An intracellular delivery vehicle for protein transduction of micro-dystrophin.
The Fv fragment of an antibody that selectively targets and penetrates skeletal muscle in vivo was produced as a fusion protein with a micro-dystrophin for use as a delivery vehicle to transport micro-dystrophin into muscle cells. Fv-micro-dystrophin was produced as a secreted protein by transient transfection of Fv-micro-dystrophin cDNA in COS-7 cells and as a non-secreted protein by permanent transfection in Pichia pastoris. Isolated Fv-micro-dystrophin was shown to be full-length by Western blot analysis. Fv-micro-dystrophin penetrated multiple cell lines in vitro, and it localized to the plasma membrane of a cell line with membrane beta-dystroglycan. In the absence of membrane beta-dystroglycan, it localized to the cytoplasm. Antibody-mediated transduction of micro-dystrophin into muscle cells is a potential therapy for dystrophin-deficient muscular dystrophies.
Weisbart
Richard H
RH
Department of Research, Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, CA 91343, USA. rweisbar@ucla.edu
Hansen
James E
JE
Nishimura
Robert N
RN
Chan
Grace
G
Wakelin
Rika
R
Chang
Sophia S
SS
Baresi
Larry
L
Chamberlain
Jeffrey S
JS
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D049030:Dystroglycans / Q000378:metabolism; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments; D008222:Lymphokines; D032446:Myoblasts / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012795:Sialoglycoproteins; D014162:Transfection
D016189:Dystrophin; C089422:Fv protein, human; D007128:Immunoglobulin Fragments; D008222:Lymphokines; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D049030:Dystroglycans