Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.

Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.

Synthesis, activity, and structure--activity relationship studies of novel cationic lipids for DNA transfer.

We have designed and synthesized original cationic lipids for gene delivery. A synthetic method on solid support allowed easy access to unsymmetrically monofunctionalized polyamine building blocks of variable geometries. These polyamine building blocks were introduced into cationic lipids. To optimize the transfection efficiency in the novel series, we have carried out structure-activity relationship studies by introduction of variable-length lipids, of variable-length linkers between lipid and cationic moiety, and of substituted linkers. We introduce the concept of using the linkers within cationic lipids molecules as carriers of side groups harboring various functionalities (side chain entity), as assessed by the introduction of a library composed of cationic entities, additional lipid chains, targeting groups, and finally the molecular probes rhodamine and biotin for cellular traffic studies. The transfection activity of the products was assayed in vitro on Hela carcinoma, on NIH3T3, and on CV1 fibroblasts and in vivo on the Lewis Lung carcinoma model. Products from the series displayed high transfection activities. Results indicated that the introduction of a targeting side chain moiety into the cationic lipid is permitted. A primary physicochemical characterization of the DNA/lipid complexes was demonstrated with this leading compound. Selected products from the series are currently being developed for preclinical studies, and the labeled lipopolyamines can be used to study the intracellular traffic of DNA/cationic lipid complexes.

Long-term, high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle.

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the local or systemic secretion of therapeutic proteins. However, current DNA delivery technologies have to be improved. We report very efficient luciferase gene transfer into muscle fibres obtained through the delivery of square-wave electric pulses of moderate field strength (100-200 V/cm) and of long duration (20 ms) to muscle previously injected with plasmid DNA. This intramuscular 'electrotransfer' method increases reporter gene expression by more than 100 times. It is noteworthy that this expression remains high and stable for at least 9 months. Moreover, intramuscular electrotransfer strongly decreases the interindividual variability usually observed after plasmid DNA injection into muscle fibres. Therefore, DNA electrotransfer in muscle possesses broad potential applications in gene therapy and for physiological, pharmacological and developmental studies.

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.

Minicircles are a new form of supercoiled DNA molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or beta-galactosidase gene under the control of the strong human cytomegalovirus immediate-early enhancer/promoter. Comparing maximal differences, these minicircles gave 2.5 to 5.5 times more reporter gene activity than the unrecombined plasmid in the NIH3T3 cell line and rabbit smooth muscle cells. Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter gene expression with minicircles than with the unrecombined plasmid or larger plasmids. Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid.

Gene transfer by naked DNA into adult mouse brain.

Nonviral gene transfer into the central nervous system could provide a basis for therapeutic uses and fundamental research. We show that naked DNA injected intracerebrally into the mouse brain can provide expression of a reporter protein. Expression is dose dependent, being maximal for 150 mu g DNA injected. We observed less than 5 days expression of the luciferase transgene, which is not improved with plasmid preparations virtually free of lipopolysaccharide. Thus, the adult brain behaves as striated muscle for naked DNA uptake and transcription, albeit at a much lower efficiency. In neither adult brain nor muscle did complexation of DNA with cationic lipid improve transgene expression. Double immunolabeling using cell-specific markers shows that both neurons and glia are transfected by naked DNA gene transfer methodology.

Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo.

Cationic lipids can deliver genes efficiently in vitro, but are generally inhibited by the presence of serum, and their efficiency in vivo is much lower than in vitro. An attractive strategy is to induce strong DNA compaction by its association with proteins, before addition of lipids. However the use of whole proteins might present both production and immunological limitations. We have devised a system in which DNA is associated with short peptides derived from human histone or protamine, before the addition of a cationic lipid or polymer. Peptides strongly associating with DNA confer to such peptide-DNA-lipid particles an enhanced in vitro transfection efficiency over that observed with classical DNA/lipid lipoplexes, and particularly confer the capacity to transfect in the presence of serum. This acquisition of serum resistance is cell type-independent, and observed with all four lipopolyamines tested and polyethylenimine. Precompacting DNA with a histone H1-derived peptide enhances cationic lipid RPR 115335-mediated gene transfer in an in vivo model of Lewis lung carcinoma. Apart from their use in peptide-DNA-lipid association, such peptides could be useful as part of chimeric gene delivery vectors presenting a DNA-binding moiety that can be easily associated with other functional domains.

High-efficiency gene transfer into skeletal muscle mediated by electric pulses.

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.