AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types

Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants

Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (per(WT)), mutant peripherin-2 (per(MT)), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to per(WT) and Rom-1. Furthermore, both mutants are preferentially forming non-covalent per(MT)-per(MT), per(WT)-per(MT), and Rom-1-per(MT) dimers. However, only per(WT)-per(MT), but not per(MT)-per(MT) or Rom-1-per(MT) complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent per(WT)-per(MT) dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of per(WT) and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals

AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types

Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants

Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (per$^{WT}$), mutant peripherin-2 (per$^{MT}$), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to per$^{WT}$ and Rom-1. Furthermore, both mutants are preferentially forming non-covalent per$^{MT}$-per$^{MT}$, per$^{WT}$-per$^{MT}$, and Rom-1-per$^{MT}$ dimers. However, only per$^{WT}$-per$^{MT}$, but not per$^{MT}$-per$^{MT}$ or Rom-1-per$^{MT}$ complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent per$^{WT}$-per$^{MT}$ dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of per$^{WT}$ and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals

dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy

Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the appropriate counterpart(s) to compensate for the missing gene function. This approach offers key advantages over conventional gene therapies because it is mutation- and gene size-independent. Here, we describe a protocol for the design, execution and evaluation of such gene therapies using dCas9-VPR. We offer guidelines on how to identify functionally equivalent genes, design and clone single guide RNAs and evaluate transcriptional activation in vitro. Moreover, focusing on inherited retinal diseases, we provide a detailed protocol on how to apply this strategy in mice using dual recombinant adeno-associated virus vectors and how to evaluate its functionality and off-target effects in the target tissue. This strategy is in principle applicable to all organisms that possess functionally equivalent genes suitable for transcriptional activation and addresses pivotal unmet needs in gene therapy with high translational potential. The protocol can be completed in 15-20 weeks