posted on 2024-01-12, 10:29authored byMarwa Elkhashab, Yeter Dilek, Morten Foss, Laura B. Creemers, Kenneth A. Howard
Antisense nucleic acid drugs are susceptible to nuclease
degradation,
rapid renal clearance, and short circulatory half-life. In this work,
we introduce a modular-based recombinant human albumin-oligonucleotide
(rHA-cODN) biomolecular assembly that allows incorporation of a chemically
stabilized therapeutic gapmer antisense oligonucleotide (ASO) and
FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker
(cODN) was conjugated to recombinant human albumin (rHA) using maleimide
chemistry, after which a complementary gapmer ASO, targeting ADAMTS5
involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO
biomolecular assembly production, fluorescence labeling, and purity
were confirmed using polyacrylamide gel electrophoresis. ASO release
was triggered by DNase-mediated degradation of the linker strand,
reaching 40% in serum after 72 h, with complete release observed following
30 min of incubation with DNase. Cellular internalization and trafficking
of the biomolecular assembly using confocal microscopy in C28/I2 cells
showed higher uptake and endosomal localization by increasing incubation
time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly
was demonstrated in FcRn-expressing human microvascular endothelial
cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was
comparable to free gapmer after 72 h incubation with human osteoarthritis
patients’ chondrocytes. This work introduces a versatile biomolecular
modular-based “Plug-and-Play” platform potentially applicable
for albumin-mediated half-life extension for a range of different
types of ODN therapeutics.