Reduction-sensitive lipopolyamines as a novel nonviral gene delivery system for modulated release of DNA with improved transgene expression.

We have designed and synthesized original cationic lipids for modulated release of DNA from cationic lipid/DNA complexes. Our rationale was that modulated degradation of the lipids during or after penetration into the cell could improve the trafficking of DNA to the nucleus resulting in increased transgene expression. The new reduction-sensitive lipopolyamines (RSL) harbor a disulfide bridge within different positions in the backbone of the lipids as biosensitive function. A useful synthetic method was developed to obtain, with very good yields and reproducibility, unsymmetrical disulfide-bridged molecules, starting from symmetrical disulfides and thiols. The new lipopolyamines are good candidates as carriers of therapeutic genes for in vivo gene delivery. To optimize the transfection efficiency in these novel series, we have carried out structure-activity relationship studies by placing the disulfide bridge at different positions in the backbone of the cationic lipid and by systematic variation of lipid chain length. Results indicate that the transfection level can be modulated as a function of the location of the disulfide bridge in the molecule. We suggest that an early release of DNA during or after penetration into the cell, probably promoted by reduction of a disulfide bridge placed between the polyamine and the lipid, implies a total loss of transfection efficiency. On the other hand, proper modulation of DNA release by inserting the disulfide bridge between one lipid chain and the rest of the molecule brings about increased transfection efficiency as compared to previously described nondegradable lipopolyamine analogues. Finally, preliminary physicochemical characterization of the complexes demonstrates that DNA release from complexes can be modulated as a function of the surrounding reducing conditions of the complexes and of the localization of the disulfide bridge within the lipopolyamine. Our results suggest that RSL is a promising new approach for gene delivery.

Synthesis and biological properties of new glycosidic cationic lipids for DNA delivery.

Lipidic glycosides with amino alkyl pendent groups were synthesized and evaluated for in vitro DNA transfection activity. The first representative of this new class of cationic lipids showed good gene delivery and low toxicity to HeLa and 3T3 cells.

Formulations which increase the size of lipoplexes prevent serum-associated inhibition of transfection.

BACKGROUND: Cationic lipids are the most widely used nonviral vectors for gene delivery. Upon complexation to DNA, they offer a nonimmunogenic alternative to viral gene transfer. Unfortunately, their in vivo application has been limited due to a serum-associated inhibition of transfection. As a result, significant research effort has focused on overcoming this deleterious effect of serum. METHODS: To better understand this phenomenon, we investigated the influence of lipoplex colloidal stability on gene transfection in the presence of serum. In addition, conditions of the reaction medium were modulated and their effects on collidal stability and subsequent in vitro transfection efficiency were studied. RESULTS: The colloidal stability of the cationic lipid-DNA complexes, which depended on the charge ratio, determined the efficiency of in vitro transfection in the presence of serum. In particular, large-sized, colloidally unstable complexes of over 700 nm mean diameter induced efficient transfection in the presence or absence of serum. Conversely, colloidally stable complexes of less than 250 nm in size resulted in efficient transfection only in the absence of serum. Furthermore, for the same charge ratio, both colloidally stable and unstable lipoplexes could be obtained depending on the degree to which various solution parameters (NaCl concentration, cationic lipid acyl chain length, pH and DNA concentration) were altered. In each case, only those complexes lacking colloidal stability resulted in high levels of in vitro transfection in the presence of serum. This phenomenon was shown to be independent of both the percent DNA internalized and of the lamellar organization of the cationic lipid/DNA lipoplexes. CONCLUSIONS: Through the modulation of various mixture conditions, large-sized lipoplexes can be formed which are resistant to the transfection-inhibiting effect of serum.

pCOR: a new design of plasmid vectors for nonviral gene therapy.

A totally redesigned host/vector system with improved properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plasmid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E. coli host strain, greatly reducing the potential for propagation in the environment or in treated patients. The pCOR backbone has several features that increase safety in terms of dissemination and selection: (1) the origin of replication requires a plasmid-specific initiator protein, pi protein, encoded by the pir gene limiting its host range to bacterial strains that produce this trans-acting protein; (2) the plasmid's selectable marker is not an antibiotic resistance gene but a gene encoding a bacterial suppressor tRNA. Optimized E. coli hosts supporting pCOR replication and selection were constructed. High yields of supercoiled pCOR monomers were obtained (100 mg/l) through fed-batch fermentation. pCOR vectors carrying the luciferase reporter gene gave high levels of luciferase activity when injected into murine skeletal muscle.

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.