Helper transfection

Despite the fact that many different cationic lipids have been synthesized and tested for transfection (25 34), relatively few systematic structure activity TE-relationship studies have been performed (35 39). As a result, no general relationship between chemical structure and TE could be drawn from these studies. One reason for this is that the chemical structure of a cationic lipid is not directly responsible for TE. TE rather depends on the biophysical characteristics of the cationic lipid aggregate (e.g., liposomes and lipoplexes), which, for its part, is dependent on the chemical structure of the lipids. In a previous study with analogs of the transfection lipid A-[l-(2,3-dioleoyloxy) propyl]-A,A,A-trimethylammoniumchloride (DOTAP) (40) which differ in their nonpolar hydrocarbon chains, it could be shown that the TE strongly depended on the biophysical properties of the resulting liposomes and lipoplexes (35). Minimal alterations of biophysical properties by using lipids with different hydrocarbon chains or by mixing the lipid with different neutral helper lipids could completely allow or prevent transfection. 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

The previous screening experiments were performed with lipoplexes containing equimolar amounts of the helper lipid DOPE. Here, the influence of different ratios of the helper lipids DOPE and Choi on TE of KL-1-14 were tested. The transfection behavior of KL-1-14 without any helper lipid was tested as well. 

TE of KL-1-14 without helper lipids was very low and reached only about twice the TE, which was found for the standard lipid DOTAP. Independent of the amount of DOPE incorporated in the lipoplexes (ratio of DOPE/KL-1-14 0.3, 0.5, 0.6, 0.7, 0.8, 0.8, 1.0, and 1.2), transfection behaviors (maximum transfection efficiencies and transfection profiles) of all mixtures were similar and comparable to the profile of KL-1-14/DOPE (1 1) as shown in Figure 2 (individual data for all mixtures are not shown). 

Using Choi as helper lipid for KL-1-14, the transfection efficiencies were no longer similar for the different Chol/KL-1-14-ratios (Fig. 7). 

We compared the TE and toxicity of KL-1-14 synthesized in the R- and S-configuration [with 0.6 mol% Choi as helper lipid (see above)]. The transfection efficiencies for both lipids were statistically similar. Thus, for further experiments, KL-1-14 was synthesized as racemat. 

It was believed that the main factors affecting transfection efficiency were the structure of the cationic lipid, the type of helper lipid used and their susceptibility to disruption by serum proteins. For gene transfer in vivo, apart from DOTMA-based liposomes, other complexes (in equimolar ratios) are also used—such as dioctade-cylamidoglicylspermidin (DLS)/DOPE (137), DOPE/DOTMA (1 1), DOPE/DOTAP (1 1) (138, 139), dimethyloctadecylammonium bromide (DDAB), and DOTAP with cholesterol (1 1) (mol/mol) (139). 

Pear, W.S., Nolan, G.P., Scott, M.L., Baltimore, D. (1993). Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. U.S.A., 90(18), 8392-8396. 

DBS and NLS stand for DNA binding signal and nuclear localization signal, respectively. Helper indicates that the transfection is efficient in the presence of additives for the cytosolic translocation. Chlo and lipo are chloroquine and cationic lipids, respectively. JTS-1 and HA are fusogenic peptides listed in Table 1. 

Anti-deoxyribonucleic acid autoantibodies from human and mice suffering from Lupus erythematosus can penetrate into cells and accumulate in the cell nucleus. Based on the characteristics of a mi-ON A autoantibodies, VAYISRGGVSTYYSDTVKGRFTRQKYNKRA peptide (P3), which exhibits a-helix, has been used as a vector for the intracytoplasmic and intranuclear translocation of macromolecules (Table 16.7) (Avrameas et al., 1998, 1999). P3 shares similar capabilities with Antenapedia peptide (Derossi et al., 1994), but in contrast P3 operates only at 37 °C by an energy dependent mechanism. P3 linked to a 19 lysine residue sequence (K19-P3) forms complexes with plasmid DNA. Efficient transfection of mouse 3T3 cells and hamster lung CCL39 cells were obtained with these complexes. This transfection was not impaired by the presence of serum and did not require helper molecules such as chloroquine. These observations suggest that peptides from cell specific anti-DNA autoantibodies may represent a source of peptide-based gene delivery system with different specificities. 

