein has an amino acid sequence selected from group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

Further aspects and embodiments of the present invention will be become apparent as this description continues. DESCRIPTION OF FIGURES

FIG. 1 : Close up view of the mobile gate region of CRALBP (closed conformation) with the position and orientation of the di-cysteine A212C:T250C mutation highlighted as sticks. Mutations were introduced using Coot and side chain rotamers arranged using the program’s rotamer library. Distances of 1.9 - 3.1 A between the terminal sulfhydryl groups of C212 and C250 are conducive with disulfide bond (2.05 A) formation under oxidizing conditions. 3-D overlay with the X-ray structural model (loiz.pdb) of the mobile gate moiety of the apo-form of alpha-TTP illustrates the extent of the conformational change between the closed and the open state.

FIG. 2: Close up view of the in silico model of the mobile gate region of the four mono di-cysteine mutants of CRALBP with the position of the formed di-sulfide bonds highlighted as sticks.

FIG. 3 : Close up view of the in silico model of the mobile gate region of the three double and the triple di -cysteine mutant of CRALBP with the position of the formed di-sulfide bonds highlighted as sticks.

FIG. 4: Superposition of all in silico di-cysteine mutant models showing four possible di-sulfide bonds across the mobile gate.

FIG. 5: Chromatogram of preparative GFC on a Superdex® 200 26/60 after loading CRALBP with 9-cz.s-retinal. Panel A shows wild-type CRALBP and panel B the di-cysteine mutant A212CT250C.

FIG. 6: UV-Vis absorption spectra of monomeric wild type CRALBP and the A212C:T250C mutant in complex with 9-cis retinal. Spectra are normalized to the maximum absorption, of the protein at approximately 280 nm. While this value corresponds to the protein concentration, the absorption maximum for 9-cis retinal bound to CRALBP is at 400 nm. The fully saturated CRALBP in complex with 9-cis retinal is reported to feature an ideal spectral ratio of g²⁸⁰/g⁴⁰⁰ = 2.2. The measured ratios for wild-type CRALBP:9-cz.s retinal and for the A212C:T250C:9-cz retinal mutant are 2.31 and 2.21, respectively.

FIG. 7: Overlay of analytical GFC traces at 280 nm of monomeric, HMW, and SHMW fractions of CRALBP after pooling and concentrating the corresponding fraction from the preparative GFC. Panel A shows GFC traces of the wild-type CRALBP and panel B of the dicysteine mutant A212CT250C.

FIG. 8: Photostability half-lives (ti/2) of both redox states of the di-cysteine CRALBP mutant in complex with czs-retinal, respectively. The inventive di-cysteine mutant A212C:T250C within CRALBP represents a redox switch that locks the visual pigment increasing its photostability 20-fold at 1000 lux. Half-lives were calculated using the standard formula for first-order reactions (ti/2) = Ln(2)/R.

FIG. 9: Photoisomerization of the oxidized mutant A212C:T250C as oxidized using 5mM oxidized glutathione. The preformed complex was oxidized overnight at 4°C using 5mM oxidized glutathione. From these data, reaction velocities for the different molecules have been determined for comparison purposes. The values are as follows: k (wild-type) = 6.324* 10'⁵s^_1, k (ox. A212C:T250C : l l-cz.s-retinal) = 0.644* 10'⁵s^_1, k (red. A212CT250C : l l -cz.s-retinal) = 5.988* 10'⁵S^_1. NO photoisomerization was detectable in the case of oxidized A212C:T250C:9- cz -retinal.

FIG. 10: Image of a 3-12% Native PAGE featuring individual fractions from the preparative GFC of CRALBP after loading with 9-cz.s-retinal. Panel A shows the wild-type CRALBP. Description of lanes: M, Marker (Invitrogen™ NativeMark™); Pl and Pl .2 SHMW fractions (C4-E8); P2 HMW Fractions (E8); P4 monomeric fraction (F7). Panel B shows the di-cysteine mutant A212C:T250C. Description of lanes: M, Marker (Invitrogen™ NativeMark™); Pl and Pl.2 SHMW fractions (5-8); P2 HMW Fractions (12-13); P4 monomeric fraction (26).

FIG. 11 : Bar chart showing the average size, including error (±SD), of different complex fractions of wild-type CRALBP and CRALBP di-cysteine mutant A212C:T250C after loading with 9-cz.s-retinal. Panel A, complex of monomeric wild-type CRALBP with 9-cz.s-retinal is 6.45 ± 0.76 nm, dimeric complex 7.07 ± 0.29 nm, HMW 12.89 ± 0.89 nm and SHMW 21.43 ± 7.07 nm respectively. Panel B, Di-cysteine mutant A212C:T250C CRALBP with 9-cz.s-retinal monomeric complex is 6.50 ± 1.50 nm, dimeric complex 8.72 ± 2.41 nm, HMW 13.50 ± 0.47 nm, and SHMW 28.28 ± 2.13 nm respectively.

FIG. 12: Representative results for ex-vivo ERGs. Ex-vivo ERGs recordings using the enclosed chamber perfused with Locke’s solution and supplemented with 2 mM L-glutamate and lOpM DL-ammino-4-phosphonobutyric acid (DL-AP4) to block postsynaptic components of the response. Additionally, 20 pM BaC12 was added to suppress the slow glial Pill component (Nymark et al., 2005). Panel A, rod-driven a-waves from retina stimulated with 20ms, 505 nm LED flashes delivering 0.5 log unit steps of light intensity. Retinas were treated with monomeric and super high molecular weight (SHMW) CRALBP wild-type preparations. The CRALBP solution contained approximately 1.62 mM of 9-cz.s-retinal and was diluted to in 1.8 ml of L15 cell culture solution (13.6mg/ml, pH 7.4, Sigma) containing 1% BSA and suspended thoroughly. The final concentration of the retinoid was 162 pM. An isolated RPE65- I- retina was incubated in a Petri dish with 2ml of diluted wilt-type CRALBP/9-cis-retinal solution in an oxygenated container for 4-4.5h in the dark. Panel B, rod-driven a- waves from retinas as described for panel B but treated with monomeric and SHMW CRALBP A212C:T250C mutant complexed to 9-cis-retinal. All error bars represent mean values ± SEM using the independent two-tailed Student’s Z-test, with a significance threshold of P<0.05.

FIG. 13 : Representative results for in vivo ERGs of RPE65-/- mice treated with wild-type monomeric CRALBP/9-cis-retinal complexes. Averaged ERG responses of RPE65-/- mice injected into the vitreous of the eye. The injections were performed by hand using a Hamilton syringe under the microscope and infrared illumination. Control eyes were injected with the same volume of PBS as the treated eyes. Mice were kept all the time in the dark and allowed to recover. The animals returned after the anesthesia in their original cages with access to food and water. Recordings of intensity-response were carried out after 25-30 h (day 1) and 48-72 h (days 3-4). Panel A, Averaged ERG a-wave responses. Panel B, Averaged ERG b-wave responses. All error bars represent mean values ± SEM using the independent two-tailed Student’s Z-test, with a significance threshold of P<0.05.

FIG. 14: Representative results for in vivo ERGs of RPE65-/- mice treated with wild-type super high molecular weight (SHMW) CRALBP/9-cis-retinal complexes. Averaged ERG responses of RPE65-/- mice injected into the vitreous of the eye. The injections were performed by hand using a Hamilton syringe under the microscope and infrared illumination. Control eyes were injected with the same volume of PBS as the treated eyes. Mice were kept all the time in the dark and allowed to recover. The animals returned after the anesthesia in their original cages with access to food and water. Recordings of intensity-response were carried out after 25-30 h (day 1) and 48-72 h (days 3-4). Panel A, Averaged ERG a-wave responses. Panel B, Averaged ERG b-wave responses. All error bars represent mean values ± SEM using the independent two-tailed Student’s Z-test, with a significance threshold of P<0.05.

FIG. 15: Representative results for in vivo ERGs of RPE65-/- mice treated with mutant A212C:T250C monomeric CRALBP/9-cis-retinal complexes. Averaged ERG responses of RPE65-/- mice injected into the vitreous of the eye. The injections were performed by hand using a Hamilton syringe under the microscope and infrared illumination. Control eyes were injected with the same volume of PBS as the treated eyes. Mice were kept all the time in the dark and allowed to recover. The animals returned after the anesthesia in their original cages with access to food and water. Recordings of intensity-response were carried out after 25-30 h (day 1) and 48-72 h (days 3-4). Panel A, Averaged ERG a-wave responses. Panel B, Averaged ERG b-wave responses. All error bars represent mean values ± SEM using the independent two-tailed Student’s /-test, with a significance threshold of P<0.05.

FIG. 16: Representative results for in vivo ERGs of RPE65-/- mice treated with mutant A212C:T250C super high molecular weight (SHMW) CRALBP/9-cis-retinal complexes. Averaged ERG responses of RPE65-/- mice injected into the vitreous of the eye. The injections were performed by hand using a Hamilton syringe under the microscope and infrared illumination. Control eyes were injected with the same volume of PBS as the treated eyes. Mice were kept all the time in the dark and allowed to recover. The animals returned after the anesthesia in their original cages with access to food and water. Recordings of intensityresponse were carried out after 25-30 h (day 1) and 48-72 h (days 3-4). Panel A, Averaged ERG a-wave responses. Panel B, Averaged ERG b-wave responses. All error bars represent mean values ± SEM using the independent two-tailed Student’s /-test, with a significance threshold ofP<0.05.

FIG. 17 : Representative results for dark adaptation in vivo ERGs of RPE65-/- mice treated with wild-type monomeric and SHMW CRALBP/9-cis-retinal complexes. Dark adaptation ERGs were performed, and results were reported as sensitivity as a function of time. A, Ratio of a-wave amplitudes normalized to the pre-bleach a-wave amplitude. 7>, Ratio of a-wave amplitudes normalized to the pre-bleach a-wave amplitude and per photon as described in the following formula: Sf=A/(Amax*I), I is the bleaching intensity (1.3xl0^A8 photons pm-2 s-1). All error bars represent mean values ± SEM using the independent two-tailed Student’s /-test, with a significance threshold of P<0.05.

Fig. 18 : Representative results for dark adaptation in vivo ERGs of RPE65-/- mice treated with mutant A212C:T250C monomeric and SHMW CRALBP/9-cis-retinal complexes. Dark adaptation ERGs were performed, and results were reported as sensitivity as a function of time. A, Ratio of a-wave amplitudes normalized to the pre-bleach a-wave amplitude. 7>, Ratio of a-wave amplitudes normalized to the pre-bleach a-wave amplitude and per photon as described in the following formula: Sf=A/(Amax*I), I is the bleaching intensity (1.3xl0^A8 photons pm-2 s-1). All error bars represent mean values ± SEM using the independent two- tailed Student’s /-test, with a significance threshold of P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The herein described and disclosed embodiments, preferred embodiments and very preferred embodiments should apply to all aspects and other embodiments, preferred embodiments and very preferred embodiments irrespective of whether is specifically again referred to or its repetition is avoided for the sake of conciseness. The articles “a” and “an”, as used herein, refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The term “or”, as used herein, should be understood to mean “and/or”, unless the context clearly indicates otherwise. As used herein, the terms "about" when referring to any numerical value are intended to mean a value of ±10% of the stated value. In a preferred embodiment, said “about” when referring to any numerical value are intended to mean a value of ±5% of the stated value. In another preferred embodiment, said “about” when referring to any numerical value are intended to mean a value of ±3% of the stated value.

CRALBP protein: As used herein, the term "CRALBP protein" refers to a protein comprising or consisting of a wild-type CRALBP protein, or comprising or consisting of a CRALBP mutant protein, as defined herein.

Wild-type CRALBP protein: As used herein, the term "wild-type CRALBP protein" refers to a cellular retinal binding protein (CRALBP) as occurred in nature for an animal, preferably a mammal, and further preferably for a human. Thus, preferably wild-type CRALBP protein refers to human wild-type CRALBP protein of SEQ ID NO:3.

CRALBP mutant protein: As used herein, the term “CRALBP mutant protein” refers to a mutein of a wild-type CRALBP protein, preferably of human wild-type CRALBP protein of SEQ ID NO:3. Preferably, the term “CRALBP mutant protein” refers to a mutein of human wild-type CRALBP protein of SEQ ID NO: 3 having a sequence identity of at least 90% with SEQ ID NO:3.

The term "mutein", as used herein, refers to a protein or polypeptide differing by one or more amino acids from a given reference (e.g. natural, wild-type, etc. protein or polypeptide, wherein such difference is caused by addition, substitution or deletion of at least one amino acid or a combination thereof. Preferred embodiments comprise mutations derived from substitution of at least one amino acid, preferably derived from conservative substitution of at least one amino acid. Conservative substitutions include isosteric substitutions, substitutions where the charged, polar, aromatic, aliphatic or hydrophobic nature of the amino acid is maintained. For example, substitution of a cysteine residue with a serine residue is a conservative substitution. In preferred embodiment, the term "mutein" refers to a mutein of a wild-type CRALBP protein having a sequence identity of at least 90 % with said wild-type CRALBP protein, or to a mutein of a wild-type CRALBP protein differing by at most 30, typically and preferably by at most 20 or 10 amino acids from said wild-type CRALBP protein. In a further preferred embodiment, the term "mutein" refers to a mutein of a wild-type CRALBP protein, preferably of SEQ ID NO:3, having a sequence identity of at least 90 %, 91%, 92%, 93%, 94, 95%, 96% with said wild-type CRALBP protein, preferably of SEQ ID NO:3, or to a mutein of a wild-type CRALBP protein, preferably of SEQ ID NO:3, differing by at most 30, typically and preferably by at most 20, or 10, 9, 8, 7, 6 amino acids from said wild-type CRALBP protein, preferably of SEQ ID NO:3.

Position corresponding to amino acid residues... : The position on an amino acid sequence, which is corresponding to given residues of another amino acid sequence can be identified by sequence alignment, typically and preferably by using the BLASTP algorithm, most preferably using the standard settings. Typical and preferred standard settings are: expect threshold: 10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gap costs: existence 11, extension 1; compositional adjustments: conditional compositional score matrix adjustment. By way of example, in a preferred embodiment of the present invention, one cysteine of each pair of amino acid mutations by cysteines is a mutation of an amino acid within the amino acid residues corresponding to amino acids 204-229 of SEQ ID NO:3, wherein the other mutated amino acid by cysteine of said pair is a mutation of an amino acid within the amino acid residues corresponding to amino acids 244-261 of SEQ ID NO: 3. Taking into account that SEQ ID NO: 3 refers to human wild-type CRALBP, the corresponding specific animal or mammal CRALBP positions are therefore corresponding to said human wild-type CRALBP.

