A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

Ablation of inhibitory neurons that produce AgRP, NPY and GABA in the arcuate nucleus (ARC) of adult mice results in starvation within ∼ 6 days. Viral-mediated inactivation of GABA biosynthesis in the ARC also promotes anorexia. Chronic subcutaneous delivery of bretazenil (a GABAA receptor partial agonist) for 11 days maintains feeding and survival following ablation of AgRP neurons, an effect that persists following cessation of drug delivery. Moreover, direct delivery of bretazenil into the parabrachial nucleus (PBN), a nucleus that relays gustatory and visceral sensory information, is sufficient to maintain feeding after AgRP neuron ablation, whereas delivery into other post-synaptic targets of AgRP neurons is ineffective. These results suggest that hyperactivity within the PBN following loss of GABAergic input from AgRP neurons promotes anorexia. We suggest that suppression of neuronal excitability within the PBN permits feeding and instatement of compensatory mechanisms that eventually allow mice to eat without GABA signaling from AgRP neurons.

Insulin-like peptides (ILPs) exist in insects and are encoded by multigene families that are expressed in the brain and other tissues. Upon secretion, these peptides likely serve as hormones, neurotransmitters, and growth factors, but to date, few direct functions have been demonstrated. In Drosophila melanogaster, molecular genetic studies have revealed elements of a conserved insulin signaling pathway, and as in other animal models, it appears to play a key role in metabolism, growth, reproduction, and aging. This review offers (a) an integrated summary of the efforts to characterize the distribution of ILPs in insects and to define this pathway and its functions in Drosophila and (b) a few considerations for future studies of ILP endocrinology in insects.

Animals display stereotyped behavioral modifications during development, but little is known about how genes and neural circuits are regulated to turn on/off behaviors. Here we report that Drosophila neuropeptide F (dNPF), a human NPY homolog, coordinates larval behavioral changes during development. The brain expression of npf is high in larvae attracted to food, whereas its downregulation coincides with the onset of behaviors of older larvae, including food aversion, hypermobility, and cooperative burrowing. Loss of dNPF signaling in young transgenic larvae led to the premature display of behavioral phenotypes associated with older larvae. Conversely, dNPF overexpression in older larvae prolonged feeding, and suppressed hypermobility and cooperative burrowing behaviors. The dNPF system provides a new paradigm for studying the central control of cooperative behavior.