Phoborhodopsin, a repellent phototaxis receptor in Halobacterium halobium, exhibits vibrational fine structure, a feature that has not been identified for any other rhodopsin pigment at physiological temperatures. This conclusion follows form analysis of the absorption properties of the pigment in H. halobium membranes containing native retinal and an array of retinal analogues. The absorption spectrum of the native pigment has a maximum at 487 nm with a pronounced shoulder at 460 nm; however, the bandwidth is that expected for a single retinylidene species. Gaussian band-shape simulation with a spacing corresponding to the vibrational frequencies of polyene stretching modes reproduces the structured absorption spectra of native pigment as well as of analogue phoborhodopsin. Absorption shifts produced by a series of dihydroretinal and other retinal analogues strongly indicate that the dominant factor regulating the color of the pigment is planarization of the retinal ring with respect to the polyene chain.

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The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20–35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.