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168. Peptide Aldehyde Inhibitors of the Proteasome as Improved Gene Transfer Agents
Abstract   Open access   Peer reviewed

168. Peptide Aldehyde Inhibitors of the Proteasome as Improved Gene Transfer Agents

Molly Martin, Ji-seon Kim and Kevin Rice
Molecular therapy, Vol.13(S1), pp.S65-S65
05/01/2006
DOI: 10.1016/j.ymthe.2006.08.192
url
https://doi.org/10.1016/j.ymthe.2006.08.192View
Published (Version of record) Open Access

Abstract

Proteasomes are multisubunit complexes that are responsible for the degradation of many cytosolic proteins. The barrel-like 20S catalytic core of the enzyme is composed of four heptameric rings that contain the proteolytic sites. The proteasome has multiple peptidase activities that can be classified into three main groups: cleavage after hydrophobic side chains (chymotrypsin-like), cleavage after basic residues (trypsin-like), and cleavage after acidic residues (peptidylglutamyl peptide hydrolysis or PGPH). The proteasome has been shown to be a key route of metabolism for peptide-based non-viral gene delivery systems 1 . The gene transfer efficiency of peptide-DNA condensates can be enhanced by the addition of commercially available proteasome inhibitors that prevent premature degradation of the gene delivery peptide. MG115 is a tripeptide with a C-terminal aldehyde that binds reversibly to the N-terminal Thr residue of the catalytic subunits thereby inhibiting proteasome activity. When MG115 was administered simultaneously with peptide-DNA condensates, an increase in luciferase expression was observed in both HepG2 and CF/T1 cells, as reported by Kim et al 1 . Previous studies have shown that MG115 can be toxic to cells in a dose dependent manner, with an LD50 of approximately 2 μM. Another difficulty was ensuring the concurrent uptake of both MG115 and peptide-DNA condensates by cells. Here we have described the synthesis and testing of gene delivery peptides containing a C-terminal aldehyde that condense DNA, mediate cellular uptake, inhibit the proteasome, and boost gene transfer efficiency. We also hypothesized that incorporation of the MG115 peptide sequence into a longer peptide could decrease its cytotoxic effects on the cell. The length and sequence of the cationic peptide was varied with attachment of the tripeptide aldehyde at the C-terminus. Incorporation of the peptide inhibitor sequence into the DNA condensate was compared to co-administration of MG115 and peptide-DNA condensates in assays for transfection efficiency and cell viability.

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