Generation of an efficiently secreted, cell penetrating NF-κB inhibitor.
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.
2Bone and Joint Research Unit, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK. d.j.gould@qmul.ac.uk.
Apostolos
A
Koutsokeras
Nirupam
N
Purkayastha
Nirupam
N
Purkayashta
Anne
A
Rigby
Maria C
MC
Subang
Michelle
M
Sclanders
Sandrine
S
Vessillier
Lisa
L
Mullen
Yuti
Y
Chernajovsky
David
D
Gould
D002460:Cell Line; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D020781:Matrix Metalloproteinase 1
MMP; gene therapy; inflammation; protein transduction domain