Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor ß (TGFß) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

Cell Aggregate Assembly through Microengineering for Functional Tissue Emergence.

Compared to cell suspensions or monolayers, 3D cell aggregates provide cellular interactions organized in space and heterogeneity that better resume the real organization of native tissues. They represent powerful tools to narrow down the gap between in vitro and in vivo models, thanks to their self-evolving capabilities. Recent strategies have demonstrated their potential as building blocks to generate microtissues. Developing specific methodologies capable of organizing these cell aggregates into 3D architectures and environments has become essential to convert them into functional microtissues adapted for regenerative medicine or pharmaceutical screening purposes. Although the techniques for producing individual cell aggregates have been on the market for over a decade, the methodology for engineering functional tissues starting from them is still a young and quickly evolving field of research. In this review, we first present a panorama of emerging cell aggregates microfabrication and assembly technologies. We further discuss the perspectives opened in the establishment of functional tissues with a specific focus on controlled architecture and heterogeneity to favor cell differentiation and proliferation.

3D printing of biocompatible low molecular weight gels: Imbricated structures with sacrificial and persistent N-alkyl-d-galactonamides.

HYPOTHESIS: We have shown earlier that low molecular weight gels based on N-heptyl-d-galactonamide hydrogels can be 3D printed by solvent exchange, but they tend to dissolve in the printing bath. We wanted to explore the printing of less soluble N-alkyl-d-galactonamides with longer alkyl chains. Less soluble hydrogels could be good candidates as cell culture scaffolds. EXPERIMENTS: N-hexyl, N-octyl and N-nonyl-d-galactonamide solutions in dimethylsulfoxide are injected in a bath of water following patterns driven by a 2D drawing robot coupled to a z-platform. Solubilization of the gels with time has been determined and solubility of the gelators has been measured by NMR. Imbricated structures have been built with N-nonyl-d-galactonamide as a persistent ink and N-hexyl or N-heptyl-d-galactonamide as sacrificial inks. Human mesenchymal stem cells have been cultured on N-nonyl-d-galactonamide hydrogels prepared by cooling or by 3D printing. FINDINGS: The conditions for printing well-resolved 3D patterns have been determined for the three gelators. In imbricated structures, the solubilization of N-hexyl or N-heptyl-d-galactonamide occurred after a few hours or days and gave channels. Human mesenchymal stem cells grown on N-nonyl-d-galactonamide hydrogels prepared by heating-cooling, which are stable and have a fibrillar microstructure, developed properly. 3D printed hydrogels, which microstructure is made of micrometric flakes, appeared too fragile to withstand cell growth.

Wet spinning and radial self-assembly of a carbohydrate low molecular weight gelator into well organized hydrogel filaments.

In this work, we describe how a simple single low molecular weight gelator (LMWG) molecule - N-heptyl-d-galactonamide, which is easy to produce at the gram scale - is spun into gel filaments by a wet spinning process based on solvent exchange. A solution of the gelator in DMSO is injected into water and the solvent diffusion triggers the supramolecular self-assembly of the N-heptyl-d-galactonamide molecules into nanometric fibers. These fibers entrap around 97% of water, thus forming a highly hydrated hydrogel filament, deposited in a well organized coil and locally aligned. This self-assembly mechanism also leads to a very narrow distribution of the supramolecular fiber width, around 150 nm. In addition, the self-assembled fibers are oriented radially inside the wet-spun filaments and at a high flow rate, fibers are organized in spirals. As a result, this process gives rise to a high control of the gelator self-assembly compared with the usual thermal sol-gel transition. This method also opens the way to the controlled extrusion at room temperature of these very simple, soft, biocompatible but delicate hydrogels. The gelator concentration and the flow rates leading to the formation of the gel filaments have been screened. The filament diameter, its internal morphology, the solvent exchange and the velocity of the jet have been investigated by video image analysis and electron microscopy. The stability of these delicate hydrogel ropes has been studied, revealing a polymorphic transformation into macroscopic crystals with time under some storage conditions. The cell viability of a neuronal cell line on the filaments has also been estimated.

