Key Points AAV- and ZFN-mediated targeting of the albumin locus corrects disease phenotype in mouse models of hemophilia A and B. Robust expression from the albumin locus provides a versatile platform for liver-directed protein replacement therapy.

Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumorsand is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845-and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII. The epidermal growth factor receptor (EGFR)2 is overexpressed in many cancers including breast cancers, where its overexpression is associated with a poor prognosis (1, 2), yet EGFR alone when overexpressed in fibroblasts is a weak oncogene (3). The non-receptor-tyrosine kinase, c-Src, is also overexpressed in ϳ70% of breast cancers (4), suggesting that EGFR and c-Src may function cooperatively in cancers that co-overexpress both kinases (1). Investigations in murine fibroblasts and in human breast cancer cells have revealed synergistic increases in DNA synthesis, soft agar colony growth, and tumor formation in nude mice when EGFR and c-Src are co-overexpressed as compared with cells overexpressing EGFR or c-Src alone (5, 6). These synergistic increases are dependent upon EGF stimulation, the kinase activity of c-Src, and c-Src phosphorylation of tyrosine 845 (Tyr-845) (7), a highly conserved residue in the activation loop of the catalytic domain of the EGFR. Substitution of a phenylalanine for a tyrosine at position 845 (Y845F) ablates EGF-induced DNA synthesis (even in the presence of overexpressed c-Src) without affecting the intrinsic kinase activity of the EGFR or its ability to phosphorylate canonical downstream substrates (13). Combined, these results suggest that phosphorylated Tyr-845 (Tyr(P)-845) elicits a critical mitogenic signal when EGFR and c-Src are co-overexpressed that is mediated through unconventional substrates.Subsequent efforts to identify mediators of the Tyr(P)-845 EGFR mitogenic signal revealed that phosphorylation and transcriptional activation of Stat5b are dependent on Tyr(P)-845 and that Stat5b is required for EGF-induced DNA synthesis (8). Phage display screening of a human breast cancer tissue library also identified cytochrome c oxidase subunit II (CoxII) as a protein that binds the EGFR in a Tyr(P)-845-and EGF-dependent...

Key Points AAV delivery of ZFNs and corrective Donor vectors to adult mouse liver results in stable human factor IX levels, normalizing hemophilic clotting times.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.