RLBP1 Antibody

What is RLBP1/CRALBP and what is its role in retinal physiology?

RLBP1 (Retinaldehyde-binding protein 1), also known as cellular retinaldehyde-binding protein (CRALBP), is a 36-kDa aqueous protein expressed primarily in the retinal pigment epithelium (RPE) and retinal Müller cells. In the RPE, CRALBP functions in the visual cycle by serving as a carrier for 11-cis-retinol and 11-cis-retinal, the chromophore of rod and cone opsins that is delivered to photoreceptors . In Müller cells, CRALBP supports the recycling of chromophore that helps cone cells function in high light intensities .

The protein's significance is demonstrated in knockout models where Rlbp1-/- mice produce 11-cis retinal at a rate 10-fold slower than normal, resulting in significantly delayed dark adaptation . This functional role makes CRALBP a critical component in vision research and a target for therapeutic interventions in related retinal diseases.

What are the validated applications for RLBP1 antibodies in retinal research?

RLBP1 antibodies have been validated for multiple experimental applications:

These applications are particularly valuable for localizing RLBP1 expression in tissue sections, quantifying protein levels in various samples, and validating gene therapy approaches in both animal models and cell cultures.

What are the recommended dilutions and protocols for optimal RLBP1 antibody performance?

For optimal results with RLBP1 antibodies, the following application-specific dilutions are recommended:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative . It's important to note that these reagents should be titrated in each testing system to obtain optimal results, as performance can be sample-dependent . For storage, maintain antibodies at -20°C in PBS with 0.02% sodium azide and 50% glycerol pH 7.3 for up to one year after shipment .

How can RLBP1 antibodies be used to validate gene therapy approaches for retinal diseases?

RLBP1 antibodies play a crucial role in validating gene therapy approaches for RLBP1-associated retinal diseases:

First, they can detect CRALBP expression in neural retinal lysates from Rlbp1-/- mouse eyes following injection with AAV vectors carrying the human RLBP1 gene . This allows researchers to confirm successful transduction and expression of the therapeutic gene.

Second, RLBP1 antibodies enable researchers to compare protein expression between untreated Rlbp1-/- mice (negative control), wild-type mice (positive control), and treated mice to assess therapy efficacy . This comparison helps quantify the degree of restoration achieved through gene supplementation.

Third, though technical challenges exist in analyzing RPE protein lysates by Western blotting, indirect evidence of successful RPE transduction can be correlated with functional rescue as measured by electroretinogram (ERG) recovery .

Finally, in more recent advances, RLBP1 antibodies have been used to evaluate gene therapy efficacy in patient-specific iPSC-derived RPE models, providing a human-relevant platform for preclinical testing .

What genetic disorders are associated with RLBP1 mutations and how are they characterized?

Mutations in the RLBP1 gene are associated with several inherited retinal disorders:

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa characterized by abnormally slow recovery of retinal sensitivity after light exposure, eventually progressing to blindness . More specifically, variants in RLBP1 lead to three distinct clinical subtypes :

• Bothnia dystrophy (BD)

• Retinitis punctata albescens (RPA)

• Newfoundland rod-cone dystrophy (NFRCD)

In a screening of 324 unrelated patients with recessive or isolate retinitis pigmentosa, retinitis punctata albescens, Leber congenital amaurosis, or related diseases, four novel mutations were identified in three unrelated patients with recessively inherited retinitis punctata albescens . These mutations included two missense mutations, one frameshift, and one affecting a canonical splice donor site .

Research indicates that recessive mutations in the RLBP1 gene are an uncommon cause of retinal degeneration in humans, with the primary phenotype appearing to be a form of retinitis punctata albescens .

How do RLBP1-deficient animal models compare to human RLBP1-associated diseases?

There are notable differences between RLBP1-deficient animal models and human RLBP1-associated disorders:

Rlbp1-/- knockout mice exhibit a 10-fold reduction in 11-cis retinal production rate compared to wild-type mice, resulting in significantly slower dark adaptation . After bleaching more than 10% of rhodopsin, these mice require many hours to regain full light sensitivity, in contrast to the substantial recovery observed in Rlbp1+/+ mice within approximately 3 hours .

Interestingly, after prolonged dark adaptation periods (≥24 hours), the electroretinograms (ERGs) of Rlbp1-/- mice show normal amplitudes in response to single light flashes . This suggests that the primary defect in these models is in the kinetics of visual cycle regeneration rather than in absolute photoreceptor function.

