Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.

Retinitis Pigmentosa (RP) is a progressive, debilitating visual disorder caused by mutations in a diverse set of genes. In both humans with RP and mouse models of RP, rod photoreceptor dysfunction leads to loss of night vision, and is followed by secondary cone photoreceptor dysfunction and degeneration, leading to loss of daylight color vision. A strategy to prevent secondary cone death could provide a general RP therapy to preserve daylight color vision regardless of the underlying mutation. In mouse models of RP, cones in the peripheral retina survive long-term, despite complete rod loss. The mechanism for such peripheral cone survival had not been explored. Here, we found that active retinoic acid (RA) signaling in peripheral Muller glia is necessary for the abnormally long survival of these peripheral cones. RA depletion by conditional knockout of RA synthesis enzymes, or overexpression of an RA degradation enzyme, abrogated the extended survival of peripheral cones. Conversely, constitutive activation of RA signaling in the central retina promoted long-term cone survival. These results indicate that RA signaling mediates the prolonged peripheral cone survival in the rd1 mouse model of retinal degeneration, and provide a basis for a generic strategy for cone survival in the many diseases that lead to loss of cone-mediated vision.

Retinitis Pigmentosa (RP) is a wide array of progressive, debilitating visual disorders caused by mutations in a diverse set of genes. In both human patients and mouse models of RP, rod photoreceptor dysfunction leads to loss of night vision, and is followed by secondary cone photoreceptor dysfunction and degeneration, leading to loss of daylight color vision. A strategy to prevent secondary cone death could provide a generalized RP therapy to preserve daylight color vision regardless of the underlying mutation. In mouse models of RP, cones in the far peripheral retina survive long-term, despite complete rod loss. The mechanism for such peripheral cone survival had not been explored. Here, we found that active retinoic acid (RA) signaling in peripheral Muller glia is both sufficient and necessary for the extended cone survival. RA depletion by conditional knockout of RA synthesis enzymes, or overexpression of an RA degradation enzyme, abrogated peripheral cone survival. Conversely, constitutive activation of RA signaling in the central retina promoted long-term cone survival. These results indicate that RA signaling mediates the prolonged peripheral cone survival in the rd1 mouse model of retinal degeneration, and provide a basis for a generic strategy for cone survival in the many diseases that lead to loss of cone-mediated vision.

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