The terms “able of forming a disulfide bond” and “capable of forming a disulfide bond”, as interchangeably used herein and referring to pairs of cysteines being amino acid mutations as compared to the wild-type CRALBP protein, typically and preferably refer to the ability and capability, respectively, of the mutated cysteines to form disulfide bonds as typically and preferably determined in a manner as described in Example 3.

Sequence identity: The sequence identity of two given amino acid sequences is determined based on an alignment of both sequences. Algorithms for the determination of sequence identity are available to the artisan. Preferably, the sequence identity of two amino acid sequences is determined using publicly available computer homology programs such as the “BLAST” program (http://blast.ncbi.nlm.nih. ov/Blast.c i) or the “CLUSTALW” (http ://www. enome. i p/tool s/ clustal w/), and hereby preferably by the “BLAST” program provided on the NCBI homepage at http://blast.ncbi.nlm.nih.gov/Blast.cgi, using the default settings provided therein. Typical and preferred standard settings are: expect threshold: 10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gap costs: existence 11, extension 1; compositional adjustments: conditional compositional score matrix adjustment. The term “amino acid exchange” refers to the exchange of a given amino acid residue in an amino acid sequence by any other amino acid residue having a different chemical structure, preferably by another proteinogenic amino acid residue. Thus, in contrast to insertion or deletion of an amino acid, the amino acid exchange does not change the total number of amino acids of said amino acid sequence. In case of an amino acid exchange within the present invention and referring to typically and preferably non-functional amino acid substitutions, conservative amino acid substitutions are preferred. Conservative amino acid substitutions, as understood by a skilled person in the art, include, and typically and preferably consist of isosteric substitutions, substitutions where the charged, polar, aromatic, aliphatic or hydrophobic nature of the amino acid is maintained. Typical conservative substitutions are substitutions between amino acids within one of the following groups: Gly, Ala; Vai, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr, Cys; Lys, Arg; and Phe and Tyr.

The term “polypeptide” as used herein refers to a polymer composed of amino acid monomers which are linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. The term “polypeptide” as used herein should also refer, typically and preferably to a polypeptide as defined before and encompassing modifications such as post- translational modifications, including but not limited to glycosylations. In a preferred embodiment, said term “polypeptide” as used herein should refer to a polypeptide as defined before and not encompassing modifications such as post-translational modifications such as glycosylations. In particular, for said biologically active peptides, said modifications such as said glycosylations can occur even in vivo thereafter, for example, by bacteria.

A cognate ligand of CRALBP: The term “a cognate ligand of CRALBP”, as used herein, refers to a molecule that binds the binding pocket of wild-type CRALBP protein, preferably human wild-type CRALBP protein of SEQ ID NO:3, with at least nanomolar affinity of typically and preferably 1-200 nM, further preferably of 1-100 nM, further typically and preferably with an affinity of 6-80 nM (wherein said affinity is typically and preferably determined as described in Golovleva I., et al., 2003, J. Biol. Chem. 278(14)), and which molecule is typically and preferably functionally associated with the wild-type CRALBP protein. Functionally associated: The term “functionally associated”, as used herein, refers to the physiological function of the wild-type CRALBP protein, preferably of human wild-type CRALBP protein of SEQ ID NO:3.

The term “cA-retinoid”, as used herein, refers to a natural or synthetic molecule, typically and preferably a Vitamin A derivative, comprising a cyclohexen or a phenyl moiety, typically and preferably substituted by one or more methyl and/or methoxy substituents, which cyclohexen or phenyl moiety is (further) substituted by a Ce-Cu linear or branched alkenyl group having at its terminal carbon atom (terminal carbon atom in relation to its attachment to the cyclohexen or phenyl moiety) an alcohol, aldehyde, carboxy or an ester functionality, wherein said linear or branched alkenyl group have at least one carbon-carbon double bond, typically and preferably one or more, most preferably four carbon-carbon double bonds, wherein at least one of said carbon-carbon double bond is in the cis configuration. Said cis- retinoid includes retinol, retinaldehyde, and tretinoin, isotretinoin, alitretinoin, etretinate, acitretin as well as its esters, of which retinol and retinaldehyde are preferred. C/.s-retinoids have been described (Mukherjee S et al., 2006, Clin Interv Aging. l(4):327-48; Kiser PD et al., 2014, Chem. Rev. 114: 194-232).

The term “complex”, as used herein, in particular if referred to a complex comprising a CRALBP protein and a cognate ligand of CRALBP, refers to (i) a 1 : 1 monomeric complex of said CRALBP protein and said cognate ligand of CRALBP, wherein said cognate ligand of CRALBP binds said CRALBP protein, typically and preferably with at least nanomolar affinity of 1-200 nM, further preferably of 1-100 nM (wherein said affinity is typically and preferably determined as described in Golovleva I., et al., 2003, J. Biol. Chem. 278(14)); and/or (ii) to a dimeric and/or oligomeric complex of said CRALBP protein and said cognate ligand of CRALBP, wherein it is believed, without being bound by this theory, that typically and preferably said 1 : 1 monomeric complexes dimerizes and/or oligomerizes forming said dimeric and/or oligomeric complexes of said CRALBP protein and said cognate ligand of CRALBP. In a preferred embodiment, said complex comprises, preferably consists of, a wild-type CRALBP protein and a cognate ligand of CRALBP, and hereby refers to (i) a 1 : 1 monomeric complex of said wild-type CRALBP protein and said cognate ligand of CRALBP, wherein said cognate ligand of CRALBP binds said wild-type CRALBP protein, typically and preferably with at least nanomolar affinity of 1-200 nM, further preferably of 1-100 nM (wherein said affinity is typically and preferably determined as described in Golovleva I., et al., 2003, J. Biol. Chem. 278(14)); and/or (ii) to a dimeric and/or oligomeric complex of said wild-type CRALBP protein and said cognate ligand of CRALBP, wherein it is believed, without being bound by this theory, that typically and preferably said 1 : 1 monomeric complexes dimerizes and/or oligomerizes forming said dimeric and/or oligomeric complexes of said wild-type CRALBP protein and said cognate ligand of CRALBP. In another preferred embodiment, said complex comprises, preferably consists of, a CRALBP mutant protein and a cognate ligand of CRALBP, and hereby refers to (i) a 1 : 1 monomeric complex of said CRALBP mutant protein and said cognate ligand of CRALBP, wherein said cognate ligand of CRALBP binds said CRALBP mutant protein, typically and preferably with at least nanomolar affinity of 1-200 nM, further preferably of 1- 100 nM (wherein said affinity is typically and preferably determined as described in Golovleva I., et al., 2003, J. Biol. Chem. 278(14)); and/or (ii) to a dimeric and/or oligomeric complex of said CRALBP mutant protein and said cognate ligand of CRALBP, wherein it is believed, without being bound by this theory, that typically and preferably said 1 : 1 monomeric complexes dimerizes and/or oligomerizes forming said dimeric and/or oligomeric complexes of said CRALBP mutant protein and said cognate ligand of CRALBP. Typically and preferably, the number of said cognate ligands within said dimeric and/or oligomeric complexes of said CRALBP protein, wild-type CRALBP protein or CRALBP mutant protein and said cognate ligand of CRALBP are equal to the number of said CRALBP proteins, wild-type CRALBP proteins or CRALBP mutant proteins. Further, without being bound, it is believed that the interaction between said CRALBP protein, wild-type CRALBP protein or CRALBP mutant protein and said cognate ligand of CRALBP corresponds to the stereospecific interaction as reported in the prior art, in particular for wild-type CRALBP protein and said cognate ligand of CRALBP such as 9-cv.s-retinal and 11-cA-retinal, representing reversible non-covalent interactions typical and preferably including hydrogen bonds, hydrophobic forces, van der Waals forces, electrostatic interactions or the like (He, X., et al., 2009, PNAS, 106(44): 18545— 18550); Bolze, C.S., et al., 2014, JACS, 136(1): 137-146).

The term “retinal disease or disorder”, as used herein, refers to a disease or disorder that cause damage to any part of the retina.

The term “an inherited retinal disease or disorder”, as used herein, refers to a retinal disease or disorder that can cause severe vision loss or even blindness typically by altering the structure and function of the retina, and is caused by at least one gene that is not working as it should. An inherited retinal disease or disorder can affect individuals of all ages, can progress at different rates, and are rare. However, many inherited retinal diseases or disorders are degenerative, in particular with respect to retina, meaning that the symptoms of the disease or disorder will get worse over time.

The term "maintaining or improving visual function”, as used herein shall be understood as the maintenance of substantially the same level or an improvement in the level of vision as assessed by one or more test of visual function, when the vision in a treated eye is compared before and after the methods of the invention have been performed. Preferably, the inventive treatment of the endogenous retinoid deficiencies and/or retinal diseases, disorders and dystrophies including retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age- related macular degeneration (AMD), preferably dry AMD, Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB) enables maintenance or improvement in visual function. Visualisation of the appearance of a retina and assessment of visual function may be readily carried out by the skilled person. For example, visual function tests that might be carried out by the skilled person include best corrected visual acuity, visual field testing, micro perimetry, colour vision, dark adaptometry, electroretinography and cone flicker fusion tests. In a preferred embodiment, said maintaining or improving visual function, as used herein, refers to the maintenance of substantially the same level or an improvement in the level of vision as assessed by one or more test of visual function, when the vision in a treated eye is compared before and after the methods of the invention have been performed, and wherein said one or more test of visual function comprises, preferably consists of, ERG measurements as effected and described in Examples 12 to 14, preferably as effected and described in Example 13 and 14.

In a preferred embodiment of the present invention, visual function is improved, preferably substantially restored, or maintained in the treated eye. Visual function, for example as determined by a test of visual function as described herein, may, for example, be improved, preferably restored to about the same level in an affected eye as existed before the onset of the endogenous retinoid deficiencies, the retinoid deficiencies of the retina, and/or retinal diseases, disorders and dystrophies including RP, LCA, AMD, preferably dry AMD, STGD, COD, CRD, RPA, fundus albipunctatus and CSNB. In a preferred embodiment of the present invention, visual function is improved as compared to a level in an affected eye as existed before the onset of the endogenous retinoid deficiencies and/or retinal diseases, disorders and dystrophies including RP, LCA, AMD, preferably dry AMD, STGD, COD, CRD, RPA, fundus albipunctatus and CSNB and as compared before the methods of the invention have been performed. In a preferred embodiment of the present invention, visual function is maintained at about the same level in a healthy animal or subject at risk of developing an endogenous retinoid deficiency and/or a retinal disease, disorder or dystrophy including RP, LCA, AMD, preferably dry AMD, STGD, COD, CRD, RPA, fundus albipunctatus and CSNB, or in a subject or animal already suffering from an endogenous retinoid deficiency and/or a retinal disease, disorder or dystrophy including RP, LCA, AMD, preferably dry AMD, STGD, COD, CRD, RPA, fundus albipunctatus and CSNB.

Subject: The term “subject”, as used herein refers to an animal, preferably to a human. In a very preferred embodiment said subject is a human.

The term “animal”, as used herein, refers to an animal (e.g., a non human animal), a vertebrate animal, a mammal, a rodent (e.g., a guinea pig, a hamster), a canine (e.g., a dog), a feline (e.g., a cat), a porcine (e.g., a pig), an equine (e.g., a horse), a primate, or a human. In a preferred embodiment said animal is a mammal. In another preferred embodiment said animal is a human or a non-human mammal preferably selected from a dog, a cat, and a horse. In a very preferred embodiment said animal is a human.

The term "treatment" of a disorder or disease as used herein (e.g., "treatment" of a retinal disease) is well known in the art. "Treatment" of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in an animal, subject or patient. An animal or subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease). The "treatment" of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the animal, subject or patient suffering from the disorder or disease. Accordingly, the "treatment" of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that an animal or subject may experience a broad range of responses to a treatment. The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and/or eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief). The "amelioration" of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. The term “prevention”, as used herein refers to a prophylactic treatment.

The treatment with the compositions for use in the methods of the present invention, and in particular, the effectiveness of the treatment with the compositions for use in the methods of the present invention, may be assessed by different measures of visual and ocular function and structure, as referred herein and including, among others, best corrected visual acuity (BCVA), retinal sensitivity to light as measured by perimetry or microperimetry in the dark and light- adapted states, full-field, multi-focal, focal or pattern elecroretinography ERG, contrast sensitivity, reading speed, color vision, clinical biomicroscopic examination, fundus photography, optical coherence tomography (OCT), fundus auto- fluorescence (FAF), infrared and multicolor imaging, fluorescein or ICG angiography, and additional means used to evaluate visual function and ocular structure. The treatment or prevention with the compositions for use in the methods of the present invention, as described herein, may result in protection of the photoreceptor cells, such as the cone cells, from degeneration. Preferably, the treatment protects both cone and rod cells from degeneration. Numbers of rods and cones can be estimated by the skilled person in the clinic using techniques such as adaptive optics, autofluorescence and optical coherence tomography (OCT) scans.

The term "effective amount", as used herein, refers to an amount necessary or sufficient to realize a desired biologic effect. Preferably, the term “effective amount” refers to an amount of an inventive composition or an inventive pharmaceutical composition that (i) treats the particular disease or disorder, and/or (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease or disorder, and/or (iii) or (iii) prevents or delays the onset of one or more symptoms of the particular disease or disorder, described herein. In the case of endogenous retinoid deficiencies and/or retinal diseases, disorders and dystrophies including RP, LCA, AMD, preferably dry AMD, STGD, COD, CRD, RPA, fundus albipunctatus and CSNB, the effective amount of the inventive compositions is capable of maintaining, restoring or improving visual function of the animal or subject, preferably human, and/or preventing or reducing chromphore depletion in an animal or subject, preferably human, and/or reducing clinical symptoms. The "effective amount" may vary depending on the disease and its severity and the age, weight, medical history, susceptibility, and preexisting conditions, of the animal or subject, preferably human to be treated.

The present invention relates to the use of CRALBP based compositions and complexes for the treatment of multiple groups of diseases and disorders, preferably retinal diseases and disorders that are, in particular caused by endogenous retinoid deficiency and/or by retinoid deficiency of the retina. Thus, we have surprisingly found that treatment using compositions comprising mono-, di- or oligomeric complexes of either wild-type or engineered CRALBP mutant protein with its cognate ligand, preferably with its cognate cv.s-retinoid, can overcome endogenous retinoid deficiency and/or by retinoid deficiency of the retina. In detail, we have found that the application of these CRALBP based compositions and complexes to the vitreous of the eye through intravitreal injection, under scotopic conditions, surprisingly revealed rod photoreceptor light sensitivity recovery to near physiologic levels. CRALBP has dual function in that it sequesters the cz.s-retinoid, in particular the l l -cz.s retinal or the 9-cis retinal, and prevents it from premature photoisomerization as well as preventing the cellular context from aldehyde toxicity. CRALBP represents in the vision cycle the most downstream protein component, and has high affinity for the bound cz.s-retinoid, in particular the l l -cz.s retinal or the 9-cis retinal, and chaperones the reloading process of the opsin receptor molecules within the rod and cone photoreceptor cells.

