DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety

Episode von Chemie und Pharmazie - Open Access LMU - Teil 02/02 von Ludwig-Maximilians-Universität München · 1. Januar 1991

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules

for the introduction of genes into eucariotic cells. We describe here a more specific method for conjugation

of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate

moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNAbinding

domains. Successful transferrin-receptor-mediatedd elivery and expression of the Photinus

pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity

of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine

conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with

TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing

agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine

conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation

method facilitates the synthesis of large quantities (>lo0 mg) of conjugates.

INTRODUCTION

Transferrin-polycation conjugates are efficient carriers

for the uptake of DNA into eucariotic cells (I). This gene

transfer technique, termed tramferrinfection, is based

on receptor-mediated endocytosis of DNA complexed with

polycation-transferrin conjugates (2,3). Our initial conjugate

synthesis (1) involved the modification of one to

two amino groups on the transferrin molecule with the

bifunctional reagent succinimidyl34 2-pyridy1dithio)propionate

(SPDP), followed by ligation to similarly modified

polycations (polylysine or protamine) through the formation

of disulfide bonds. Because there are more than

50 lysines on the large (about 80 kDa) transferrin protein,

the actual site (or sites) of ligation to the polycation is

unknown with this method.

In this paper we describe the synthesis of new transferrin

conjugates that are ligated with DNA-binding

compounds in a specific manner through modification of

the transferrin carbohydrate moiety. The conjugates thus

obtained are free of any groups derived from chemical

linking agents, since the connecting atoms are already

present within the starting compounds. The carbohydrate

group acts as anatural spacer that puts a 32-atom distance

between the transferrin and the DNA binding moiety. This

spacer effect may be important for appropriate presentation

of the ligand to its receptor. As a DNA-binding

compound, the polycation polylysine was used, similar to

the use described in ref 1 or to the asialo-orosomucoid

conjugates prepared by Wu and Wu (4). We have also

prepared a novel type of transferrin conjugate that contains

the DNA intercalator ethidium homodimer (5) as the DNAbinding

group and demonstrate successful receptormediated

gene delivery with these conjugates.

EXPERIMENTAL PROCEDURES

Human transferrin (iron-free), conalbumin (iron-free),

and poly(L-lysine) were obtained from Sigma. Liquid chro-

Abbreviations used: FITC, fluorescein ieothiocyenate; TfEtD,

traneferrin-ethidium homodimer conjugate; TfpL, traneferrinpolytL-

lysine) conjugate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic

acid.

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