DNA-polylysine complexes

Mamounas, M., Leavitt, M., Yu, M., Wong-Staal, F. (1995). Increased titer of recombinant AAV vectors by gene transfer with adenovirus coupled to DNA-polylysine complexes. Gene Ther., 2, 429 -32. 

Trubetskoy et al. (1999). Preformed DNA/polylysine complexes were cross-linked by the addition of an heterobifimctional agent, dimethyl-3,3 -dithiobispropionimidate. In addition, the cross linking of DNA complexes made with polylysine prevented aggregation of compact particles, which occurs naturally at physiological salt concentration. Such DNA particles were active in transfecting cells in culture. 

Figure 2. ATA inhibition of DNA-polylysine complex formation. The concentrations of the ATA fractions were as follows SATA, 10 ygm/ml MATA, 10 ugm/ml LATA 6.7 ygm/ml.

The two classes of non-viral vector that are being investigated most actively are the cationic lipid-based vectors and the cationic polymer-based vectors. The cationic polymer-based vectors represent the newest class of non-viral delivery system to be developed and there are a number of groups working with these vectors. As this is such a new area of research there has yet to be any standardised definition of the terms involved in this field. Complexes formed between cationic polymers and DNA have been referred to as interpolyelectrolj1 e complexes, molecular conjugates, polylysine-DNA complexes, DNA-polylysine complexes, polyplexes and so on. In this review complexes formed between poly-(L-lysine) (pLL) and DNA are referred to as polylysine/DNA complexes or abbreviated to pLL/DNA complexes. Where polymers other dian poly(L-lysine) have been used the term polymer/DNA complex is used. The forward slash (/) is used to denote a non-covalent interaction, while a hyphen (-) is used to denote a covalent link. 

Erbacher, P., Roche, A.C., Monsigny, M. and Midoux, P. (1996a) Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. Exp. Cell Res., 225, 186-194. 

Erbacher, P., Bousser, M.-T., Raimond, J., Monsigny, M., Midoux, P. and Roche, A.C. (1996b) Gene transfer by DNA/ glycosylated polylysine complexes into human blood monocyte-derived macrophages. Hum. Gene Ther., 7, 721-729. 

Folate-targeted LPD-II particles were generated by mixing anionic liposomes composed of DOPE/cholesteryl hemisuccinate (CHEMS)/ folate-polyethyleneglycol (PEG)-DOPE and the cationic DNA-polylysine (1 0.75, w/w) complexes. Structural analysis of LPD-II by negative-stain EM showed that the DNA-polylysine (which appears individually as rod shaped) and lipid complex seemed to be a highly electron dense, spherical core with a low-density coating. The mean diameter of these particles was 74 14 nm, i.e., smaller than the empty liposomes. 

In order to incorporate targeting molecules in the DNA/carrier complex, usually the ligand is chemically coupled to polylysine, such as by treatment with 3-mercaptopropionate or succinimidyl 3-(2-pyridyldithio)proprionate (SPDP) to form a disulfide linkage, followed by purification by column chromatography and dialysis (67) (see Note 14). 

The ligand-polylysine complex can then be mixed with plasmid DNA (1 1 w w), or with adenoviral particles then plasmid DNA. 

For example, the approximate ratios useful for transfection in a report using this particular quaternary complex are 2 x 1010 adenoviral particles 1.25 pg antibody-polylysine complex 6 pg plasmid DNA, added to 6 pg polylysine or 8 pg polylysine-transferrin complex (74). Simple mixing of the components results tin rapid and spontaneous formation of the macrocomplex. Carbohydrates have also been used as targeting ligands (84,85). 

Synthesis of poly(L-lysine)-grafted hyaluronic acid (PLL-g-HA) comb-type copolymers has recently been described. HA chains were covalently coupled at the E-amino groups of polylysine by reductive amination reaction (66). These PLL-g-HA derivatives were shown to form complexes with plasmid DNA. The complex showed stability against nuclease degradation and was internalized by liver cells (67-70). 

Peptides have been used for the delivery of gene-based drugs. Several rationales have been utilized. Cationic peptides are used to neutralize the charges of DNA or oligonucleotide. These peptides include polylysines and polyarginines [241,244,245]. They form complexes with DNA or oligonucleotide, thereby resulting in enhanced cellular uptake. 

Wagner E, Zatloukal K, Cotten M, Kirlappos H, Mechtler K, Curiel DT, Bimstiel ML (1992) Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-... 

In common with the polylysine DNA-condensing peptide, the p peptide has also been shown to have nuclear localization properties. Confocal microscopy revealed that p peptide in a complex containing fluorescent lipid- and dye-labelled DNA associates with the nuclei and nucleoli of both dividing and nondividing cells within 15 minutes of exposure to the complex, thus suggesting an NLS function. However, this property was not detectable when the peptide became incorporated into a 3p-[N-(N, N -dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol)/DOPE cationic liposome (149). It may be the case that the lipids may mask the critical residues. 

Wagner E, Plank C, Zatloukal K, et al. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes toward a synthetic virus-like gene-transfer vehicle. Proc Natl Acad Sci USA 1992 89(17) 7934-7938. 

There is a wide variety of vectors used to deliver DNA or oligonucleotides into mammalian cells, either in vitro or in vivo. The most common vector systems are based on viral [retroviruses (9, 10), adeno-associated virus (AAV) (11), adenovirus (12, 13), herpes simplex virus (HSV) (14)] andnonviral [cationic liposomes (15,16), polymers and receptor-mediated polylysine-DNA] complexes (17). Other viral vectors that are currently under development are based on lentiviruses (18), human cytomegalovirus (CMV) (19), Epstein-Barr virus (EBV) (20), poxviruses (21), negative-strand RNA viruses (influenza virus), alphaviruses and herpesvirus saimiri (22). Also a hybrid adenoviral/retroviral vector has successfully been used for in vivo gene transduction (23). A simplified schematic representation of basic human gene therapy methods is described in Figure 13.1. 

It has been reported that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing NLS. NLS are short karyophilic peptides on proteins that bind to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus. Examples of these peptides will be given later in this section. DNA can also be presented to cells in culture as a complex with polycations such as polylysine, or basic proteins such as protamine, total histones or specific histone fractions (110), cationized albumin, and others. These molecules increase the transfection efficiency. In addition histone HI is identified as transfection-enhancing protein in cell culture (111). 

Curiel, D.T. (1994). High-efficiency gene transfer employing adenovirus-polylysine-DNA complexes. Nat. Immun., 13, 141-164. 

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