The collection of in vitro and in vivo data using a chromophore deficient RPE65-/- mouse model yielded consistent data of rod receptor recovery and increased bleaching resistance. The RPE65-/- mouse model was chosen because the enzyme termed RPE65 catalyses the conversion of the light-insensitive all-/zzzzz.s retinyl ester to light-sensitive l l -cz.s retinol for promoting cz.s-retinoid regeneration within the pigment epithelium of the mouse retina (RPE) (Moiseyev, G., et al., 2005, Proc. Nat. Acad. Sci. 102: 12413-12418). The abrogation of the enzyme’ s activity in the RPE65-/- mouse is characterized by the virtual absence of 11 -cis retinal chromophore regeneration and related absence of light-sensitive rod photoreceptors i.e. a hallmark of impaired vision (Maeda, T., et al., 2009, Invest Ophthalmol Vis Sci. 50(9):4368- 78). In vitro and in vivo restoration of light-sensitive rod photoreceptors in such mouse model is a strong indicator for the high biological potency of the treatment.

Thus, the provided inventive methods and uses of the described drugs, i.e. the described compositions and complexes, are able to produce meaningful improvement of vision in a subject having an endogenous retinoid deficiency and/or a retinoid deficiency of the retina. Without being bound, from a mechanistic point of view, the treatment is also novel in that it is believed to be not targeting a specific gene defect but instead is meant to circumvent all components of the visual cycle to directly replenish in vitro and in vivo chromophore depleted rod and cone photoreceptors with fresh cz.s-retinoid, in particular cz -retinal. This mechanism thus represents a so far unknown shunt and may fulfil the requirements for a broader, i.e. nontargeted, therapeutic approach.

In a first aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, preferably of said human, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cz.s-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein preferably said disease or disorder is a retinal disease or disorder, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cv.s-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a retinal disease or disorder of an animal, preferably of a human, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a c/.s-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, preferably of said human, and wherein said disease or disorder is a retinal disease or disorder, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a c/.s-retinoid.

In a further aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is an inherited retinal disease or disorder, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cv.s-retinoid.

In a preferred emdodiment, said disease or disorder is a retinal disease or disorder. In another preferred emdodiment, said disease or disorder is an inherited retinal disease or disorder. In another preferred emdodiment, said disease or disorder is an inherited retinal disease or disorder associated with a retinoid deficiency of the retina of said animal, preferably of said human. In a preferred emdodiment, said disease or disorder is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said disease or disorder is RP. In a preferred emdodiment, said retinal disease or disorder is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said disease or disorder is RP. In a preferred emdodiment, said inherited retinal disease or disorder is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said disease or disorder is RP.

In a preferred embodiment, the disease or disorder is retinitis pigmentosa (RP). In a preferred embodiment, the disease or disorder is moderate to severe RP. In a preferred embodiment, the disease or disorder is mild RP. In a preferred embodiment, the disease or disorder is early onset or juvenile RP. In a preferred embodiment, the disease or disorder is Leber congenital amaurosis (LCA). In a preferred embodiment, the disease or disorder is age- related macular degeneration (AMD), preferably dry AMD. In a preferred embodiment, the disease or disorder is Stargardt disease (STGD). In a preferred embodiment, the disease or disorder is cone dystrophy (COD). In a preferred embodiment, the disease or disorder is conerod dystrophy (CRD). In a preferred embodiment, the disease or disorder is retinitis punctata albesciens (RPA). In a preferred embodiment, the disease or disorder is fundus albipunctatus. In a preferred embodiment, the disease or disorder is cone stationary night blindness (CSNB). In a preferred embodiment, the disease or disorder is a RPE65 gene mutation. In a preferred embodiment, the disease or disorder is a LRAT gene mutation. In a preferred embodiment, the retinal disease or disorder is retinitis pigmentosa (RP). In a preferred embodiment, the retinal disease or disorder is moderate to severe RP. In a preferred embodiment, the retinal disease or disorder is mild RP. In a preferred embodiment, the retinal disease or disorder is early onset or juvenile RP. In a preferred embodiment, the retinal disease or disorder is Leber congenital amaurosis (LCA). In a preferred embodiment, the retinal disease or disorder is age-related macular degeneration (AMD), preferably dry AMD. In a preferred embodiment, the retinal disease or disorder is Stargardt disease (STGD). In a preferred embodiment, the retinal disease or disorder is cone dystrophy (COD). In a preferred embodiment, the retinal disease or disorder is cone-rod dystrophy (CRD). In a preferred embodiment, the retinal disease or disorder is retinitis punctata albesciens (RPA). In a preferred embodiment, the retinal disease or disorder is fundus albipunctatus. In a preferred embodiment, the retinal disease or disorder is cone stationary night blindness (CSNB). In a preferred embodiment, the retinal disease or disorder is a RPE65 gene mutation. In a preferred embodiment, the retinal disease or disorder is a LRAT gene mutation. In a preferred embodiment, the inherited retinal disease or disorder is retinitis pigmentosa (RP). In a preferred embodiment, the inherited retinal disease or disorder is moderate to severe RP. In a preferred embodiment, the inherited retinal disease or disorder is mild RP. In a preferred embodiment, the inherited retinal disease or disorder is early onset or juvenile RP. In a preferred embodiment, the inherited retinal disease or disorder is Leber congenital amaurosis (LCA). In a preferred embodiment, the inherited retinal disease or disorder is age-related macular degeneration (AMD), preferably dry AMD. In a preferred embodiment, the inherited retinal disease or disorder is Stargardt disease (STGD). In a preferred embodiment, the inherited retinal disease or disorder is cone dystrophy (COD). In a preferred embodiment, the inherited retinal disease or disorder is cone-rod dystrophy (CRD). In a preferred embodiment, the inherited retinal disease or disorder is retinitis punctata albesciens (RPA). In a preferred embodiment, the inherited retinal disease or disorder is fundus albipunctatus. In a preferred embodiment, the inherited retinal disease or disorder is cone stationary night blindness (CSNB). In a preferred embodiment, the inherited retinal disease or disorder is a RPE65 gene mutation. In a preferred embodiment, the inherited retinal disease or disorder is a LRAT gene mutation.

In a further aspect, the present invention provides a composition for use in a method of maintaining or improving visual function in an animal, preferably in a human, having a retinoid deficiency of the retina, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

In another aspect, the present invention provides a composition for use in a method of reducing or preventing chromphore depletion in an animal, preferably in a human, having an endogenous retinoid deficiency, wherein said method comprises administering, preferably by way of intravitreal administration, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP protein; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

In a preferred emdodiment, said endogenous retinoid deficiency is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), preferably dry AMD, Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation wherein preferably said endogenous retinoid deficiency is RP. In another preferred emdodiment, said retinoid deficiency of the retina is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), preferably dry AMD, Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said endogenous retinoid deficiency is RP. In a preferred embodiment, said animal is a human.

In a preferred embodiment, the endogenous retinoid deficiency is retinitis pigmentosa (RP). In a preferred embodiment, the endogenous retinoid deficiency is moderate to severe RP. In a preferred embodiment, the endogenous retinoid deficiency is mild RP. In a preferred embodiment, the endogenous retinoid deficiency is early onset or juvenile RP. In a preferred embodiment, the endogenous retinoid deficiency is Leber congenital amaurosis (LCA). In a preferred embodiment, the endogenous retinoid deficiency is age-related macular degeneration (AMD), preferably dry AMD. In a preferred embodiment, the endogenous retinoid deficiency is Stargardt disease (STGD). In a preferred embodiment, the endogenous retinoid deficiency is cone dystrophy (COD). In a preferred embodiment, the endogenous retinoid deficiency is conerod dystrophy (CRD). In a preferred embodiment, the endogenous retinoid deficiency is retinitis punctata albesciens (RPA). In a preferred embodiment, the endogenous retinoid deficiency is fundus albipunctatus. In a preferred embodiment, the endogenous retinoid deficiency is cone stationary night blindness (CSNB). In a preferred embodiment, the endogenous retinoid deficiency is a RPE65 gene mutation. In a preferred embodiment, the endogenous retinoid deficiency is a LRAT gene mutation.

In a preferred embodiment, the retinoid deficiency of the retina is retinitis pigmentosa (RP). In a preferred embodiment, the retinoid deficiency of the retina is moderate to severe RP. In a preferred embodiment, the retinoid deficiency of the retina is mild RP. In a preferred embodiment, the retinoid deficiency of the retina is early onset or juvenile RP. In a preferred embodiment, the retinoid deficiency of the retina is Leber congenital amaurosis (LCA). In a preferred embodiment, the retinoid deficiency of the retina is age-related macular degeneration (AMD), preferably dry AMD. In a preferred embodiment, the retinoid deficiency of the retina is Stargardt disease (STGD). In a preferred embodiment, the retinoid deficiency of the retina is cone dystrophy (COD). In a preferred embodiment, the retinoid deficiency of the retina is conerod dystrophy (CRD). In a preferred embodiment, the retinoid deficiency of the retina is retinitis punctata albesciens (RPA). In a preferred embodiment, the retinoid deficiency of the retina is fundus albipunctatus. In a preferred embodiment, the retinoid deficiency of the retina is cone stationary night blindness (CSNB). In a preferred embodiment, the retinoid deficiency of the retina is a RPE65 gene mutation. In a preferred embodiment, the retinoid deficiency of the retina is a LRAT gene mutation.

In a preferred embodiment, the animal, preferably the human, has retinitis pigmentosa (RP). In a preferred embodiment, the animal, preferably the human, has moderate to severe RP. In a preferred embodiment, the animal, preferably the human, has mild RP. In a preferred embodiment, the animal, preferably the human has early onset or juvenile RP. In a preferred embodiment, the animal, preferably the human, has Leber congenital amaurosis (LCA). In a preferred embodiment, the animal, preferably the human, has age-related macular degeneration (AMD), preferably dry AMD. In a preferred embodiment, the animal, preferably the human, has Stargardt disease (STGD). In a preferred embodiment, the animal, preferably the human, has cone dystrophy (COD). In a preferred embodiment, the animal, preferably the human, has cone-rod dystrophy (CRD). In a preferred embodiment, the animal, preferably the human, has retinitis punctata albesciens (RPA). In a preferred embodiment, the animal, preferably the human, has fundus albipunctatus. In a preferred embodiment, the animal, preferably the human, has cone stationary night blindness (CSNB). In a preferred embodiment, the animal, preferably the human, has a RPE65 gene mutation. In a preferred embodiment, the animal, preferably the human, has a LRAT gene mutation.

In a preferred embodiment, said composition is administered to the eye of said animal, preferably of said human, by subretinal, direct retinal or intravitreal injection, wherein preferably said composition is administered to the eye of said animal, preferably said human, by intravitreal injection. The skilled person will be familiar with and well able to carry out individual subretinal, direct retinal or intravitreal injections. Subretinal injections are injections into the subretinal space, i.e. underneath the neurosensory retina. Intravitreal is a route of administration of a drug, or other substance, in which the substance is delivered into the vitreous humor of the eye, i.e. administration directly into the vitreous chamber. In a preferred embodiment, the visual function of said animal, preferably of said human, is substantially restored or maintained in the treated eye.

In a preferred embodiment, said animal is a human.

In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein. In a preferred embodiment, said CRALBP protein comprises a CRALBP mutant protein. In a preferred embodiment, said CRALBP protein consists of a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said CRALBP protein consists of a wild-type CRALBP protein. In a preferred embodiment, said CRALBP protein consists of a CRALBP mutant protein. In a preferred embodiment, said CRALBP protein is a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said CRALBP protein is a wild-type CRALBP protein. In a preferred embodiment, said CRALBP protein is a CRALBP mutant protein.

In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein. In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein, and wherein said wild-type CRALBP protein comprises human wild-type CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein comprises a wildtype CRALBP protein, and wherein said wild-type CRALBP protein consists of human wildtype CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein, and wherein said wild-type CRALBP protein is the human wild-type CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein, and wherein said CRALBP protein comprises human wild-type CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein, and wherein said CRALBP protein consists of human wild-type CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein consists of a wild-type CRALBP protein, and wherein said CRALBP protein consists of human wild-type CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein comprises a wild-type CRALBP protein, and wherein said CRALBP protein is the human wild-type CRALBP protein of SEQ ID NO:3. In a preferred embodiment, said CRALBP protein consists of a wild-type CRALBP protein, and wherein said CRALBP protein is the human wild-type CRALBP protein of SEQ ID NO:3.

In a preferred embodiment, said CRALBP protein comprises a CRALBP mutant protein, wherein said CRALBP mutant protein comprises a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond. In a preferred embodiment, said CRALBP protein comprises a CRALBP mutant protein, wherein said CRALBP mutant protein consists of a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond. In a preferred embodiment, said CRALBP protein comprises a CRALBP mutant protein, wherein said CRALBP protein comprises a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond. In a preferred embodiment, said CRALBP protein comprises a CRALBP mutant protein, wherein said CRALBP protein consists of a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond. In a preferred embodiment, said CRALBP protein consists of a CRALBP mutant protein, wherein said CRALBP protein consists of a mutein of a wild-type CRALBP protein, wherein said mutein consists of at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond.

Thus, in another aspect, the present invention provides a composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, wherein said method comprises administering, preferably by intravitreal injection, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein is a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a c/.s-retinoid.

In another aspect, the present invention provides a complex for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, wherein said method comprises administering, preferably by intravitreal injection, said complex to said animal, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein is a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

In another aspect, the present invention provides a composition for use in a method of preventing or treating a retinal disease or disorder of an animal, preferably of a human, wherein said method comprises administering, preferably by intravitreal injection, said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein is a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond, and wherein one cysteine of each pair of amino acid mutations by cysteines is a mutation of an amino acid within the amino acid residues corresponding to amino acids 204-229 of SEQ ID NO:3, wherein the other mutated amino acid by cysteine of said pair is a mutation of an amino acid within the amino acid residues corresponding to amino acids 244-261 of SEQ ID NO: 3; and (b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cz.s-retinoid, wherein further preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11-cz -retinal; (iv) 11-cz -retinol; (v) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, again further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9,13-di- cz -retinal; and (vi) 9,13-di-cz.s-retinol, and again further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal and (ii) 11-cz -retinal.

In a preferred embodiment, said CRALBP mutant protein has an amino acid sequence selected from group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26, and wherein preferably said CRALBP mutant protein has an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NO: 24, and wherein further preferably said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 11.

In again a further aspect, the present invention provides for a pharmaceutical composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, wherein said method comprises administering, preferably by intravitreal injection, said composition to said animal, wherein said composition comprises, preferably consists of, wherein said pharmaceutical composition comprises (a) the composition of the present invention; and (b) a pharmaceutically acceptable carrier.

