Preparation of Asialoorosomucoid-Polylysine Conjugates - American

Center, Division of Gastroenterology AM-044, #1845, 263 Farmington Avenue, Farmington, Connecticut 06030. Received September 29, 1993®...
0 downloads 0 Views 2MB Size
Bioconjugate Chem. 1994, 5, 306-3 11

306

Preparation of Asialoorosomucoid-Polylysine Conjugates Timothy D. McKee,+ Mary E. DeRome,+ George Y. Wu,* and Mark A. Findeis*p+ TargeTech, Inc., 290 Pratt Street, Meriden, Connecticut 06450, and University of Connecticut Health Center, Division of Gastroenterology AM-044, 51’1845, 263 Farmington Avenue, Farmington, Connecticut 06030. Received September 29, 1993”

Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently developed as carriers of electrostatically bound DNA for targeted delivery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therapy. Using acid-urea gel electrophoresis we have found that previously reported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. DNA complexes prepared with these conjugates have low solubilities, which limits their usefulness for subsequent experimentation, particularly in uiuo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective to remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid-urea gel electrophoresis was used to isolate crude ASOR-PL fractions free of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-functionalizedcolumns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution acid-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.

INTRODUCTION The transfer to human cells of DNA containing therapeutic genes (gene therapy) has undergone a transition in recent years from a laboratory technique to a variety of experimental applications in the clinic ( 1 , 2 ) . Most of the gene-transfer protocols that have been developed are based on the use of retroviral vectors to transfect cells outside of the patient. The modified cells are subsequently transferred back to the patient after transfection. Amidst concern over the complexity and efficacyof these strategies, researchers have investigated ways to introduce therapeutic DNAs directly into patients in the manner of a more traditional pharmaceutical agent such as by intravenous injection. To improve the efficiency of this strategy techniques have been developed to use receptor-mediated endocytosis to carry DNA into cells (3, 4 ) . The liver has been recognized as a useful site for highly hepato-selective targeted delivery via the asialoglycoprotein receptor (ASGPr)’ for over a decade (5). Early experiments focused on the delivery of agents covalently bound to asialoglycoproteins such as asialofetuin (6-8) and asialoorosomucoid (9)or galactose-terminated neoglycoproteins ( 1 0 , l l ) . ASGPr-mediated endocytosis of the glycoproteins carried the bound agent into cells. More

* Author to whom correspondenceshould be addressed. Tel: (203) 235-5600. Fax: (203) 237-8539. + TargeTech,Inc., a wholly owned subsidiary of The Immune Response Corporation of Carlsbad, CA 92008. * University of Connecticut Health Center. Abstract publishedin Advance ACS Abstracts, May 15,1994. 1 Abbreviations used: ASGPr, asialoglycoprotein receptor; ASOR, asialoorosomucoid; ASOR-PL, asialoorosomucoid-polylysine conjugate; AUGE,acid-urea gel electrophoresis; CEHPLC, cation-exchange high-performanceliquid chromatography;DEAE, (diethy1amino)ethyl;EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide;MW, molecular weight;MWCO,molecular weight cutoff; NaOAc, sodium acetate; OR, orosomucoid; PEAUGE, preparative elution acid-urea gel electrophoresis; PL, poly-Llysine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEMED, N,N,”,N’-tetramethylethylenediamine. @

