Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).

The p53 DNA binding domain (DBD) contains a single bound zinc ion that is essential for activity. Zinc remains bound to wild-type DBD at temperatures below 30 degrees C; however, it rapidly dissociates at physiological temperature. The resulting zinc-free protein (apoDBD) is folded and stable. NMR spectra reveal that the DNA binding surface is altered in the absence of Zn(2+). Fluorescence anisotropy studies show that Zn(2+) removal abolishes site-specific DNA binding activity, although full nonspecific DNA binding affinity is retained. Surprisingly, the majority of tumorigenic mutations that destabilize DBD do not appreciably destabilize apoDBD. The R175H mutation instead substantially accelerates the rate of Zn(2+) loss. A considerable fraction of cellular p53 may therefore exist in the folded zinc-free form, especially when tumorigenic mutations are present. ApoDBD appears to promote aggregation of zinc-bound DBD via a nucleation-growth process. These data provide an explanation for the dominant negative phenotype exhibited by many mutations. Through a combination of induced p53 aggregation and diminished site-specific DNA binding activity, Zn(2+) loss may represent a significant inactivation pathway for p53 in the cell.

The spectra of equilibrium chain conformation fluctuations of apomyoglobin (apoMb) as a function of folding, from the aciddenatured state at pH 2.6 through the stable molten globule state pH Ϸ 4.1 to the folded state at pH 6.3, are reported, as measured by fluorescence correlation spectroscopy. The conformational fluctuations, which are detected by quenching of an N-terminal fluorescent label by contact with various amino acids, can be represented by superpositions of decaying exponentials with time scales ranging from Ϸ3 to Ϸ200 s. Both the time scales and amplitudes of the fluctuations increase with the degree of acid denaturation, with principal shifts associated with the transition across the molten globule state. Measurements of the diffusion of apoMb confirm theoretical values showing a Ϸ40% increase in the hydrodynamic radius upon acid denaturation. This study uses the model protein apoMb to illustrate the complex scope of foldingassociated structural dynamics.

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