A Hepatocyte Targeted Antisense Oligonucleotide Delivery System Using Copolymers of N-(2-hydroxypropyl)methacrylamide.
Keith D. Jensen, Pavla Kopeckova, Jindrich Kopecek.
12th Annual Meeting of the American Association of Pharmaceutical Scientists, Boston, Massachusetts, November 2-6, 1997, Pharmaceutical Research 14 (11) supplement, S-55, Abstract #1169.
Presented Monday, November 3, 1997 in the Polynucleotides, Oligonucleotides/Vaccines Poster Session.


Objective. To design a novel targeted antisense oligonucleotide delivery system to study and improve antisense therapy of hepatitis B. The hepatocytes, affected by this disease, have asialoglycoprotein receptors, which recognize galactose residues on the asialoglycoproteins. The water soluble polymer delivery system is targeted by incorporation of galactosamine to the polymer, which results in internalization by receptor mediated endocytosis. Cleavable spacers allow release of the oligonucleotide in the endosome or lysosome. A fluorescent tag on the oligonucleotide allows tracking via confocal microscopy.

Methods. A 21-mer oligodeoxynucleotide phosphorothioate, complementary to nucleotides 1903-1923 of the human hepatitis B virus (ayw subtype) was synthesized using both regular and ExpediteTM phosphoramidites (which allow room temperature cleavage and deprotection). Fluorescein was attached to the oligonucleotide via a fluorescein labeled phosphoramidite. The oligonucleotide was terminated with a primary amine at either the 3' or the 5' end to allow coupling to the polymer delivery system. The delivery system consisted of copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), and comonomers containing N-acylated galactosamine and 4-nitrophenylate groups on the termini of the side-chains. The oligonucleotide was conjugated to the polymer via aminolysis of 4-nitrophenylate. Conjugation was attempted in solution and in a two-phase reaction with the oligonucleotide remaining attached to the CPG beads on which it was synthesized. Conjugation was detected by PAGE.

Results. Several polymer were synthesized with Mw between 30,000 - 43,000 Da. The mole percent of galactose containing monomers varied between 11 - 20% depending on the monomer feed concentration. The amount of monomer with 4-nitrophenylate groups was between 3.4 - 4.5 mole %. Solution phase conjugation yielded less than half of the oligonucleotide bound to the polymer. A two phase reaction with the oligonucleotide bound to the CPG synthesis beads and the polymer in anhydrous organic solvent yielded only minute amount of conjugation of lower molecular weight polymer. This is probably due to steric hindrance of the CPG beads. An HPLC analysis of the oligonucleotide generated with the ExpediteTM amidites revealed two sets of doublets (a doublet is expected due to fluorescein isomers). The more hydrophobic second set had a molecular weight 190 Da higher than expected which indicated some type of addition-possibly by a protecting group.

Conclussions. A water soluble polymer delivery system was synthesized using HPMA copolymers. Coupling the oligonucleotide proved more challenging than expected. A solution phase reaction, which previously yielded good results in coupling both small molecular weight drugs and large molecular weight proteins, gave low yields of oligonucleotide conjugation. The yield of the two phase conjugation was lower probably due to steric effects. This might be overcome by the attachment of a low molecular weight polymerizable group to the CPG bound oligonucleotide provided free radical polymerization does not change the structure. This and other work suggests that protecting groups can attach to modified phosphoramidites when DNA is synthesized with Expedite TM phosphoramidites.

KDJ has been supported an AFPE fellowship and NIH Grant GM08573.

back to CV