Generation of an efficiently secreted, cell penetrating NF‐κB inhibitor

Abstract

Gene therapy is a powerful approach to treat disease locally. However, if the therapeutic target is intracellular, the therapeutic will be effective only in the cells where the therapeutic gene is delivered. We have engineered a fusion protein containing an intracellular inhibitor of the transcription factor NF-κB pathway that can be effectively secreted from producing cells. This fusion protein is cleaved extracellularly by metalloproteinases allowing release of a protein transduction domain (PTD) linked to the NF-κB inhibitor for translocation into neighboring cells. We show that engineered molecules can be efficiently secreted (>80%); are cleaved with matrix metalloprotease-1; inhibit NF-κB driven transcription in a biological assay with a human reporter cell line; and display significant inhibition in mouse paw inflammation models when delivered by lentivirus or secreting cells. No inhibition of NF-κB transcription or therapeutic effect was seen using molecules devoid of the PTD and NF-κB inhibitory domains. By creating a fusion protein with an endogenous secretion partner, we demonstrate a novel approach to efficiently secrete PTD-containing protein domains, overcoming previous limitations, and allowing for potent paracrine effects.

Polymer Capsules with Hydrophobic Liquid Cores as Functional Nanocarriers

“…The delivery of bioactive molecules like enzymes, hormones, nucleic acids and transcription factors in a targeted manner remains a challenge in the development of novel therapies. In many cases, it is effective only in the cells where the therapeutic agent is delivered [ 152 ]. As human tissues are transparent to magnetic fields, the encapsulation of magnetic nanoparticles enables magnetic navigation with the external magnetic fields and accumulation of the carriers in the desired site of action [ 153 , 154 , 155 ] due to the increased microcapsule concentration at the magnetic site [ 156 ].…”

Influence of vitamin D and estrogen receptor gene polymorphisms on calcium absorption: Bsm I predicts a greater decrease during energy restriction

“…Also, in a cross-sectional study, premenopausal women with the BAt haplotype had significantly lower levels of calcium absorption than women with the baT haplotype [9]. In addition, in a separate pilot analysis of twenty-seven women who were randomly assigned in a double blind manner to 2500 IU/d or 400 IU/d supplemental vitamin D 3 intake, the FCA response to high vitamin D intake was significantly attenuated in women with the BB compared to bb genotype [31]. These data indicate that VDR polymorphisms exhibit a modest effect on calcium absorption even under stable conditions, suggesting that weight loss is not the only contributing factor to calcium absorption.…”

Establishment of Stable and Secretable TATκ-GFP Recombinant Protein: A Preliminary Report of Promoter Methylation in 293T Cell Line

“…This could explain the absence of recombinant protein in the culture medium although it is being expressed at gene level. Contrary, Koutsokeras et al (2014) reported that PTD fused to protein of interest is the culprit that inhibit the secretion of the protein itself.…”