Protamine Sulfate Enhances Lipid-Mediated Gene Transfer
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Gene Therapy (1997) 4, 961–968 1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Protamine sulfate enhances lipid-mediated gene transfer FL Sorgi1,2, S Bhattacharya1 and L Huang1 1The Laboratory of Drug Targeting, Department of Pharmacology, The University of Pittsburgh School of Medicine, Pittsburgh, PA, USA A polycationic peptide, protamine sulfate, USP, has been USP as a condensation agent was found to be superior to shown to be able to condense plasmid DNA efficiently for poly-L-lysine as well as to various other types of protamine. delivery into several different types of cells in vitro by sev- These differences among various salt forms of protamine eral different types of cationic liposomes. The monovalent appear to be attributable to structural differences between cationic liposomal formulations (DC-Chol and lipofectin) the protamines and not due to differences in the net charge exhibited increased transfection activities comparable to of the molecule. The appearance of lysine residues within that seen with the multivalent cationic liposome formu- the protamine molecule correlate with a reduction in bind- lation, lipofectamine. This suggests that lipofectamine’s ing affinity to plasmid DNA, as well as an observed loss in superior in vitro activity arises from its ability to condense transfection-enhancing activity. This finding sheds light on DNA efficiently and that protamine’s primary role is that of the structural requirements of condensation agents for use a condensation agent, although it also possesses several in gene transfer protocols. Furthermore, protamine sulfate, amino acid sequences resembling that of a nuclear localiz- USP is an FDA-approved compound with a documented ation signal. While the use of polycations to condense DNA safety profile and could be readily used as an adjuvant to has been previously reported, the use of protamine sulfate, a human gene therapy protocol. Keywords: liposome; gene therapy; nonviral vector; DC-Chol; condensation Introduction in muscle.16 These improvements in vector efficiency translated into a 100 000-fold increase in lung expression The field of nonviral vector-mediated gene therapy, parti- over the first generation cationic lipid–DNA vectors cularly by using cationic liposomes has made great developed in the late 1980s. While these improvements 1 strides from their initial report by Felgner et al in 1987 are impressive and are approaching the efficiency (as to their use in the world’s first in vivo human gene ther- total gene expression) of the adenovirus, they are still at 2 apy clinical trial by Nabel et al in 1992. Cationic lipids least four orders of magnitude behind the adenovirus in have been shown to be a viable alternative to viral vector- terms of the transgene expression for each copy of trans- mediated gene delivery and have demonstrated an excel- gene administered. Furthermore, the time required for 2–4 lent safety profile. They are, however, hampered by development of these advances is slow. Lacking well reports of minimal efficiency as well as a poor fundamen- defined structure/function relationships and organ speci- tal understanding of the basic mechanisms of DNA com- ficity, these advances have occurred through the gross plexation, delivery and expression. While some search for screening of many lipid analogs and plasmid constructs. the ‘magic lipid’, other investigators have been studying While improvements in vector design are needed, they the structure of the DNA–lipid complex, examining the only address the issue of entry into the cell and cellular trafficking to identify barriers to efficient expression once inside the nucleus. Recent work has indi- expression, understanding the lipid coating/uncoating cated that these steps are relatively efficient (60% of the process, producing a stable ‘one-vial’ formulation, or delivered dose of DNA was found within the cell in 6 h) identifying components which may overcome known with modest room for improvement. The most critical 5–15 obstacles to transgene expression. step, which appears to be rate limiting and needs to be Recently, much work has focused on efforts to increase addressed, is the efficient delivery of the gene from the the level of transfection efficiency. New lipid formu- cytoplasm into the nucleus.10,13 It is this step in which the lations have demonstrated a 1000-fold increase in in vitro virus is very efficient and the cationic lipid vehicle transfection activity while new plasmid constructs have appears to be hampered. For the cationic lipid vehicle to resulted in a 3300-fold increase in transgene expression attain the level of gene expression as seen with the adenovirus, the process of efficient delivery of plasmid from the cytoplasm into the nucleus needs large-scale Correspondence: L Huang, University of Pittsburgh School of Medicine, improvement. W1351 Biomedical Science Tower, Pittsburgh, PA 15261, USA 2Present address: Advanced