In again a further aspect, the present invention provides for a pharmaceutical composition for use in a method of preventing or treating a disease or disorder of an animal, preferably of a human, wherein said disease or disorder is caused by an endogenous retinoid deficiency of said animal, wherein said method comprises administering, preferably by intravitreal injection, said composition to said animal, wherein said composition comprises, preferably consists of, wherein said pharmaceutical composition comprises (a) the complex of the present invention; and (b) a pharmaceutically acceptable carrier.

In a preferred embodiment, said cognate ligand, preferably said cz.s-retinoid, is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11-cz -retinal; (iv) 11-cz -retinol; (v) 11, 13-di-cz.s- retinal; (vi) 11, 13-di-cz.s-retinol; (vii) 9, 13-di-cz.s-retinal; and (viii) 9, 13-di-cz.s-retinol. In a further preferred embodiment, said cognate ligand, preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11-cz -retinal; (iv) 11-cz -retinol; (v) 9,13-di-cz.s-retinal; and (vi) 9,13-di-c/.s-retinol . In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 1 l-czs-retinal; and (iii) 9, 13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal.

In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 9- cz -retinal. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 9-cv.s-retinol. In a further preferred embodiment, said cognate ligand, preferably said cisretinoid is l l-czs-retinal. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 11-czs-retinol. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 11,13-di-cz -retinal. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 11,13-di-cz -retinol. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 9,13-di-cv.s-retinal. In a further preferred embodiment, said cognate ligand, preferably said cv.s-retinoid is 9,13-di-cv.s-retinol.

In a further preferred embodiment said mutein of a wild-type CRALBP protein has a sequence identity of at least 90 % with said wild-type CRALBP protein, preferably of at least 95%, further preferably of at least 96%, again further preferably at least 97%, again further preferably at least 98%, and again more preferably of at least 99%. In a further preferred embodiment, said mutein of a wild-type CRALBP protein has a sequence identity of at least 95% with said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein has a sequence identity of at least 96% with said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein has a sequence identity of at least 97% with said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein has a sequence identity of at least 98% with said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein has a sequence identity of at least 99% with said wildtype CRALBP protein.

In a further preferred embodiment, said mutein of a wild-type CRALBP protein differs by at most 30 amino acids from said wild-type CRALBP protein. In a further preferred embodiment, said mutein of a wild-type CRALBP protein differs by at most 20 amino acids from said wild-type CRALBP protein. In again another preferred embodiment, said mutein of a wild-type CRALBP protein differs by at most 10 amino acids from said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein differs by 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids from said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein differs by at most 8 amino acids from said wild-type CRALBP protein. In a further preferred embodiment said mutein of a wild-type CRALBP protein differs by at most 6, 4 or 2 amino acids from said wild-type CRALBP protein.

In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence having a sequence identity of at least 90% with said wild-type CRALBP protein, preferably a sequence identity of at least 90% with of SEQ ID NO: 3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence having a sequence identity of at least 93% with said wild-type CRALBP protein, preferably a sequence identity of at least 93% with of SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence having a sequence identity of at least 96% with said wild-type CRALBP protein, preferably a sequence identity of at least 96% with of SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence having a sequence identity of at least 98% with said wild-type CRALBP protein, preferably a sequence identity of at least 98% with of SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence having a sequence identity of at least 99% with said wild-type CRALBP protein, preferably a sequence identity of at least 99% with of SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 15 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 10 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 8 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 6 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 5 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 4 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 3 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3. In a preferred embodiment, said mutein of a wild-type CRALBP protein has an amino acid sequence differing by at most 2 amino acids from said wild-type CRALBP protein, preferably from said SEQ ID NO:3.

In a preferred embodiment of the present invention, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 90% with a wild-type CRALBP protein. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 90% with SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 93% with a wild-type CRALBP protein. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 93% with SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 96% with a wild-type CRALBP protein. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 96% with SEQ ID NO: 3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 98% with a wild-type CRALBP protein. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 98% with SEQ ID NO: 3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 99% with a wild-type CRALBP protein. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 15, 12, 10, 8 or 6 amino acids from a wild-type CRALBP protein. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 15 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 12 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 10 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 8 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 6 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 5 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 4 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 3 amino acids from SEQ ID NO:3. In a preferred embodiment, said CRALBP mutant protein comprises, or consists of, an amino acid sequence differing by at most 2 amino acids from SEQ ID NO:3.

In a further preferred embodiment, one cysteine of each pair of amino acid mutations by cysteines is a mutation of an amino acid within the amino acid residues corresponding to amino acids 204-229 of SEQ ID NO:3, wherein the other mutated amino acid by cysteine of said pair is a mutation of an amino acid within the amino acid residues corresponding to amino acids 244-261 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises one, two, three or four pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein preferably said mutein comprises one or two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein.

In a further preferred embodiment, each of said pair of cysteines forms a disulfide bond.

In a further preferred embodiment, said mutein comprises one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein. In another preferred embodiment, said mutein comprises two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein. In a further preferred embodiment, said mutein comprises three pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein. In a further preferred embodiment, each of said pair of cysteines forms a disulfide bond.

In a further preferred embodiment, said pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is selected from

(1) a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3;

(2) a mutation of an amino acid corresponding to amino acid 217 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 253 of SEQ ID NO:3;

(3) a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3;

(4) a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3. In a further preferred embodiment, said pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 217 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 253 of SEQ ID NO:3. In a further preferred embodiment, said pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3. In a further preferred embodiment, said pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises one or two or three pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is selected from

(i) a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3;

(ii) a mutation of an amino acid corresponding to amino acid 217 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 253 of SEQ ID NO:3; and

(iii) a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3; and wherein said two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein are selected from

(a) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3;

(b) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3; and

(c) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 217 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 253 of SEQ ID NO:3; and wherein said three pairs of amino acid mutations by cysteines as compared to said wildtype CRALBP protein is

(x) a first pair of amino acid mutations by cysteines, a second pair of amino acid mutations by cysteines, and a third pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3, said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3, and said third pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3. In a further preferred embodiment, said mutein comprises one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 217 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 253 of SEQ ID NO:3. In a further preferred embodiment, said mutein comprises one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein are a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein are a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein are a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 217 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 253 of SEQ ID NO:3.

In a further preferred embodiment, said mutein comprises three pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein said three pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein are a first pair of amino acid mutations by cysteines, a second pair of amino acid mutations by cysteines, and a third pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 212 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 250 of SEQ ID NO:3, said second pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3, and said third pair of amino acid mutations by cysteines is a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3.

In a preferred embodiment, at least one of said pair of cysteines forms a disulfide bond. In another preferred embodiment, each of said pair of cysteines forms a disulfide bond.

In a preferred embodiment, said wild-type CRALBP protein is the human wild-type CRALBP protein of SEQ ID NO:3.

In a preferred embodiment, said wild-type CRALBP protein is the human wild-type CRALBP protein of SEQ ID NO:3, and said pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is selected from

(1) a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3;

(2) a mutation of amino acid 217 of SEQ ID NO:3 and a mutation of amino acid 253 of SEQ ID NO:3;

(3) a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3;

(4) a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3; and wherein preferably, said mutein of said human wild-type CRALBP protein has a sequence identity of at least 90 % with said human wild-type CRALBP protein, preferably of at least 95%, further preferably of at least 96%, again further preferably at least 97%, again further preferably at least 98%, and again more preferably of at least 99%.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein has a sequence identity of at least 95% with said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein has a sequence identity of at least 96% with said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein has a sequence identity of at least 97% with said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein has a sequence identity of at least 98% with said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wildtype CRALBP protein has a sequence identity of at least 99% with said human wild-type CRALBP protein.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein differs by at most 30 amino acids from said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein differs by at most 20 amino acids from said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein differs by at most 10 amino acids from said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein differs by 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids from said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein differs by at most 8 amino acids from said human wild-type CRALBP protein. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein of said human wild-type CRALBP protein differs by at most 6, 4 or 2 amino acids from said human wild-type CRALBP protein.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, said pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 217 of SEQ ID NO:3 and a mutation of amino acid 253 of SEQ ID NO:3. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 220 of SEQ ID NO: 3 and a mutation of amino acid 254 of SEQ ID NO: 3. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises one or two or three pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said human wildtype CRALBP protein is selected from

(i) a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3;

(ii) a mutation of amino acid 217 of SEQ ID NO:3 and a mutation of amino acid 253 of SEQ ID NO:3; and

(iii) a mutation of amino acid 220 of SEQ ID NO: 3 and a mutation of amino acid 254 of SEQ ID NO:3; and wherein said two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein are selected from

(a) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 220 of SEQ ID NO: 3 and a mutation of amino acid 254 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3;

(b) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3; and

(c) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 217 of SEQ ID NO:3 and a mutation of amino acid 253 of SEQ ID NO:3; and wherein said three pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is

(x) a first pair of amino acid mutations by cysteines, a second pair of amino acid mutations by cysteines, and a third pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, said second pair of amino acid mutations by cysteines is a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3, and said third pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 217 of SEQ ID NO:3 and a mutation of amino acid 253 of SEQ ID NO:3. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein are a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3. In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein are a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said two pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein are a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 217 of SEQ ID NO:3 and a mutation of amino acid 253 of SEQ ID NO:3.

In a further preferred embodiment, said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises three pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said three pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein are a first pair of amino acid mutations by cysteines, a second pair of amino acid mutations by cysteines, and a third pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, said second pair of amino acid mutations by cysteines is a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3, and said third pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3.

In a further preferred embodiment, said CRALBP mutant protein has an amino acid sequence selected from group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26, and wherein preferably said CRALBP mutant protein has an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NO:24, and wherein further preferably said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 11. In another preferred embodiment of the aforementioned embodiments, each of said pair of cysteines forms a disulfide bond, and thus, within said complexes, said CRALBP mutant protein is completely in the oxidized form.

In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 11. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 14. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 16. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 18. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO:20. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO:22. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO:24. In a further preferred embodiment, said CRALBP mutant protein has the amino acid sequence of SEQ ID NO:26. In another preferred embodiment of the aforementioned embodiments, each of said pair of cysteines forms a disulfide bond, and thus, within said inventive complexes, said CRALBP mutant protein is completely in the oxidized form. In a further preferred embodiment, said CRALBP mutant protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 11, and wherein said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond.

In a further preferred embodiment, said CRALBP mutant protein consists of an amino acid sequence selected from group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26, and wherein preferably said CRALBP mutant protein consists of an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NO:24, and wherein further preferably said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11. In another preferred embodiment of the aforementioned embodiments, each of said pair of cysteines forms a disulfide bond. Thus, within said complexes, said CRALBP mutant protein is completely in the oxidized form.

In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 14. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 16. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 18. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO:20. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO:22. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO:24. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO:26. In another preferred embodiment of the aforementioned embodiments, each of said pair of cysteines forms a disulfide bond. Thus, within said complexes, said CRALBP mutant protein is completely in the oxidized form. In a further preferred embodiment, said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11, and wherein said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond.

In a further preferred embodiment, said CRALBP protein has an amino acid sequence selected from group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, wherein preferably said CRALBP protein has an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:28, and wherein further preferably said CRALBP protein has an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:27 and SEQ ID NO:28, and wherein again further preferably said CRALBP protein has the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:28.

In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:3. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:4. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO: 11. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO: 12. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO: 14. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO: 16. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO: 18. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:20. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:22. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:24. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:26. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:27. In a further preferred embodiment, said CRALBP protein has the amino acid sequence of SEQ ID NO:28.

In a further preferred embodiment, said CRALBP protein consists of an amino acid sequence selected from group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, wherein preferably said CRALBP protein consists of an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:28, and wherein further preferably said CRALBP protein consists of an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:27 and SEQ ID NO:28, and wherein again further preferably said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:28.

In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:3. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:4. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 11. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 12. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 14. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 16. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 18. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:20. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:22. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:24. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:26. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:27. In a further preferred embodiment, said CRALBP protein consists of the amino acid sequence of SEQ ID NO:28.

In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a CRALBP mutant protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wildtype CRALBP protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a CRALBP mutant protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein. In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a CRALBP mutant protein. In a further preferred embodiment, said complex is a monomeric complex of said CRALBP mutant protein and said cognate ligand. In a further preferred embodiment, said complex is a monomeric complex of said wild-type CRALBP protein and said cognate ligand.

In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wildtype CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a CRALBP mutant protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wild-type CRALBP protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a CRALBP mutant protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein. In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a CRALBP mutant protein. In a further preferred embodiment, said complex is a dimeric complex of said CRALBP mutant protein and said cognate ligand. In a further preferred embodiment, said complex is a dimeric complex of said wild-type CRALBP protein and said cognate ligand.

In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a CRALBP mutant protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wildtype CRALBP protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a CRALBP mutant protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a CRALBP mutant protein. In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand. In a further preferred embodiment, said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand. In a further preferred embodiment, said complex is an homo oligomeric complex of said CRALBP protein and said cognate ligand. In a further preferred embodiment, said complex is an homo oligomeric complex of said CRALBP mutant protein and said cognate ligand. In a further preferred embodiment, said complex is an homo oligomeric complex of said wild-type CRALBP protein and said cognate ligand. In a preferred embodiment, said oligomeric complex has a molecular weight of at least 500kDa, preferably of at least 600kDa, preferably of at least 720kDa, and preferably a molecular weight of at most 2500kDa, further preferably a molecular weight of at most 2000kDa, and further preferably a molecular weight of at most 1500kDa, or wherein said oligomeric complex has a average diameter of about 18 to 35 nm, preferably of about 20 to 33 nm, further preferably of about 24 to 33 nm, and wherein preferably said oligomeric complex has a average diameter of about 25 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said oligomeric complex has a molecular weight of at least 600kDa, preferably of at least 720kDa, and preferably a molecular weight of at most 2500kDa, further preferably a molecular weight of at most 2000kDa, and further preferably a molecular weight of at most 1500kDa. In a preferred embodiment, said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 24 to 33 nm, and wherein preferably said oligomeric complex has a average diameter of about 25 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 600kDa, preferably of at least 720kDa, and preferably a molecular weight of at most 2500kDa, further preferably a molecular weight of at most 2000kDa, and further preferably a molecular weight of at most 1500kDa, or wherein said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 24 to 33 nm, and wherein preferably said oligomeric complex has a average diameter of about 25 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 20 to 35 nm, further preferably about 23 to 33 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 10 to 18 nm, preferably about 10 tol6 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 10 to 18 nm, preferably about 10 to 16 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 10 to 18 nm, preferably about 10 to 16 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a preferred embodiment, said complex is a dimeric complex of said CRALBP protein and said cognate ligand, wherein said dimeric complex has a average diameter of about 7 to 10 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is a dimeric complex said wild-type CRALBP protein and said cognate ligand, wherein said dimeric complex has a average diameter of about 7 to 10 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is a dimeric complex said CRALBP mutant protein and said cognate ligand, wherein said dimeric complex has a average diameter of about 7 to 10 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a preferred embodiment, said complex is a monomeric complex of said CRALBP protein and said cognate ligand, wherein said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is a monomeric complex said wild-type CRALBP protein and said cognate ligand, wherein said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a preferred embodiment, said complex is a monomeric complex said CRALBP mutant protein and said cognate ligand, wherein said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 240 kDa, preferably of at least 300 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 660 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 600 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 500 kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 600kDa, preferably of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and further preferably a molecular weight of at most 1500kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2500 kDa, preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and further preferably a molecular weight of at most 1500kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 240 kDa, preferably of at least 300 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 660 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 600 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 500 kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 600kDa, preferably of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and further preferably a molecular weight of at most 1500kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2500 kDa, preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and further preferably a molecular weight of at most 1500kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said wildtype CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 240 kDa, preferably of at least 300 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 660 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 600 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 500 kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said wildtype CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 500kDa, preferably of at least 600kDa, preferably of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and further preferably a molecular weight of at most 1500kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said wildtype CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 600kDa, preferably of at least 720 kDa, preferably of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2500 kDa, furthr preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and further preferably a molecular weight of at most 1500kDa.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein the number of said cognate ligands and said CRALBP proteins within said oligomeric complex are equal or higher than 6 and equal or lower than 18, preferably equal or higher than 8 and and equal or lower than 16, and wherein further preferably said number of said cognate ligands the number of said CRALBP proteins are equal.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein the number of said cognate ligands and said CRALBP mutant proteins within said oligomeric complex are equal or higher than 6 and equal or lower than 18, preferably equal or higher than 8 and and equal or lower than 16, and wherein further preferably said number of said cognate ligands the number of said CRALBP mutant proteins are equal.