These differences highlight both the utility and limitations of animal models for studying RLBP1-associated retinal diseases and testing potential therapies.

What is known about the newly discovered smaller CRALBP isoform?

Recent research has revealed a previously unknown smaller CRALBP isoform with important implications for both basic research and therapeutic development:

This smaller isoform is naturally and differentially expressed in both human and murine retina, arising from an alternative methionine initiation site . Functional studies indicate that this isoform also plays a role in the visual cycle, suggesting it has physiological relevance .

The discovery has direct implications for gene therapy approaches. Researchers found that the AAV2/5-CAG-RLBP1 vector encodes two CRALBP isoforms, in contrast to the previously published AAV2/8-hRLBP1-RLBP1 vector that appeared to encode only the full-length isoform .

This finding provides novel insights into CRALBP expression and RLBP1-associated pathophysiology while raising important considerations for successful gene supplementation therapy . The expression of both isoforms may be necessary for complete functional rescue, which has implications for vector design and therapeutic strategies.

How have different AAV serotypes and promoters been evaluated for RLBP1 gene therapy?

Multiple AAV serotypes and promoters have been tested to optimize RLBP1 gene delivery for therapeutic applications:

Researchers have explored various recombinant adeno-associated vectors (rAAVs) with different promoters, capsid serotypes, and genome conformations . Specifically, they have generated rAAVs using promoters derived from the human RLBP1, RPE65, and BEST1 genes to drive expression of reporter genes and therapeutic transgenes .

Among these, a promoter derived from the RLBP1 gene demonstrated superior targeting, mediating expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in both wild-type mice and monkeys .

More recent studies have employed AAV2/5 with the CAG promoter (AAV2/5-CAG-RLBP1) for RLBP1 gene delivery . This vector design was functionally validated in iPSC-derived RPE models from patients with different RLBP1-associated clinical subtypes before in vivo testing in the Rlbp1-/- mouse model .

This systematic evaluation of vector designs highlights the importance of both serotype selection and promoter choice in developing effective gene therapies for RLBP1-associated retinal diseases.

How can iPSC-derived retinal models be used to study RLBP1-associated diseases?

Patient-specific induced pluripotent stem cell (iPSC) technology has revolutionized the modeling of RLBP1-associated retinal diseases:

Researchers have generated iPSC-derived retinal pigment epithelium (RPE) from patients carrying RLBP1 variants associated with the three clinical subtypes: Retinitis punctata albescens (RPA), Bothnia dystrophy (BD), and Newfoundland rod-cone dystrophy (NFRCD) . These models enable the study of disease mechanisms in a patient-specific context.

The iPSC-derived RPE models allow researchers to assay the effect of different RLBP1 variants on CRALBP expression and RPE function, helping identify pertinent read-outs to assess therapeutic rescue . Importantly, the functionality of RLBP1 iPSC-derived RPE has been found to correlate with clinical severity, providing a valuable model system that recapitulates disease features .

These models also serve as platforms for proof-of-concept studies for gene therapy approaches. Researchers have successfully performed AAV2/5-mediated RLBP1 supplementation ex vivo in the RLBP1 iPSC-derived RPE before validating their findings in vivo in the Rlbp1-/- mouse model .

This approach provides a human-relevant system for preclinical testing of therapeutic strategies and offers insights into RLBP1-associated pathophysiology that may not be evident in animal models.

What technical challenges exist in analyzing RLBP1 expression in different retinal cell types?

Several technical challenges have been reported when analyzing RLBP1 expression in retinal tissue:

Researchers have encountered specific difficulties when analyzing RLBP1/CRALBP by Western blotting in protein lysates generated from tissue containing RPE . These challenges have sometimes necessitated relying on indirect evidence of successful gene delivery, such as functional recovery measured by electroretinogram (ERG), rather than direct protein detection .

The recent discovery of a smaller CRALBP isoform adds complexity to expression analysis, as both isoforms need to be detected and quantified to fully understand RLBP1 biology and therapeutic outcomes . This requires optimized antibodies and detection methods that can distinguish between the isoforms.

The differential expression of RLBP1 in RPE and Müller cells presents challenges in isolating and analyzing these specific cell populations separately. The protein's expression in multiple cell types can complicate the interpretation of whole-tissue analyses.

Finally, the relative abundance of RLBP1 may vary between species and disease states, requiring careful optimization of detection methods and appropriate controls when comparing expression levels between different samples or experimental conditions.