Natural and Synthetic Suppressor Mutations Defy Stability-Activity Tradeoffs.

Thermodynamic stability represents one important constraint on protein evolution, but the molecular basis for how mutations that change stability impact fitness remains unclear. Here, we demonstrate that a prevalent global suppressor mutation in TEM ß-lactamase, M182T, increases fitness by reducing proteolysis in vivo. We also show that a synthetic mutation, M182S, can act as a global suppressor and suggest that its absence from natural populations is due to genetic inaccessibility rather than fundamental differences in the protein's stability or activity.

Analysis of PEG-lipid anchor length on lipid nanoparticle pharmacokinetics and activity in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) affects millions of people worldwide, yet there are currently no therapeutics that address the long-term impairments that develop in a large portion of survivors. Lipid nanoparticles (LNPs) are a promising therapeutic strategy that may address the molecular basis of TBI pathophysiology. LNPs are the only non-viral gene delivery platform to achieve clinical success, but systemically administered formulations have only been established for targets in the liver. In this work, we evaluated the pharmacokinetics and activity of LNPs formulated with polyethylene glycol (PEG)-lipids of different anchor lengths when systemically administered to a mouse model of TBI. We observed an increase in LNP accumulation and activity in the injured brain hemisphere compared to the uninjured contralateral brain hemisphere. Interestingly, transgene expression mediated by LNPs was more durable in injured brain tissue compared to off-target organs when compared between 4 and 24 hours. The PEG-lipid is an important component of LNP formulation necessary for the stable formation and storage of LNPs, but the PEG-lipid structure and content also has an impact on LNP function. LNP formulations containing various ratios of PEG-lipid with C18 (DSPE-PEG) and C14 (DMG-PEG) anchors displayed similar physicochemical properties, independent of the PEG-lipid compositions. As the proportion of DSPE-PEG was increased in formulations, blood circulation times of LNPs increased and the duration of expression increased. We also evaluated diffusion of LNPs after convection enhanced delivery (CED) in healthy brains and found LNPs distributed >1 mm away from the injection site. Understanding LNP pharmacokinetics and activity in TBI models and the impact of PEG-lipid anchor length informs the design of LNP-based therapies for TBI after systemic administration.

Engineered nanomaterials that exploit blood-brain barrier dysfunction for delivery to the brain.

The blood-brain barrier (BBB) is a highly regulated physical and functional boundarythat tightly controls the transport of materials between the blood and the brain. There is an increasing recognition that the BBB is dysfunctional in a wide range of neurological disorders; this dysfunction can be symptomatic of the disease but can also play a role in disease etiology. BBB dysfunction can be exploited for the delivery of therapeutic nanomaterials. Forexample, there can be a transient, physical disruption of the BBB in diseases such as brain injury and stroke, which allows temporary access of nanomaterials into the brain. Physicaldisruption of the BBB through external energy sources is now being clinically pursued toincrease therapeutic delivery into the brain. In other diseases, the BBB takes on new properties that can beleveraged by delivery carriers. For instance, neuroinflammation induces the expression ofreceptors on the BBB that can be targeted by ligand-modified nanomaterials, and theendogenous homing of immune cells into the diseased brain can be hijacked for the delivery ofnanomaterials. Lastly, BBB transport pathways can be altered to increase nanomaterial transport. In this review, we will describe changes that can occur in the BBB in disease, and how these changes have been exploited by engineered nanomaterials forincreased transport into the brain.