In a further preferred embodiment, said complex is an oligomeric complex of said wildtype CRALBP protein and said cognate ligand, wherein the number of said cognate ligands and said wild-type CRALBP proteins within said oligomeric complex are equal or higher than 6 and equal or lower than 18, preferably equal or higher than 8 and and equal or lower than 16, and wherein further preferably said number of said cognate ligands the number of said wildtype CRALBP proteins are equal.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein the number of said cognate ligands and said CRALBP proteins within said oligomeric complex are equal or higher than 20 and equal or lower than 75, preferably equal or higher than 22 and equal or lower than 60, and wherein further preferably said number of said cognate ligands the number of said CRALBP proteins are equal. In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein the number of said cognate ligands and said CRALBP proteins within said oligomeric complex are equal or higher than 24 and equal or lower than 75, preferably equal or higher than 26 and equal or lower than 60, and wherein further preferably said number of said cognate ligands the number of said CRALBP proteins are equal.

In a further preferred embodiment, said complex is an oligomeric complex of said wildtype CRALBP protein and said cognate ligand, wherein the number of said cognate ligands and said wild-type CRALBP proteins within said oligomeric complex are equal or higher than 20 and equal or lower than 75, preferably equal or higher than 22 and equal or lower than 60, and wherein further preferably said number of said cognate ligands the number of said wild-type CRALBP proteins are equal. In a further preferred embodiment, said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein the number of said cognate ligands and said wild-type CRALBP proteins within said oligomeric complex are equal or higher than 24 and equal or lower than 75, preferably equal or higher than 26 and equal or lower than 60, and wherein further preferably said number of said cognate ligands the number of said wild-type CRALBP proteins are equal.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein the number of said cognate ligands and said CRALBP mutant proteins within said oligomeric complex are equal or higher than 20 and equal or lower than 75, preferably equal or higher than 22 and equal or lower than 60, and wherein further preferably said number of said cognate ligands the number of said CRALBP mutant proteins are equal. In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein the number of said cognate ligands and said CRALBP mutant proteins within said oligomeric complex are equal or higher than 24 and equal or lower than 75, preferably equal or higher than 26 and equal or lower than 60, and wherein further preferably said number of said cognate ligands the number of said CRALBP mutant proteins are equal.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 8 to 20 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), and wherein preferably said oligomeric complex has a average diameter of about 10 to 18 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 18 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), and wherein preferably said oligomeric complex has a average diameter of about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 8 to 20 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), and wherein preferably said oligomeric complex has a average diameter of about 12 to 17 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a further preferred embodiment, said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 24 to 33 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), and wherein preferably said oligomeric complex has a average diameter of about 26 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a further preferred embodiment, said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 8 to 20 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), and wherein preferably said oligomeric complex has a average diameter of about 12 to 16 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a further preferred embodiment, said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a average diameter of about 18 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), and wherein preferably said oligomeric complex has a average diameter of about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand. In a further preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand.

In a further preferred embodiment, said composition comprises monomeric complexes and homo oligomeric complexes of said CRALBP mutant protein and said cognate ligand. In a further preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP mutant protein and said cognate ligand.

In a further preferred embodiment, said composition comprises monomeric complexes and homo oligomeric complexes of said wild-type CRALBP protein and said cognate ligand. In a further preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said wild-type CRALBP protein and said cognate ligand.

In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a wild-type CRALBP protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein comprises a CRALBP mutant protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a wild-type CRALBP protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein consists of a CRALBP mutant protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein or a CRALBP mutant protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a wild-type CRALBP protein. In a preferred embodiment, said complex comprises monomeric complexes and homo oligomeric complexes of said CRALBP protein and said cognate ligand, wherein said CRALBP protein is a CRALBP mutant protein.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 11; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11- czs-retinal; (iv) l l-czs-retinol; (v) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9,13-di-cz.s-retinal; and (vi) 9, 13-di-cz.s-retinol, and again further preferably wherein said cisretinoid is selected from (i) 9-cz.s-retinal and (ii) 1 l-czs-retinal; and wherein preferably said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 11; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11- czs-retinal; (iv) l l-czs-retinol; (v) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9,13-di-cz.s-retinal; and (vi) 9, 13-di-cz.s-retinol, and again further preferably wherein said cisretinoid is selected from (i) 9-cz.s-retinal and (ii) 1 l-czs-retinal; and wherein preferably said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond, and wherein further preferably said complex is a monomeric complex of said CRALBP mutant protein and said cognate ligand, and wherein preferably said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- cz.s-retinal and (ii) 1 l-czs-retinal; and wherein preferably said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond, and wherein preferably said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and wherein preferably said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- cz.s-retinal and (ii) 1 l-czs-retinal; and wherein preferably said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond, and wherein preferably said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- cz.s-retinal and (ii) 11-czs-retinal; and wherein said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 preferably forms a disulfide bond, and wherein said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP mutant protein, wherein said CRALBP mutant protein consists of the amino acid sequence of SEQ ID NO: 11; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid selected from (i) 9- cz.s-retinal and (ii) 1 l-czs-retinal; and wherein said pair of cysteines of amino acid 212 and 250 of SEQ ID NO: 11 forms a disulfide bond, and wherein said complex is an oligomeric complex of said CRALBP mutant protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a wild-type CRALBP protein, wherein said wild-type CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:3; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11- cz -retinal; (iv) l l-czs-retinol; (v) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9,13-di-cz.s-retinal; and (vi) 9, 13-di-cz.s-retinol, and again further preferably wherein said cisretinoid is selected from (i) 9-cz.s-retinal and (ii) 11-cz -retinal.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a wild-type CRALBP protein, wherein said wild-type CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:3; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11- cz -retinal; (iv) l l-czs-retinol; (v) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9,13-di-cz.s-retinal; and (vi) 9, 13-di-cz.s-retinol, and again further preferably wherein said cisretinoid is selected from (i) 9-cz.s-retinal and (ii) 11-cz -retinal, and wherein further preferably said complex is a monomeric complex of said wild-type CRALBP protein and said cognate ligand, and wherein preferably said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a wild-type CRALBP protein, wherein said wild-type CRALBP protein consists of the amino acid sequence of SEQ ID NO:3; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- c/.s-retinal and (ii) 11-czs-retinal; and wherein preferably said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and wherein preferably said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a wild-type CRALBP protein, wherein said wild-type CRALBP protein consists of the amino acid sequence of SEQ ID NO:3; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- c/.s-retinal and (ii) 11-czs-retinal; and wherein preferably said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a wild-type CRALBP protein, wherein said wild-type CRALBP protein consists of the amino acid sequence of SEQ ID NO:3; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- c/.s-retinal and (ii) 11-czs-retinal; and wherein said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a wild-type CRALBP protein, wherein said wild-type CRALBP protein consists of the amino acid sequence of SEQ ID NO:3; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a c/.s-retinoid selected from (i) 9- c/.s-retinal and (ii) 11-czs-retinal; and wherein said complex is an oligomeric complex of said wild-type CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 12; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11-cz -retinal; (iv) 11- cz -retinol; (v) 9,13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, and again further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal and (ii) 11-czs-retinal; and wherein preferably said pair of cysteines of amino acid 232 and 270 of SEQ ID NO: 12 forms a disulfide bond.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 12; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11-cz -retinal; (iv) 11- cz -retinol; (v) 9,13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, and again further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal and (ii) 11-cz -retinal; and wherein preferably said pair of cysteines of amino acid 232 and 270 of SEQ ID NO: 12 forms a disulfide bond, and wherein further preferably said complex is a monomeric complex of said CRALBP protein and said cognate ligand, and wherein preferably said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 12; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid selected from (i) 9-cz.s-retinal and (ii) 11-cz -retinal; and wherein preferably said pair of cysteines of amino acid 232 and 270 of SEQ ID NO: 12 forms a disulfide bond, and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and wherein preferably said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 12; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv'.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 1 l-czs-retinal; and wherein preferably said pair of cysteines of amino acid 232 and 270 of SEQ ID NO: 12 forms a disulfide bond, and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 12; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv'.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said pair of cysteines of amino acid 232 and 270 of SEQ ID NO: 12 preferably forms a disulfide bond, and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 12; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv'.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said pair of cysteines of amino acid 232 and 270 of SEQ ID NO: 12 forms a disulfide bond, and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:28; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cA-retinal; (ii) 9-cA-retinol; (iii) 11-cz -retinal; (iv) 11- czs-retinol; (v) 9,13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, and again further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal and (ii) 11-czs-retinal; and wherein preferably said pair of cysteines of amino acid 215 and 253 of SEQ ID NO:28 forms a disulfide bond.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:28; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid, wherein preferably said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 9-cz.s-retinol; (iii) 11-cz -retinal; (iv) 11- czs-retinol; (v) 9,13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal; (ii) 11-czs-retinal; and (iii) 9, 13-di-cz.s-retinal; and (vi) 9,13-di-cz.s-retinol, and again further preferably wherein said cz.s-retinoid is selected from (i) 9-cz.s-retinal and (ii) 11-czs-retinal; and wherein preferably said pair of cysteines of amino acid 215 and 253 of SEQ ID NO:28 forms a disulfide bond, and wherein further preferably said complex is a monomeric complex of said CRALBP protein and said cognate ligand, and wherein preferably said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:28; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid selected from (i) 9-cz.s-retinal and (ii) 11-cz -retinal; and wherein preferably said pair of cysteines of amino acid 215 and 253 of SEQ ID NO:28 forms a disulfide bond, and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and wherein preferably said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:28; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein preferably said pair of cysteines of amino acid 215 and 253 of SEQ ID NO:28 forms a disulfide bond, and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:28; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said pair of cysteines of amino acid 215 and 253 of SEQ ID NO:28 forms a disulfide bond, and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:28; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cz.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said pair of cysteines of amino acid 215 and 253 of SEQ ID NO:28 forms a disulfide bond, and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:4; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid, wherein preferably said cv.s-retinoid is selected from (i) 9-cA-retinal; (ii) 9-cA-retinol; (iii) 1 l-czs-retinal; (iv) 11- czs-retinol; (v) 9, 13-di-cv.s-retinal; and (vi) 9, 13-di-cv.s-retinol, further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 1 l-czs-retinal; and (iii) 9, 13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, and again further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:4; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid, wherein preferably said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 9-cv.s-retinol; (iii) 11-czs-retinal; (iv) 11- czs-retinol; (v) 9,13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 11-czs-retinal; and (iii) 9, 13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, and again further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal, and wherein further preferably said complex is a monomeric complex of said CRALBP protein and said cognate ligand, and wherein preferably said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:4; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and wherein preferably said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5. In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:4; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 1 l-czs-retinal; and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:4; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO:4; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:27; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid, wherein preferably said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 9-cv.s-retinol; (iii) 11-czs-retinal; (iv) 11- czs-retinol; (v) 9,13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 11-czs-retinal; and (iii) 9, 13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, and again further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 27; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid, wherein preferably said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 9-cv.s-retinol; (iii) 1 l-czs-retinal; (iv) 11- czs-retinol; (v) 9,13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal; (ii) 1 l-czs-retinal; and (iii) 9, 13-di-cv.s-retinal; and (vi) 9,13-di-cv.s-retinol, and again further preferably wherein said cv.s-retinoid is selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal, and wherein further preferably said complex is a monomeric complex of said CRALBP protein and said cognate ligand, and wherein preferably said monomeric complex has a average diameter of about 5 to 7 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 27; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa, and wherein preferably said oligomeric complex has a average diameter of about 18 to 35 nm, preferably about 20 to 33 nm, further preferably about 20 to 30 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS), preferably wherein said average diameter is determined by DLS as described in Example 11 and Table 5.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 27; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein preferably said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 27; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv'.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 1 l-czs-retinal; and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 720 kDa, preferably of at least 800 kDa, and wherein preferably said oligomeric complex has a molecular weight of at most 2500 kDa, further preferably wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In a further preferred embodiment, said composition comprising, preferably consisting of, a complex, wherein said complex comprises (a) a CRALBP protein, wherein said CRALBP protein consists of the amino acid sequence of SEQ ID NO: 27; and (b) a cognate ligand of CRALBP, wherein said cognate ligand is a cv'.s-retinoid selected from (i) 9-cv.s-retinal and (ii) 11-czs-retinal; and wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 800 kDa, and wherein said oligomeric complex has a molecular weight of at most 2000 kDa.

In another aspect, the present invention provides a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, an amino acid sequence selected from group consisting of SEQ ID NO:4, SEQ ID NO: 12, SEQ ID NO:27 and SEQ ID NO:28. In another aspect, the present invention provides a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:4. In another aspect, the present invention provides a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 12. In another aspect, the present invention provides a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:27. In another aspect, the present invention provides a CRALBP protein, wherein said CRALBP protein comprises, preferably consists of, the amino acid sequence of SEQ ID NO:28.

