Bertrand Schwartz

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 λ integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or β-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.

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.

Human α-Iduronidase Gene Transfer Mediated by Adeno-Associated Virus Types 1, 2, and 5 in the Brain of Nonhuman Primates: Vector Diffusion and Biodistribution

We have previously demonstrated that delivery of a recombinant adeno-associated virus (rAAV) encoding human alpha-iduronidase (hIDUA) in the putamen and centrum semiovale was feasible and beneficial in a dog model of Hurlers syndrome. In the present study, we investigated the safety and vector diffusion profile of three rAAV serotypes (rAAV2/1, rAAV2/2, and rAAV2/5), encoding hIDUA in the central and peripheral nervous systems of nonhuman primates. Six macaques received the same vector dose injected into the right putamen and the homolateral internal capsule. Neurological examinations were done regularly and showed no detectable clinical consequence of the intracerebral injections. Because transgene IDUA was indistinguishable from endogenous enzymatic activity, we looked for vector diffusion by performing quantitative polymerase chain reaction on serial sections from the brain and spinal cord. We found that global diffusion throughout the brain was not significantly different between the three serotypes. However, rAAV2/1 and rAAV2/5 resulted in higher vector copy numbers per cell than did rAAV2/2, respectively, in the brain and the distal neuronal structures (spinal cord and peripheral nerves).

Human FGF‐1 gene transfer promotes the formation of collateral vessels and arterioles in ischemic muscles of hypercholesterolemic hamsters

Acidic fibroblast growth factor (FGF‐1) has been identified as a potent mitogen for vascular cells, inducing formation of mature blood vessels in vitro and in vivo and represents one of the most promising approaches for the treatment of ischemic cardiovascular diseases by gene therapy. Nevertheless, and most probably due to the few experimental models able to address the issue, no study has described the therapeutic effects of FGF‐1 gene transfer in subjects with peripheral arterial disease (PAD) exhibiting a clinically relevant cardiovascular pathology.

Catheter-based plasmid-mediated transfer of genes into ischemic myocardium using the pCOR plasmid.

Background Direct transfer of genes holds promise for the sustained delivery of therapeutic proteins to treat cardiovascular diseases. This can be accomplished by several approaches, including use of adenoviral vectors and naked plasmid DNA vectors. We previously demonstrated achieval of effective delivery of genes into the myocardium with a left ventricular-guided catheter-based approach using an adenoviral vector. Objective To evaluate the levels and duration of expression of genes induced after injection of a specific plasmid vector, using the same delivery platform as that in our previous work. Methods The pCOR plasmids are narrow-host-range plasmid vectors designed for nonviral gene therapy. We tested the ability of the pCOR plasmid vector to express its transgene after injection into the myocardium of pigs with chronic experimental ischemia using a catheter-based transendocardial delivery system. Four animals were subjected to transendocardial injections of the luciferase reporter pCOR gene into ischemic and nonischemic zones using the Biosense intramyocardial injection catheter. Injections (1 mg per animal, 50 μg per injection site) were performed at 20 sites in ischemic and nonischemic zones. Measurements of luciferase activity were performed 3 and 7 days thereafter. Results We observed high levels of expression of luciferase gene in ischemic and nonischemic regions (on days 3 and 7, respectively, in ischemic zone 58 237 and 33 709 pg; in nonischemic zone 39 928 and 46 036 pg). Control noninjected samples from the left and right ventricles contained no detectable luciferase activity. Conclusions With a catheter-based approach, the pCOR plasmid was successfully used to deliver genes into designated myocardial regions, and provides sustained expression of protein for at least 7 days, of roughly similar magnitudes in ischemic and nonischemic myocardium.

Novel nonviral vectors for gene delivery: Synthesis and applications

We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer.

Functional Expression of Multidrug Resistance P-Glycoprotein in Cellular Models of Physiological Barriers

The P-glycoprotein mdr (P-gp) is expressed not only in tumoral cells, but also in several non-transformed cells, specially in the apical plasma membrane of the intestinal, kidney or hepatic epithelium or of the blood-brain barrier endothelial cells. This apical localization is thought to be responsible for the net flux of hydrophobic compounds toward the intestinal lumen or from the brain to the blood, reflecting the protective role of P-gp at the epithelial and endothelial barriers. Many efforts are presently spent by several groups for the obtention of cellular models of intestinal epithelium or of the blood-brain barrier, in order to determine drug absorption by a predictive in vitro assay.