Therapies, Inc, 371 Bel Marin Keys Blvd, It has been hypothesized that the condensation of DNA Suite 210, Novato, CA 94949, USA into a toroidal-like structure is essential for the protection Received 4 February 1997; accepted 9 May 1997 of DNA from enzymatic degradation as well as to facili- Protamine sulfate enhances gene transfer FL Sorgi et al 962 tate entry of the DNA into the nucleus.17–23 Freeze-frac- known.17–22 In order to condense DNA before its associ- ture electron microscopy of DNA–cationic liposome com- ation with the cationic lipid, increasing amounts of prota- plexes has revealed the presence of elongated lipid- mine sulfate, USP or PLL were added to 1 mg of DNA coated structures. These structures have been shown to followed by subsequent addition of 7.5 nmol of DC-Chol be lipid-coated DNA and have been seen with several liposomes. As seen in Figure 1a and b, increasing monovalent cationic liposome formulations.9 However, amounts of PLL resulted in an increase in transfection, multivalent cationic liposome formulations, upon com- reaching a constant level of luciferase gene expression at plexation with DNA did not result in the production of approximately 1 mg of poly-l-lysine per microgram of elongated structures, rather they produced small DNA. Poly-l-lysine was able to increase the expression (approximately 20 nm), highly condensed lipidic struc- 3.5- to 10-fold (in 293 and CHO cells, respectively) over tures.24 It is felt that this efficient condensation or packag- DNA–liposome complexes without polycation. These ing of plasmid DNA accounts for the observation that the data are in agreement with previously published find- polyvalent liposome formulations generally result in a ings.15 Increasing amounts of protamine sulfate, USP also more efficient rate of in vitro gene transfer than the mono- resulted in an increase in transfection, reaching a constant valent cationic liposome formulations. level of luciferase gene expression at approximately 2 mg Recently, several investigators have been using a cat- of protamine sulfate, USP per microgram of DNA. How- ionic polymer, poly-l-lysine (PLL) in an effort to con- ever, the level of expression achieved was seven- to 45- dense DNA into an artificial virus-like structure to facili- fold (in 293 and CHO cells, respectively) over DNA–lipo- tate entry into the cell.15,25–31 However, PLL has several some complexes without polycation, a two- to four-fold qualities which could potentially render it a poor candi- increase over the levels seen with poly-l-lysine. date for human use. Firstly, PLL is a synthetic polymer which is not well defined. The polymerization reaction is Effect on transfection activity of different types of difficult to control resulting in a wide range of molecular protamine weights (the PLL used in this work has a molecular The results shown in Figure 1a and b do not agree with weight range of 18 000–19 200). Further, PLL is not ident- that of Gao and Huang15 in which the transfection ified as a ‘generally regarded as safe (GRAS)’ compound enhancement of activity of PLL was several-fold higher and much testing will be needed before this substance is than that of a protamine. However, this earlier work was ready to be introduced into humans. With these obvious done with a different salt form of protamine, ie prota- hurdles in mind, there may exist other cationic sub- stances which have not been adequately explored that may be capable of producing a similar condensation effect, without the unwanted characteristics associated with PLL. One such cationic compound could be protamine. Pro- tamines are small peptides (MW 4000–4250) which are very basic due to their high arginine content. They are naturally occurring substances found only in sperm and are purified from the mature testes of fish, usually sal- mon. Protamine’s role in sperm is to bind with DNA, assist in forming a compact structure, and deliver the DNA to the nucleus of the egg after fertilization. This unique role overcomes a major obstacle in gene therapy by nonviral vectors, the efficient delivery of DNA from the cytoplasm into the nucleus.10–13 Furthermore, prota- mine sulfate is a USP compound, FDA-approved and is used clinically as an antidote to heparin-induced anti- coagulation. Protamine sulfate has also been complexed with insulin (also known as NPH) and serves as a long- acting delivery system which is administered to patients on a daily basis. Due to the extensive history of adminis- tration of NPH insulin to diabetics, issues of toxicity and immunogenicity are minimal. For these reasons, we hypothesize that protamine sulfate may be a safer and more appropriate alternative to PLL for condensation, as well as the delivery of plasmid DNA to the nucleus. In this report, we describe the ability of several different protamine species in enhancing the transfection activity of cationic lipids. Figure 1 Comparison of the ability of protamine sulfate, USP and poly- Results l-lysine to increase transfection activity in (a) CHO cells and (b) 293 cells.