EXAMPLES

EXAMPLE 1

Cloning of RLBP1 and expression of wild-type CRALBP

Cloning of RLBP1 and expression of wild-type CRALBP was analogously effected as described in the prior art (He, X., et al., 2009, PNAS, 106(44): 18545-18550; WO 2017/129555. In brief, human RLBP1 cDNA (JRAUp969D1020D, SEQ ID NO: 1) was obtained from the German Center for Genomic Research GmbH. The CRALBP overexpression vector construct was obtained by cloning the RLBP1 cDNA into the Ndel and Xhol sites of the pET-28a(+) vector following the protocol of “StreamLined Restriction Digestion, Dephosphorylation and Ligation” (Promega) leading to the pET-28a(+) CRALBP overexpression plasmid (SEQ ID NO:2). Incubations were carried out at 37°C and 750 rpm (Thermomixer Compact, Eppendorf) for 2.25h, inactivation at 74°C for 15min, separation via 1% agarose gel and extraction of the desired cutting fragments with the “Wizard SV Gel and PCR Clean-Up System” from Promega according to the user manual. Thermosensitive Alkaline Phosphatase was used only for the digestion of the pET-28a(+) target vector but not for the digestion of the RLBP1 cDNA (JRAUp969D1020D). BL21(DE3) strains of E. coli (Invitrogen) were transformed with the pET-28a(+) CRALBP overexpression plasmid (SEQ ID NO:2), and were cultured overnight with agitation at 37 °C in 120 mL LB medium containing 30 mg/mL kanamycin. The overnight culture was used to inoculate 6 L of LB medium (30 mg/mL kanamycin). The culture was grown at 20 °C to an ODeoo of 0.7 and then was induced with 1 mM isopropyl-thiogalacto- pyranoside for 16 h. Cells were harvested by centrifugation at 5000 g for 45 min and were resuspended in 250 mL of ice-cold lysis buffer (20 mM imidazole; 100 mMNaCl; 20 mM Tris- HC1, pH 7.4; 1% wt/vol Triton X-100). The cells were disrupted by ultrasonication for 20 min, and the lysate comprising human wild-type CRALPB (SEQ ID NO:3) in the form of its His-tag adduct (SEQ ID NO:4) which was centrifuged at 20,000g for 35 min to remove debris.

EXAMPLE 2

Cloning and expression of di-cys mutant CRALBP A212C:T250C

Point mutations were introduced sequentially by site-directed mutagenesis in pET-28a(+) CRALBP overexpression plasmid (SEQ ID NO:2) according to the instructions described in the QuikChange kit from Stratagene. The corresponding primer sequences, the PCR reaction mixtures and the temperature protocol used to produce the di-cys mutant CRALBP A212C:T250C are depicted in Table 1 and Table 2 respectively. Thus, in Table 1 mutated positions are identified by underlined-blocks. In the case of T250C, the point mutation directly leads to the formation of a novel restriction site. In the case of the A212C point mutation, an additional silent mutation was introduced (see underlined and cursive).

Table 1. Primer sequences

Table 2. PCR reaction mixtures and conditions

Thus, nucleotide sequence of SEQ ID NO:2 was used as starting template in the site- directed PCR. After sequential PCR reactions the resulting expression plasmid pET-28a(+) mutant RLBP1 (SEQ ID NO:9) comprising the nucleotide sequence of SEQ ID NO: 10 and encoding for the amino acid SEQ ID NO: 11 of the di-cys mutant CRALBP A212C:T250C was obtained.

Thus, after PCR reaction, 2 pl of Dpnl restriction enzymes were added to the reaction mixture and the sample incubated for two hours at 37°C. Following this, lOpl of the reaction mixture was directly transformed into competent E. coli XLlOGold cells (Stratagene). Clones obtained after transformation and plating were checked for the presence of freshly introduced restriction sites by analytical restriction digestion. Clones containing both restriction sites were used for isolating the expression plasmid pET-28a(+) mutant RLBP1 (SEQ ID NO:9) comprising the nucleotide sequence of di-cys mutant CRALBP A212C:T250C (SEQ ID NO: 10). The presence of the A212C:T250C point mutations in the plasmid was verified by sequencing (Microsynth AG, Balgach).

BL21(DE3) cells transformed with and containing the expression plasmid pET-28a(+) mutant RLBP1 (SEQ ID NO:9) comprising the nucleotide sequence of di-cys mutant CRALBP A212C:T250C (SEQ ID NO: 10) were cultured overnight with agitation at 37 °C in 120 mL LB medium containing 30 mg/mL kanamycin. The overnight culture was used to inoculate 6 L of LB medium (30 mg/mL kanamycin). The culture was grown at 20 °C to an ODeoo of 0.7 and then was induced with 1 mM isopropyl -thiogalacto-pyranoside for 16 h. Cells were harvested by centrifugation at 5000 g for 45 min and were resuspended in 250 mL of ice-cold lysis buffer (20 mM imidazole; 100 mM NaCl; 20 mM Tris-HCl, pH 7.4; 1% wt/vol Triton X-100). The cells were disrupted by ultrasonication for 20 min, and the lysate comprising the di-cys mutant CRALBP A212C:T250C of SEQ ID NO: 11 in the form of its His-tag adduct (SEQ ID NO: 12) was centrifuged at 20,000g for 35 min to remove debris.

EXAMPLE 3

In silica mutagnesis of CRALBP

Native CRALBP binds both 1 l -c/.s-retinal and 9-cv.s-retinal ligands with high affinity in the low nanomolar range. Upon ligand binding, CRALBP’ s mobile gate moiety adopts its ‘closed’ conformational state with helixl2 and helixlO being within van der Waals distance of one another. In this conformation, the side-chain sulphur atoms of the mutated residues C212 and C250 oppose each other at 2.4 A distance within the interface formed by the two helices (see Figure 1).

In silica cysteine mutagenesis was carried out in order to identify possibly stabilizing disulfide bridges in CRALBP, other than the above described di-cysteine mutant A212C:T250C (SEQ ID NO: 11). For this, a truncated model was created from the X-ray structural model of wild-type CRALBP with bound 1 l -c/.s-retinal (PDB entry 3HY5). Two sequence regions of wild-type CRALBP (SEQ ID NO:3), namely amino acid residues 204-229 of SEQ ID NO:3 and amino acid residues 244-261 of SEQ ID NO: 3 were defined by editing the original model. Cysteine modifications were introduced systematically into both chains using the FoldX V5 computer algorithm (Schymkowitz J., et al., 2005, Nucleic Acids Res. l;33:W382-8. doi: 10.1093/nar/gki387). Using the algorithm, the truncated model of wild-type CRALBP was minimized at 298K, pH 7, 0.1 M ionic strength to generate an in silico reference structure (“Reference - WT”). The FoldX V5 “BuildModel” command, which implements a probabilitybased rotamer library of its own, was used to create in silico eight CRALBP mutant proteins comprising one, two or three pairs of cysteines mutations containing models including the di- cys mutant A212C:T250C. Table 3 lists said identified CRALBP mutant proteins as well as their amino acid and nucleic acid sequences.

Table 3. Overview of di-cysteine mutants of CRALBP including amino acid and nucleotide sequences

The mutant models from FoldX were submitted to phenix. dynamic (Adams P.D., et al., 2010, Acta Crystallogr. D Biol. Crystallogr. 66(Pt 2):213-21) for simple model perturbation and flexible backbone modeling respectively, thereby de-biasing the models and simultaneously forming the disulfide bonds. Molecular dynamics calculations were performed at 300K, 200 iterations of 0.005 fs each, and the threshold maximum for bond formation set to 3.1 A. Subsequently, the change in free energy of the interface was assessed with the EMBL PISA serverl7. The solvation free energy gain upon formation of the interface between the two layers of the mobile gate (residues 204-229 of SEQ ID NO: 3 and residues 244-261 of SEQ ID NO:3) was calculated as A‘G in kcal/mol. A compilation of the calculations for the described inventive CRALBP mutants including the wild-type CRALBP (SEQ ID NO:3) as reference is shown in Table 4.

Table 4. Overview of in silico mutant analysis

Distance SS-

Variant Chain A Chain B interface area Bonds AiG

Surface A² Surface A² A² A kcal/mol

Reference wt CRALBP 2837 2060 472.4 NA -11.2

V224C:F257C 2853 2099 490.8 2.021 -9.7

T217C:V253C 2815 2085 487.7 2.052 -12.6

L220C:V254C 2923 2096 481.0 2.084 -12.8

A212C:T250C 2861 2120 524.9 2.042 -15.3

L220C:V254C - V224C:F257C 2921 2122 487.1 2.030/2.037 -12.6

A212C:T250C - V224C:F257C 2810 2109 531.8 2.043/2.049 -13.7

A212C-T250C - T217C-V253C 2885 2138 569.0 2.062/2.056 -17.4

212-220-224C:250-254-257C 2864 2127 536.4 2.062/2.053/2.001 -17.3

Surface A² is the total solvent accessible surface area in square Angstroms; Interface area in A², calculated as difference in total accessible surface areas of isolated and interfacing structures divided by two. A'G indicates the solvation free energy gain upon formation of the interface, in kcal/mol. Any A‘G value that is reported to be more negative than the reference value of -11.2 kcal/mol is considered to effect a further stabilization of the corresponding mutant in comparison to wild-type CRALBP.

Figure 2 shows individually the close up view of the in silico model of the mobile gate region of the four described mono di-cysteine mutants of CRALBP with the position of the formed disulfide bonds highlighted as sticks, while in Figure 3 the close up view of the in silico model of the mobile gate region of each of the three double and the triple di-cysteine mutants of CRALBP with the position of the formed disulfide bonds highlighted as sticks are shown. The superposition of all in silico di-cysteine mutant models showing four possible described disulfide bonds across the mobile gate of CRALBP is shown in Figure 4.

EXAMPLE 4

Cloning and expression of the in silico generated di-cys CRALBP mutants

The nucleic acid sequences as well as the amino acid sequences of the further identified di-cys CRALBP mutants are described in Table 3. The cloning of the corresponding mutant RLBP 1 gene sequences and the expression of the corresponding di-cys CRALBP mutant proteins is effected analogously as described in Example 1 and 2. The purification of these further identified di-cys CRALBP mutants is effected as described in Example 5 below.

EXAMPLE 5

Purification of wild-type CRALBP and of di-cys CRALBP mutants

The lysates comprising the wild-type CRALBP of SEQ ID NO:3 in the form of its His- tag adduct (SEQ ID NO:4) obtained in Example 1 and the di-cys mutant CRALBP A212C:T250C of SEQ ID NO: 11 in the form of its His-tag adduct (SEQ ID NO: 12) obtained in Example 2 were both purified from supernatant by affinity chromatography on 10 mL of Ni- NTA SUPERFLOW (Qiagen) according to the manufacturer’s instructions. Briefly, the lysates were loaded on the column previously equilibrated in lysis buffer, washed with lysis buffer, and were eluted in 35 mL of elution buffer (20 mM Tris-HCl, pH 7.4; 200 mM imidazole; 100 mM NaCl). Typical yields of 35-40 mg of pure wild-type CRALBP and of pure di-cys mutant CRALBP A212C:T250C respectively were obtained as judged by SDS/PAGE and determined using the colorimetric bicinchoninic acid assay (Pierce Chemical Company). Either the N- terminal his-tag constructs or the his-tag-cleaved CRALPB proteins were used for the complex formation” as described in Example 6. The (His)e-tag was cleaved by adding 20 units of thrombin protease (GE Healthcare) and subsequent incubation at 4 °C overnight. The protein solution was then passed through a Ni-NTA column previously equilibrated in lysis buffer to remove uncleaved material. The flowthrough was concentrated by Centriprep-10 (Millipore) to 20 mg/mL. Cleavage of the His-tag led to CRALPB protein of SEQ ID NO:27 in case of the wild-type CRALPB, and to CRALPB protein of SEQ ID NO:28 in case of the A212GT250C CRALBP mutant protein. Cognate ligand-complexes of CRALBP were separated on a Superdex® 200 26/60 gel filtration column (GE Healthcare).

EXAMPLE 6

Ligand loading and gel permeation chromatography

All procedures involving cz.s-retinal were performed under dim red illumination (40-W ruby bulbs) at 4 °C unless specified. Ligand loading of the affinity purified wild-type CRALBP and of the di-cysteine mutant A212GT250C of CRALBP respectively was carried out in elution buffer (20 mM Tris-HCl, pH 7.4; 200 mM imidazole; 100 mM NaCl) by adding aliquots of 9- cz.s-retinal or 1 l -cz.s-retinal stocks (40mM) dissolved in ethanol to the protein solutions at a 1.5 molar excess and a final ethanol concentration of 2% vol/vol. The samples were incubated for 30 min at 4°C and then centrifuged at 15,000 g for 10 min. The samples were concentrated using a Vivaspin 15R Hydrosart (Sartorius) to 30-50 mg/ml of protein with three washes in buffer (10 mM Tris-HCl, 100 mM NaCl, pH 7.5). Finally, unbound retinoid was removed from the ligand complexes by gel filtration chromatography (GFC).

EXAMPLE 7

Gel filtration chromatography of wild-type CRALBP and of di-cys mutants of CRALBP

Purification of the protein cz.s-retinal complexes of wild-type CRALBP and of the dicysteine mutant A212C:T250C of CRALBP was performed on a Superdex® 200 26/60 column in Gel filtration chromatography (GFC) buffer (lOmM Hepes, 100 mMNaCl, pH 7.5). The total elution volume was 330 ml, and fractions were collected at 5 ml intervals. Preparative gel filtration reveals essentially the same chromatogram for the cz.s-retinal complex of the dicysteine mutant A212GT250C of CRALBP as obtained for the cz.s-retinal complex of wildtype CRALBP (Figure 5, Panel A and Panel B).

Four significant peaks with similar elution volumes were observed when the apo dicysteine mutant A212C:T250C of CRALBP was loaded with cz -retinal. Figure 5B shows the typical UV/Vis absorption trace at 280 nm featuring said four peaks. The order of the peaks is as follows: the first peak from left to right corresponds to the super high molecular weight (SHMW) fractions, the second peak corresponds to the high molecular weight (HMW) fraction, the third peak corresponds to dimeric, and the fourth peak to monomeric fractions, respectively. UV-Vis absorption spectra of monomeric wild type CRALBP and of the A212C:T250C mutant in complex with 9-cis retinal were used to determine the ligand loading of the monomeric complexes. For this, the absorption spectra were normalized to the maximum absorption, of the protein at approximately 280 nm. According to Crabb et al. (Crabb JW. et al., (1998), Protein Sci. 7(3):746-57) the absorption maximum at 280 nm corresponds to the protein concentration, and the one at 400 nm to bound 9-cis retinal. The fully saturated CRALBP in complex with 9- cis retinal has been reported by the later to have an ideal spectral ratio of _s ²⁸⁰/_s ⁴⁰⁰ = 2.2 In our experiments, the ratios for wild-type CRALBP:9-cA retinal and for the A212C:T250C:9-cA retinal mutant are 2.31 and 2.21, respectively, indicating the formation of fully saturated 1 : 1 complexes (see Figure 6).

