The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

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(2011) A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity, mAbs, 3:3, 273-288,

The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.

Background: VERVE-101 is an investigational in vivo CRISPR base editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower LDL-cholesterol (LDL-C). We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of non-human primates and a murine F1 progeny study. Methods: Cynomolgus monkeys were dosed with a single intravenous infusion of a vehicle control (N=10) or VERVE-101 at a dose of 0.75 mg/kg (N=4) or 1.5 mg/kg (N=22) with subsequent follow-up out to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male non-human primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu). Results: Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 of 67% and 83% and reductions of LDL-C of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days following dosing. Liver safety monitoring noted a transient rise in ALT and AST concentrations following infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male non-human primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals. Conclusions: VERVE-101 was well-tolerated in in non-human primates and led to 83% lower blood PCSK9 protein and 69% lower LDL-C with durable effects up to 476 days following dosing. These results have supported initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.