Fig. 6 Transfection activity of polyamine cholesterol cationic derivatives with linear (a-c) and T-shaped (d—f) structures. The activity of their mixtures with the helper lipid DOPE was measured in vivo as chloramphenicol acetyl transferase (CAT) expression in mouse lung [36]...

Fig. 8 Transfection of HeLa cells using cholesterol-based ether linked cationic lipids with different headgroups at N/P ratio 3 1, in the presence of 10% serum the formulations were optimized for the helper lipid DOPE content [38]...

Cationic lipids are often combined with neutral and zwitterionic lipids in formulations for gene therapy. The most frequent colipids are cholesterol, DOPE and dioleoylphosphatidylcholine (DOPC) (or other PCs) (Fig. 28). These neutral lipids may play a role in transfection by increasing the level of DNA protection against DNases or facilitating the destabilization of the endosomes [35,103]. The optimum cationic lipid/helper lipid stoichiometry varies for the different cationic lipids, nucleic acids, and cells. 

Fig. 29 Optimum mole fraction of 0.5 for the helper DOPE in cationic lipid transfection formulations (reproduced from [35] copyright by the American Society for Biochemistry and Molecular Biology)...

The helper effects of DOPE and cholesterol appear to be hydrocarbon chain-specific. This is demonstrated in studies of their mixtures with a series of alkyl acyl carnitine esters (alkyl 3-acyloxy-4-trimethylammonium butyrate chloride) tested with CV-1 cell culture (monkey fibroblast) [127]. The influence of the aliphatic chain length (n - 12-18) on transfection in vitro was determined using cationic liposomes prepared from these lipids and their mixtures with the helper lipids DOPE and cholesterol (Fig. 30). Both helper lipids provided for significant transfection enhancements in an apparently chain-specific manner, with the highest effects found for short-chain lipids with diC12 0 and diC14 0 chains in 1 1 mixtures with the respective helper lipid. 

Fig. 30 Transfection activity of lipoplexes consisting of alkyl/acyl carnitine esters, alone and with helper lipid (DOPE or cholesterol), on [1-galactosidase expression in CV-1 cell culture (monkey fibroblast) cationic lipid/DNA charge ratio 4 1 [127]...

Fig. 31 Chain-specific effects of the helper lipids DOPE and cholesterol on the transfection activity of lipoplexes comprising TAP lipids with different hydrocarbon chains for COS-7 cells at cationic lipid/DNA charge ratio 2.5 1. Transfection activity was expressed as rel. units per pg of cellular protein [128]...

Zuhom IS, Oberle V, Visser WH et al (2002) Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape, DNA translocation, and transfection efficiency. Biophys J 83 2096-2108... 

Several reports in the literature state that DOPE, while successfully used for in vitro gene delivery, is a poor helper lipid for in vivo applications [28-32], Instead, for reasons that are not understood, lipid mixtures for successful transfection in vivo seem to require cholesterol [33]. In fact, an equimolar mixture of cholesterol and DOTAP is widely used for in vivo experiments and clinical trials. Cholesterol has also been included in liposomes along with cationic DOTAP and fusogenic DOPE to form a potent mixture used to study the treatment of ovarian cancer by delivery of the p53 tumor suppressor gene [34, 35]. 

Production of vector virions requires transfection of the vector construct in plasmid form together with the introduction on separate plasmids and/or helper viruses of both the AAV genes and the helper virus genes required for a productive AAV infection (Xiao et al., 1998) (Fig. 1.1). Variations have included producing vectors... 

Transfection methods that use a mini-Ad plasmid DNA system (Grimm et al., 1998 Grimm, 2002 Zolotukhin et al., 2002) to supply Ad helper functions and AAV genes are incapable of... 

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