Analytical GFC of corresponding peaks from each complex were concentrated (approx. 4 mg/ml) and characterized with a Superose® 6 Increase 10/300 GL column. Figure 7 (Panel A and Panel B shows overlayed traces at 280 nm of the SHMW, HMW and the monomeric analytical GFC runs for each complex.

EXAMPLE 8

Gel filtration chromatography of di-cys mutants of CRALBP under oxidizing conditions

Purification of the protein cv.s-retinal complex of di-cysteine mutant A212C:T250C of CRALBP under oxidizing conditions was performed on a Superdex® 200 26/60 column in Gel filtration chromatography (GFC) buffer (lOmM Hepes, 100 mM NaCl, 5 mM GSSG, pH 7.5). The total elution volume was 330 ml, and fractions were collected at 5 ml intervals. Preparative gel filtration revealed essentially identical chromatograms when using the reduced cv.s-retinal complex of the di-cysteine mutant A212C:T250C of CRALBP or the cv.s-retinal complex of wild-type CRALBP (see Example 7, Figures 5 to 7). Oxidized glutathione (GSSG) is used during Gel filtration chromatography to in-situ generate oxidized di-cysteine CRALBP/cv.s- retinal complexes exhibiting about 20-fold increased photoprotection of the bound cv.s-retinal compared to the corresponding reduced complexes, see Figure 8. EXAMPLE 9

Photoisomerization Assay

Photoisomerization assays of c/.s-retinal and its complexes with wild-type CRALBP or with di-cysteine mutant A212C:T250C of CRALBP were performed essentially as described by Saari et al. (Saari JC and Bredberg DL, (1978), J Biol Chem. 262(16):7618-22). For this, the gel permeation chromatography purified ligand complexes were diluted to 26 pM in (GFC) buffer (lOmM Hepes, 100 mM NaCl, pH 7.5) and equimolar amounts of BSA were added to avoid protein precipitation through the light induced formation of free all-/ra//.s-retinal. The samples were exposed with a 100-W daylight bulb to an illuminance of 380 lux (Voltcraft MS- 1300 Luxmeter) at room temperature in the darkroom. Simultaneously UV/Vis absorption spectra were collected every 180 seconds for 3600 sec in total using the Evolution array UV/Vis spectrophotometer (Thermo Scientific). For photoisomerization experiments of the oxidized mutant A212C:T250C or the wild-type CRALBP, 5mM oxidized glutathione was added to the preformed complexes and the mixtures were kept overnight at 4°C. For repeated reduction, the oxidized glutathion was first removed from the samples using a Vivaspin 15R Hydrosart (Sartorius) by three times concentrating the samples to a tenth of the volume and subsequent redilution in buffer (10 mM Hepes, 100 mM NaCl, pH 7.5) to the original volume. The washed samples were then reduced by adding 5mM DTT and kept at 4°C for 1 hour. As can be seen from Figure 9, there was blockage of the mobile gate in the oxidized state, slowing down the rate of photoisomerization by at least 10-fold. Repeat reduction of the sample in 5 mM DTT reestablished wild-type like behavior of the protein as captured by the assay. Thus, the introduction of the A212CT250C double mutation represents an engineered redox-sensitive on-off switch into human CRALBP allowing for a reversible turning on and off of CRALBP’ s mobile gate functionality. The two mutations are located at adjacent positions in the mobile gate’s interface of CRALBP allowing for the formation of an intramolecular disulfide bond under oxidizing conditions and to regain the gate’s native functionality under reducing conditions. Accordingly, the photo-isomerization assay of the oxidized state of the A212C:T250C: l l-cz -retinal complex reveals increased photoprotection for bound l l -cv.s- retinal, while the reduced state restores CRALBP’ s native in vitro photosensitivity.

Our experimental data indicate that both distance and orientation of the two opposing cysteine side-chains lend themselves to the formation of a covalent disulfide bond. As a direct consequence, both the exposure to atmospheric oxygen and the addition of suitable oxidizing agents induce the formation of a covalent disulphide linkage via the loss of two electrons (one for each sulphur atom). The resulting immobilization of the mobile gate in A212C:T250C provides enhanced photoprotection for the bound ligand ( l l -cv.s-retinal or 9-cv.s-retinal) under persisting oxidizing conditions, e.g. upon entry into extracellular compartments. In relation to the activity profile of native CRALBP, stabilization of the photosensitive c/.s-retinoids not only impairs the formation, but also the premature release of /ra/z.s-retinal from the CRALBP binding-pocket. It is concluded that the wild-type CRALBP proteins as well as the redoxsensitive CRALBP mutant proteins allow production of complexes of CRALBP proteins with their cognate ligands, in particular cv.s-retinoids and preferably c/.s-retinals. In particular the complexes comprising the engineered CRALBP mutant proteins exhibit significantly increased photo resistance to daylight in their oxidized states in comparison to the respective wild-type CRALBP protein ligand-complexes. The oxidized CRALBP mutant protein ligand complexes are further shown to possess similar biophysical properties compared to wild-type CRALBP protein ligand complexes under reducing conditions.

EXAMPLE 10

Native page of wild-type and di-Cys mutants of CRALBP

Native-PAGE of the di -cysteine mutants of CRALBP in complex with cv.s-retinal on a 4- 16% polyacrylamide gel revealed similar bands as for wild-type CRALBP in complex with cis- retinal (Figure 10, Panel A and Panel B). After preparative GFC, the Native PAGE of the dicysteine mutant A212C:T250C CRALPB samples showed the same protein band pattern for fractions derived from Peaks 1,2,3 and 4 as for wild-type CRALBP. The lanes containing fractions 5, 6, 7, and 8 from peak one and its right-sided shoulder (Pl and P1.2) revealed continuous protein bands starting at a molecular weight of 720 kDa and ranging beyond 1048 kDa, as shown in Figure 10. These bands represent a A212C:T250C CRALBP oligomeric complex class, we call super-high molecular weight (SHMW). Lanes of the following smaller CRALBP oligomeric complex fraction are called high molecular weight CRALBP (HMW). These includes samples of the second peak (P2), fractions 12 and 13, and do feature a continuous band with a molecular weight between 242 kDa and 720 kDa, respectively.

EXAMPLE 11

Characterization of inventive complexes by dynamic light scattering

The same fractions derived from the preparative GFC that were characterized by analytical GFC, were also analyzed by dynamic light scattering (DLS) on a Malvern Zetasizer -n -

S®. Each DLS experiment contains was performed following the settings compiled in Table 5, the run duration and number of runs per measurement was set according to the autooptimization of the Malvern Zetasizer software.

Table 5. Instruments settings for characterization by DLS

Application CRALBP A212C:T250C + 9-cis-retinal complexes

Type: Protein Size

Material: Protein

Refractive. Index RI: 1.40

Absorbance (Abs): 0.001

Dispersant: H2O

Temperature: 25 °C

Viscosity: 0.8872 cP

Refracting Index RI: 1.33

Equilibration time: 15 sec

Detection angle: 173° (backscatter)

Run duration: Automatic

Number of run per measurement: Automatic (10 - 15)

The dynamic light scattering (DLS) measurements indicate that the sample peaks obtained from gel filtration represent discrete populations of inventive compositions and complexes, respectively. The average size distribution in diameter for the monomeric complex of wild-type CRALBP with 9-cv.s-retinal is 6.45 ± 0.76 nm, for the dimeric complex 7.07 ± 0.29 nm, for the HMW 12.89 ± 0.89 nm, and for the SHMW 21.43 ± 7.07 nm, respectively and of di-cysteine mutant A212C:T250C CRALBP with 9-cv.s-retinal is 6.50 ± 1.50 nm, for the dimeric complex 8.72 ± 2.41 nm, for the HMW 13.50 ± 0.47 nm, and for the SHMW 28.28 ± 2.13 nm, respectively (see Figure 11, Panel A and Panel B).

EXAMPLE 12

Use of RPE65-/- mouse model for monitoring RP treatments

Mice with a conventional knockout of the retinal pigmented epithelium protein 65 kDa gene (Rpe65~^/~) were described previously (Redmond, T.M., et al. 1998, Nat Genet. 20(4):344- 51). Young adult animals of either sex (6-10-week-old) were used. Animals were provided with standard chow (LabDiet 5053; LabDiet, Purina Mills) and maintained under a 12 h light / 12 h dark cycle. Mice were dark-adapted overnight before physiological recordings.

The RPE65-/- model chosen in this study for readout represents the most widely used mouse model for monitoring retinitis pigmentosa (RP) as well as for leber congenital amaurosis (LCA) related defects of the retina. In addition, the genetic defect abolishes the trans- o-cis re- isomerization activity of RPE65 in the visual cycle resulting in a very low c/.s-retinoid background. RPE65-/- thus is considered the prototypic animal model for chromophore deficiencies in retinal dystrophies. Six to ten week old mice are typically chosen for the ex vivo electroretinogram (ERG) experiments because at this age the rod and cone photoreceptors are, despite the lack of endogenously produced cis-retinoids, still viable and most suitable for reloading experiments using exogeneosly added czs-retinal.

EXAMPLE 13

Ex vivo Experiments

Acute application of wild-type and of mutant CRALBP monomer and SHMW 1:1 complexes loaded with 9-cis-retinal to mouse retinas for ex-vivo ERG recordings 200 pl aliquotes of concentrated (about 55-65 mg/ml) wild-type or mutant monomeric or super high molecular weight (SHMW) CRALBP complexes in the form of their His-tag adducts (SEQ ID NO:4 and SEQ ID NO: 12) loaded 1 : 1 with 9-cis retinal in 100 mM NaCl in 10 mM HEPES solution (pH 7.5) containing about 1.62 mM of 9-cv.s-retinal were diluted in 1.8 ml of L15 cell culture solution (13.6 mg/ml, pH 7.4, Sigma) containing 1% BSA and suspended thoroughly, in the dark. The final concentration of the 9-cis retinal was estimated to be about 162 pM. A whole isolated RpedS ^ mouse retina on filter paper was incubated in a Petri dish with 2 ml of this solution in oxygenated dark container for 4-4.5 h in the dark, at RT. Some untreated control retinas from the same mouse line were incubated 4-4.5 h in the same L15 solution without CRALBP and retinoid. In a separate control experiment, a comparable amount of pure 9-cis retinal in L15 (about 135 pM, dissolved in 0.1% EtOH) was applied to the retina for 1-1.5 h. The tissue was then transferred to the perfusion chamber for ex vivo ERG recordings, as described below.

Ex vivo ERG recordings from isolated mouse retinas

RPE65-deficient mice were dark-adapted overnight, sacrificed by CO2 asphyxiation, and a whole retina was removed from each mouse eyecup under infrared illumination. The retina was mounted on filter paper with the photoreceptor side up and placed in a perfusion chamber between two electrodes connected to a differential amplifier (Vinberg, F., et al., 2014, Vision Res. 2014;101 : 108-17). The tissue was perfused with Locke’s solution containing 112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCh, 1.2 mM CaCh, 10 mM HEPES, pH 7.4, 20 mM NaHCCh, 3 mMNa succinate, 0.5 mM Na glutamate, 0.02 mM EDTA, and 10 mM glucose. This solution was supplemented with 2 mM L-glutamate and 10 pM DL-2-amino-4-phosphonobutyric acid (DL-AP4) to block postsynaptic components of the photoresponse (Sillman, A.J., 1969, Vision Res. 9: 1435-1442), and with 20 pM BaCh to suppress the slow glial Pill component (Nymark, S., 2005, J Physiol. 15:567(PT3), 923-938). MEM vitamins and MEM amino acid solutions (Sigma) were also added to improve retina viability. The perfusion solution was continuously bubbled with a 95% O2 / 5% CO2 mixture and heated to 36-37 °C.

Light stimulation was applied in 20 ms test flashes of calibrated 505 nm LED light. The stimulating light intensity was controlled by a computer in 0.5 log unit steps. Intensity-response relationships were fitted with Naka-Rushton hyperbolic functions, as follows:

D . / l n > ^Kmax ¹ ^K ~

¹ jn + j ' ⁿ ’ 1/2 where R is the transient-peak amplitude of the response, R_max is the maximal response amplitude, I is the flash intensity, n is the Hill coefficient (exponent), and I is the halfsaturating light intensity. Photoresponses were amplified by a differential amplifier (DP-311, Warner Instruments), low-pass filtered at 30 Hz (8-pole Bessel), digitized at 1 kHz, and stored on a computer for further analysis. Data were analyzed with Clampfit 10.4 and Origin 8.5 software.

For ex vivo drug treatments RpedS ^ mice have to be dark adapted as described in Example 12, sacrificed and the retinae isolated from the eye-balls and mounted on filterpaper. The later are then transferred into 2ml of LI 5 cell culture solution containg the drug. In all experiments the concentrations for 9-cis retinal and CRALBP are equimolar at 162 pM and all experiments are carried out under infrared light illumination as described in the Example 13. Retinae are incubated in the oxygenated dark container for 4-4.5 h in the dark, at RT. After the incubation with the drug the retinae are transferred into a perfusion chamber containing Locke’s solution to improve retina viability and, for the ERG measurements, placed with the photoreceptor side up between two electrodes connected to a differential amplifier.

As diagnostic tool light-evoked intraretinal field potentials (electroretinograms, ERGs) are recorded in vivo to assess the electrical activity of the retina in response to the light stimulus. The a-wave, sometimes called the “late receptor potential,” reflects the general physiological health of the photoreceptors in the outer retina. In contrast, the b-wave reflects the health of the inner layers of the retina, including the ON bipolar cells and the Muller cells (Miller, R.F., 1970, J Neurophysiol. 33(3):323-41).

The mounted retinae are stimulated with short light flashes with the electric responses of the retinae being monitored in dependency of the applied intensities of the light flashes. In Figure 12 the obtained ex vivo ERG photoresponse curves for RPE65-/- and wild-type retinae are shown as semi-logarithmic graph where the y-axis represents the amplitude of the photoresponse in pV while the x-axis (logarithmic scale) represents the light intensity in photons per square pm. The position of the inflection point of each curve in relation to the x- axis thus correlates with the light-sensitivity of the retina, while the maximum of the curve with realation to the y-axis correlates with the amount of light sensitive photoreceptors of the retina.