1043-1802/94/2905-0306$04.50/0

recently, this strategy has been expanded to the use of asialoorosomucoid-polylysine (ASOR-PL) conjugates to carry DNA into hepatocytes as a strategy for gene therapy (5). DNA is bound in a tight electrostatic complex by ASOR-PL. The ASGPr recognizes the ASOR in the complex, binds and internalizes the ASOR, and brings the DNA into the cell at the same time. This technique has been used successfully for the delivery of DNAs containing a variety of genes in vitro (12-15) and in uiuo (16-20) including the gene for the low-density lipoprotein receptor (LDLr) in Watanabe rabbits. Expression of the delivered LDLr gene resulted in transiently lowered serum cholesterol levels (19). ASOR is readily available by isolation of orosomucoid (OR, also referred to as al-acid glycoprotein) from human plasma (21) followed by desialylation to expose penultimate galactosyl groups. Orosomucoid is quite a robust protein and can be desialylated by acid treatment with heating thus avoiding the expense of using neuraminidase to cleave sialic acid residues (22). Poly-L-lysine (PL) is available commercially with varying molecular weight ranges. Conjugates formed by crosslinking PL with proteins have been reported using carbodiimide-mediated amide bond formation (14,151 and thiol reagents (12,13, 23). The product mixtures obtained in the formation of conjugates of this type are both very heterogeneous and highly charged. Previously described methods for the preparation of ASOR-PL for targeted delivery to the ASGPr-bearing cells have used dialysis (91,gel filtration (9, 12), and ion-exchange HPLC (14, 23) for purification of the conjugate from reaction byproducts and unconsumed starting materials. To be able to conduct gene delivery experiments in uiuo, both larger quantities of targeting conjugates and higher concentrations of complexed DNA are needed in comparison with the usual experiments conducted in vitro. We have found ASORPL conjugates prepared by the previously detailed methods to be of variable quality with respect to the solubility of DNA complexes made with them. When solubility of complexed DNA is low it is not practical to inject animals with sufficient DNA to observe gene expression in a 0 1994 American Chemical Society

Preparatlon of Aslaloorosomucoid-Polylysine Conjugates

reproducible manner. In this study we report our investigation of the use of the combination of acid-urea gel electrophoresis and cation-exchange chromatography to analyze and purify ASOR-PL conjugates to be used in the formation of soluble targetable DNA complexes. EXPERIMENTAL PROCEDURES

General. Human plasma was obtained from the American Red Cross Blood Services, Farmington, CT. DEAE-cellulose was purchased from Sigma. Poly-L-lysine was obtained from Sigma, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) was from Aldrich. MWs of PL are expressed as the average MW determined by viscosity measurements. Agarose for electrophoresis was from International Biotechnologies, Inc. Water was purified with a Barnstead Nanopure System. DNA plasmid pSVHBV2 containing the gene for hepatitis B virus surface antigen was provided by Dr. Henry Chiou (TargeTech, Inc.). Assays for surface antigen expression were performed using the correspondng Abbott Auszyme monoclonal antibody assay kit according to the manufacturer's instructions. Concentrations of DNA were based on an extinction coefficient of 20 mL m g ' cm-' at 260 nm, and agarose gel electrophoresis of DNA was performed as described by Sambrook (24). Dialysis tubing (12-14 kDa MWCO) was from Spectrum. Dialyses were performed in 20-L tanks of water for 2 days at 4 "C with one change of water after 1 day. Orosomucoid (a1-Acid Glycoprotein). OR was isolated using an adaptation of a previous report (21). Buffers used in this isolation were made up to the stated concentration in sodium acetate (NaOAc) with glacial acetic acid added to obtain the desired pH: buffer 1,0.05 M NaOAc, pH 4.5; buffer 2,O.lO M NaOAc, pH 4.0; buffer 3, 0.05 M NaOAc, pH 3.0. DEAE-cellulose (84 g) was suspended in water, allowed to swell for 1 2 h, and then washed successively with 0.5 N HC1, 0.5 N NaOH, and 0.01 M EDTA (25). The DEAE-cellulose was poured to a bed volume of 5 cm X 25 cm in a Waters AP-5 column. Using a peristalic pump (10 mL/min flow rate) the column was equilibrated with buffer 1until the pH of the column eluate was 4.5. Pooled human plasma (4 units, 1.2 L) was transferred to dialysis tubing (12-14 kDa MWCO) and dialyzed overnight at 4 "C against 20 L of buffer 1. The dialyzed plasma was then centrifuged at 14 000 rpm in a Beckman JA-14 rotor (30000g)for 10 min at 4 "C. The supernatant was then filtered through Whatman 514 paper, and the precipitate was discarded, followed by an additional filtration through a 0.2-pm filter (Zapcap, Schleicher & Schuell). The dialyzed and filtered plasma was applied to the DEAE-cellulose column which was then washed with buffer 1until the eluate had an absorbance at 280 nm of less than 0.10. The column was then eluted with buffer 2. The eluate was collected starting when the A280 began to increase and ending after the A2w had peaked and was