Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.

The rhodopsin crystal structure provides a structural basis for understanding the function of this and other G protein–coupled receptors (GPCRs). The major structural motifs observed for rhodopsin are expected to carry over to other GPCRs, and the mechanism of transformation of the receptor from inactive to active forms is thus likely conserved. Moreover, the high expression level of rhodopsin in the retina, its specific localization in the internal disks of the photoreceptor structures [termed rod outer segments (ROS)], and the lack of other highly abundant membrane proteins allow rhodopsin to be examined in the native disk membranes by a number of methods. The results of these investigations provide evidence of the propensity of rhodopsin and, most likely, other GPCRs to dimerize, a property that may be pertinent to their function.

Absorption of a photon by an opsin pigment causes isomerization of the chromophore from 11-cis-retinaldehyde to all-trans-retinaldehyde. Regeneration of visual chromophore following light exposure is dependent on an enzyme pathway called the retinoid or visual cycle. Our understanding of this pathway has been greatly facilitated by the identification of disease-causing mutations in the genes coding for visual cycle enzymes. Defects in nearly every step of this pathway are responsible for human-inherited retinal dystrophies. These retinal dystrophies can be divided into two etiologic groups. One involves the impaired synthesis of visual chromophore. The second involves accumulation of cytotoxic products derived from all-trans-retinaldehyde. Gene therapy has been successfully used in animal models of these diseases to rescue the function of enzymes involved in chromophore regeneration, restoring vision. Dystrophies resulting from impaired chromophore synthesis can also be treated by supplementation with a chromophore analog. Dystrophies resulting from the accumulation of toxic pigments can be treated pharmacologically by inhibiting the visual cycle, or limiting the supply of vitamin A to the eyes. Recent progress in both areas provides hope that multiple inherited retinal diseases will soon be treated by pharmaceutical intervention.

The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors.

Photoreceptor cells maintain a roughly constant length by continuously generating new outer segments from their base while simultaneously releasing mature outer segments engulfed by the retinal pigment epithelium (RPE). Thus postmitotic RPE cells phagocytose an immense amount of material over a lifetime, disposing of photoreceptor cell waste while retaining useful content. This review focuses on current knowledge of outer segment phagocytosis, discussing the steps involved along with their critical participants as well as how various perturbations in outer segment (OS) disposal can lead to retinopathies.