All response curves have solid statistics with sigmoidal shape and clear inflection points. The highest responses (see Figure 12, Panel A and Panel B) are observed with the untreated wild-type mouse retinae (round open circles) representing the positive control of the experiments and having a maximum response plateau slightly below 300 pV and a maximum shift of the inflection point towards the left, representing the maximum photo-response achievable ex-vivo with a healthy retina under nearly physiologic conditions. It should be mentioned here that the wild-type mouse retinae contain the naturally occurring chromophore 1 1 -cv.s retinal while our drug treatment is based on the less abundant 9-cis retinal chromophore. Due to the quantum yield of the later being half the one of 11 -cis retinal the maximally achievable amplitudes in our treatments must be significantly below the ones of the wild-type.

Our drug treatments using wild-type CRALBP monomer complexes (open triangles, FIG. 12, Panel A) and mutant CRALBP monomer complexes (open triangles, FIG. 12B) and using the corresponding SHMW complexes (open squares in FIG. 12, Panel A and FIG. 12, Panel B) clearly indicate that the SHMW complexes are about twofold superior in their response compared to the monomeric complexes with respect to the maximum of the amplitude and also in terms of light-sensitivity of the activated photoreceptors. Keeping in mind that all complexes are loaded at equimolar ratios with 9-cis-retinal it is concluded that SHMW complexes are more potent than the monomeric ones in the ex-vivo ERG setup. In contrary the comparison between wild-type and mutant complexes does not reveal significant differences in the response curves within the time frame of the treatment (4 - 4.5 h). This, however, was not unexpected due to settings of the scotopic measurements for which the mice had to be kept in the dark prior to the measurements, but rather these results again confirm the earlier established similar biophysical properties of the wild-type and mutant complexes (WO2022/243386). In view of the also earlier established significantly increased photoresistance to daylight of the mutant complexes in their oxidized state as compared to the wild-type complexes, superiority of the mutant complexes in case of prolongated light exposure is expected. For the untreated RPE65-/- retinae (rhombic bullets) almost no light response at all is observed as indicated by the flat line even at highest light intensities and confirming severe chromophore deficiency in RPE65-/- retinae (see Figure 12, Panel A and Figure 12, Panel B).

EXAMPLE 14

In vivo Experiments

Intravitreal injection o f of wild-type and o f mutant CRALBP monomer and SHMW 1:1 complexes loaded with 9-cis-retinal

With the room lights off, 2 pl of concentrated (55-65 mg/ml) and carefully mixed monomeric or SHMW CRALBP complexes in the form of their His-tag adducts (SEQ ID NO:4 and SEQ ID NO: 12) in 100 mM NaCl in 10 mM HEPES solution (pH 7.5) containing about 1.62 mM of 9-cv.s-retinal were injected into a vitreous of the eye of each anesthetized Rpe65 ^/~ mouse, and an equal volume of PBS solution was injected into control eyes (same or different animals were used for control injections). The injections were performed by hand using a Hamilton syringe under the microscope and infrared illumination. Before each injection, a small hole was made in the cornea with a 27 G x 1/2 needle, to facilitate the insertion of the syringe needle. The total amount of injected retinoid was estimated to be about 3.2 nmol, which exceeds the average rhodopsin content in the adult mouse eye by a factor of 5-6. Animals were then allowed to recover from the anesthesia and returned to their original cages with access to food and water and kept in the dark for 25-30 h (day 1) prior to recordings of intensity-response families by ERG in vivo, as described below Mice were then recovered and re-tested after additional 48-72 h in the dark (days 3-4).

In vivo ERG analysis

Dark-adapted RpedS^ mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (4 mg/kg). Pupils were dilated with a drop of 1% atropine sulfate. Mouse body temperature was maintained at 37 °C with a heating pad. ERG responses were recorded from both eyes by corneal contact electrodes held in place by a drop of Gonak solution. Full-field ERGs were performed with the UTAS BigShot apparatus (LKC Technologies) using Ganzfeld-derived test flashes of calibrated green 530 nm LED light (within a range from 2.2xl0'⁴ cd-s nr² to 23.5 cd-s nr²) or white light generated by the Xenon Flash tube (from 80.7 cd-s m-2 to 700 cd-s m-2) (Kolesnikov, A.V., 2010, J Neurosci. 18;30(33): 11222-31). Both scotopic ERG a-waves and b-waves were measured on days 1 and 3-4 after injection of CRALBP nanoparticles.

For rod dark adaptation tests (performed on days 3-4 after injections), rod ERG a-wave flash sensitivity was first determined in the dark, as follows: 5f - A/(A_ma.^ I) , where A is the rod a-wave dim flash response amplitude, A_max is the maximal response amplitude for that eye produced with the brightest green light stimulus (23.5 cd-s/m²), and I is the flash strength of dim flash response (in cd-s m'²). The rod pigment was then near completely bleached by a 35-s exposure to bright light delivered by a 520 nm LED focused at the surface of the mouse eye cornea. The bleaching fraction was estimated by the following formula:

F = 1 - exp(-/ 7), where F is the fraction of pigment bleached, t is the duration of the light exposure (in seconds), /is the bleaching light intensity of 520 nm LED light (1.3 x 10⁸ photons pm'² s'¹), and P is the photosensitivity of mouse rods at the wavelength of peak absorbance (5.7 x 10-9 pm², Woodruff, M.L., et al., 2004, The Journal of Physiology, 557:821-828). After the bleach, the recovery of rod response was followed in darkness for up to 1 h. Mice were re-anesthesized with a lower dose of ketamine (~ 1/3 of the initial dose) in the middle of that period. If necessary, a 1 : 1 mixture of PBS and Gonak solutions was gently applied to eyes with a plastic syringe to protect them from drying and maintain contacts with the recording electrodes.

Statistical analysis

All electrophysiological data were expressed as means ± SEM and analyzed using the independent two-tailed Student’s /-test, with an accepted significance level of P < 0.05.

In all our in-vivo drug treatments Rpe65-/- mice are dark adapted, anesthetized and mouse body temperature has to be maintained at 37 °C as described in Examples 12 and 13 respectively. The intravitreal injections are performed by hand using a Hamilton syringe under the microscope and infrared illumination with 2 pl of the drug complex containing ~ 3.2 nmol of 9-cv.s-retinal being injected in one eye and an equal volume of PBS solution, as a control, into the other eye. The total amount of injected retinoid is estimated to exceed the average rhodopsin content of a healthy adult mouse eye by at least a factor of 5-6.

Averaged in vivo ERG responses are recorded with both eyes by corneal contact electrodes. The number n of the used eyes for averaging the recordings is given in parentheses. Both scotopic ERG a- waves and b-waves are measured on days 1 and 3-4 after injection of CRALBP complexes (see Figures 13-16).

For all our in-vivo ERG experiments we have added, on one hand, equal numbers of untreated Rpe65-/- mous eye experiments for providing averaged in-vivo ERG recordings representing the corresponding averaged background and, on the other hand, we have provided in-vivo ERG experiments using healthy wild-type mice for providing averaged positive control ERG recordings (see Figures 13-18). Our averaged in vivo ERG readouts for the treatment of Rpe65-/- mice with the wild-type CRALBP monomer complex with 9-cis retinal (Figure 13, Panel A; open and filled triangles) after one day and after 3-4 days respectively indicate recovery to a significant degree of both rod maximal ERG response (a-wave) and sensitivity. The rescue somewhat increases after 3-4 days post-injection (mice kept in the dark between the recordings). Similar results are obtained for averaged b-wave recordings (Figure 14, Panel A; open and filled triangles), but a somewhat higher increase after 3-4 days post-injection is observable. The results obtained from the averaged recordings of the treatments of Rpe65-/- mice with the wild-type CRALBP SHMW complex with 9-cis retinal (Figurel3, Panel B amd 14, Panel B; open and filled squares) do not significantly differ from our previous wild-type data. When recapitulating all treatments and averaged recordings by using instead of the parent wild-type the corresponding mutant CRALBP complexes (see Figures 15-16) no significant differences are observed either.

In order to assess the potential of RPE65-/- mice treated with our drugs to recover after being bleached with strong light intensities dark adaptation experiments are carried out. For these dark adaptation experiments (see Figures 17-18) averaged in-vivo ERGs are reported as sensitivity over time. A, Ratio of a-wave amplitudes normalized to the pre-bleach a-wave amplitude. 7>, Ratio of a-wave amplitudes normalized to the pre-bleach a-wave amplitude and per photon. This normalization is used because it allows to compare single recordings of independent dark adaptaion experiments. Initially, rod pigment are near completely bleached by a 35-s exposure to bright light ( >90% bleach). After the bleach, the recovery of rod response is recorded in darkness for up to 1 h.

Dark adaptation in vivo ERGs are performed with RPE65-/- mice treated with wild-type monomeric and SHMW CRALBP/9-cis-retinal complexes (see Figure 17, Panel A and Figure 17, Panel B). Results are reported as recovery of sensitivity over time. In FIG. 17, Panel A the RPE65-/- eye treated with the wild-type CRALBP SHMW complex with 9-cis retinal (filled squares) shows exponential recovery from bleaching and is significantly faster than the corresponding monomeric CRALBP complex (filled triangles). The velocity of recovery is significantly higher in both cases compared with the velocity of recovery of the wild-type eye (filled circles in FIG. 17, Panel A and FIG. 17, Panel B).

Dark adaptation in vivo ERGs are also performed with RPE65-/- mice treated with monomeric and SHMW mutant CRALBP/9-cis-retinal complexes (see Figure 18, Panel A and Figure 18, Panel B). Results are reported as recovery of sensitivity over time. In FIG. 18, Panel A the RPE65-/- eye treated with the mutant CRALBP SHMW complex with 9-cis retinal (filled squares) shows again exponential recovery from bleaching but this time is significantly slower than the corresponding monomeric complex (filled triangles). The velocity of recovery is again significantly higher in both treatments compared with the one of the wild-type eye (filled circles in FIG. 18, Panel A and FIG. 18, Panel B). These results may indicate that the kinetics of recovery in the treated eyes is different and most likely occurs an the basis of a different mechanism than the one in the wild-type eye.

Claims

1. A composition for use in a method of preventing or treating a retinal disease or disorder of an animal, preferably of a human, wherein said method comprises administering said composition to said animal, wherein said composition comprises, preferably consists of, a complex, wherein said complex comprises

(a) a CRALBP protein; and

(b) a cognate ligand of CRALBP, wherein preferably said cognate ligand is a cisretinoid.

2. The composition for use of claim 1, wherein said CRALBP protein comprises a wild-type CRALBP protein or a CRALBP mutant protein.

3. The composition for use of claim 1 or claim 2, wherein said CRALBP protein comprises a wild-type CRALBP protein.

4. The composition for use of claim 3, wherein said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3.

5. The composition for use of claim 1 or claim 2, wherein said CRALBP protein comprises a CRALBP mutant protein, wherein said CRALBP mutant protein comprises a mutein of a wild-type CRALBP protein, wherein said mutein comprises at least one pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, wherein each of said pair of cysteines is able of forming, preferably forms, a disulfide bond, wherein one cysteine of each pair of amino acid mutations by cysteines is a mutation of an amino acid within the amino acid residues corresponding to amino acids 204-229 of SEQ ID NO:3, wherein the other mutated amino acid by cysteine of said pair is a mutation of an amino acid within the amino acid residues corresponding to amino acids 244-261 of SEQ ID NO:3.

6. The composition for use of claim 5, wherein said mutein comprises one, two, three or four pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein, and wherein preferably said mutein comprises one or two pairs of amino acid mutations by cysteines as compared to said wild-type CRALBP protein. The composition for use of claim 5 or claim 6, wherein said pair of amino acid mutations by cysteines as compared to said wild-type CRALBP protein is selected from

(3) a mutation of an amino acid corresponding to amino acid 220 of SEQ ID NO: 3 and a mutation of an amino acid corresponding to amino acid 254 of SEQ ID NO:3;

(4) a mutation of an amino acid corresponding to amino acid 224 of SEQ ID NO:3 and a mutation of an amino acid corresponding to amino acid 257 of SEQ ID NO:3. The composition for use of any one of the claims 5 to 7, wherein said wild-type CRALBP protein is the human CRALBP protein of SEQ ID NO:3, and said mutein comprises one or two or three pairs of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein, and wherein said one pair of amino acid mutations by cysteines as compared to said human wild-type CRALBP protein is selected from

(a) a first pair of amino acid mutations by cysteines and a second pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3, and said second pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3;

(x) a first pair of amino acid mutations by cysteines, a second pair of amino acid mutations by cysteines, and a third pair of amino acid mutations by cysteines, wherein said first pair of amino acid mutations by cysteines is a mutation of amino acid 212 of SEQ ID NO:3 and a mutation of amino acid 250 of SEQ ID NO:3, said second pair of amino acid mutations by cysteines is a mutation of amino acid 220 of SEQ ID NO:3 and a mutation of amino acid 254 of SEQ ID NO:3, and said third pair of amino acid mutations by cysteines is a mutation of amino acid 224 of SEQ ID NO:3 and a mutation of amino acid 257 of SEQ ID NO:3. The composition for use of any one of the claims 5 to 8, wherein said CRALBP mutant protein has an amino acid sequence selected from group consisting of SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26, and wherein preferably said CRALBP mutant protein has an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NO:24, and wherein further preferably said CRALBP mutant protein has the amino acid sequence of SEQ ID NO: 11. The composition for use of any one of the preceding claims, wherein said CRALBP protein has an amino acid sequence selected from group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

11. The composition for use of any one of the preceding claims, wherein said cognate ligand of CRALBP is a cz.s-retinoid selected from (i) 9-cz -retinal; (ii) 9-cz -retinol; (iii) l l -cz.s- retinal; (iv) 11-czs-retinol; (v) 11, 13-di-cz -retinal; (vi) 11, 13-di-cz -retinol; (vii) 9, 13- di-cz.s-retinal; and (viii) 9, 13-di-cz.s-retinol; and wherein preferably said cognate ligand of CRALBP is 9-cz.s-retinal or 11-czs-retinal.

12. The composition for use of any one of the preceding claims, wherein said complex is a monomeric complex of said CRALBP protein and said cognate ligand.

13. The composition for use of any one of the claims 1 to 11, wherein said complex is an oligomeric complex of said CRALBP protein and said cognate ligand, wherein said oligomeric complex has a molecular weight of at least 600kDa, preferably of at least 720kDa, and preferably a molecular weight of at most 2500kDa, further preferably a molecular weight of at most 2000kDa, or wherein said oligomeric complex has a average diameter of about 24 to 33 nm, and wherein preferably said oligomeric complex has a average diameter of about 25 to 32 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS).

14. The composition for use of any one of the preceding claims, wherein said disease or disorder is selected from retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), retinitis punctata albesciens (RPA), fundus albipunctatus and cone stationary night blindness (CSNB), a RPE65 gene mutation and a LRAT gene mutation, wherein preferably said disease or disorder is RP.

15. The composition for use of any one of the preceding claims, wherein said composition is administered to the eye of said animal, preferably of said human, by subretinal, direct retinal or intravitreal injection, wherein preferably by intravitreal injection.