Simple Synthetic Molecular Hydrogels from Self-Assembling Alkylgalactonamides as Scaffold for 3D Neuronal Cell Growth.

In this work, we demonstrated that the hydrogel obtained from a very simple and single synthetic molecule, N-heptyl-galactonamide was a suitable scaffold for the growth of neuronal cells in 3D. We evidenced by confocal microscopy the presence of the cells into the gel up to a depth of around 200 µm, demonstrating that the latter was permissive to cell growth and enabled a true 3D colonization and organization. It also supported successfully the differentiation of adult human neuronal stem cells (hNSCs) into both glial and neuronal cells and the development of a really dense neurofilament network. So the gel appears to be a good candidate for neural tissue regeneration. In contrast with other molecular gels described for cell culture, the molecule can be obtained at the gram scale by a one-step reaction. The resulting gel is very soft, a quality in accordance with the aim of growing neuronal cells, that requires low modulus substrates similar to the brain. But because of its fragility, specific procedures had to be implemented for its preparation and for cell labeling and confocal microscopy observations. Notably, the implementation of a controlled slow cooling of the gel solution was needed to get a very soft but nevertheless cohesive gel. In these conditions, very wide straight and long micrometric fibers were formed, held together by a second network of flexible narrower nanometric fibers. The two kinds of fibers guided the neurite and glial cell growth in a different way. We also underlined the importance of a tiny difference in the molecular structure on the gel performances: parent molecules, differing by a one-carbon increment in the alkyl chain length, N-hexyl-galactonamide and N-octyl-galactonamide, were not as good as N-heptyl-galactonamide. Their differences were analyzed in terms of gel fibers morphology, mechanical properties, solubility, chain parity, and cell growth.

Regenerative potential of primary adult human neural stem cells on micropatterned bio-implants boosts motor recovery.

BACKGROUND: The adult brain is unable to regenerate itself sufficiently after large injuries. Therefore, hopes rely on therapies using neural stem cell or biomaterial transplantation to sustain brain reconstruction. The aim of the present study was to evaluate the improvement in sensorimotor recovery brought about by human primary adult neural stem cells (hNSCs) in combination with bio-implants. METHODS: hNSCs were pre-seeded on implants micropatterned for neurite guidance and inserted intracerebrally 2 weeks after a primary motor cortex lesion in rats. Long-term behaviour was significantly improved after hNSC implants versus cell engraftment in the grip strength test. MRI and immunohistological studies were conducted to elucidate the underlying mechanisms of neuro-implant integration. RESULTS: hNSC implants promoted tissue reconstruction and limited hemispheric atrophy and glial scar expansion. After 3 months, grafted hNSCs were detected on implants and expressed mature neuronal markers (NeuN, MAP2, SMI312). They also migrated over a short distance to the reconstructed tissues and to the peri-lesional tissues, where 26% integrated as mature neurons. Newly formed host neural progenitors (nestin, DCX) colonized the implants, notably in the presence of hNSCs, and participated in tissue reconstruction. The microstructured bio-implants sustained the guided maturation of both grafted hNSCs and endogenous progenitors. CONCLUSIONS: These immunohistological results are coherent with and could explain the late improvement observed in sensorimotor recovery. These findings provide novel insights into the regenerative potential of primary adult hNSCs combined with microstructured implants.

Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

INTRODUCTION: Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. METHODS: A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. RESULTS: Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. CONCLUSION: The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

A shear-induced network of aligned wormlike micelles in a sugar-based molecular gel. From gelation to biocompatibility assays.

A new low molecular weight hydrogelator with a saccharide (lactobionic) polar head linked by azide-alkyne click chemistry was prepared in three steps. It was obtained in high purity without chromatography, by phase separation and ultrafiltration of the aqueous gel. Gelation was not obtained reproducibly by conventional heating-cooling cycles and instead was obtained by shearing the aqueous solutions, from 2 wt% to 0.25 wt%. This method of preparation favored the formation of a quite unusual network of interconnected large but thin 2D-sheets (7nm-thick) formed by the association side-by-side of long and aligned 7nm diameter wormlike micelles. It was responsible for the reproducible gelation at the macroscopic scale. A second network made of helical fibres with a 10-13nm diameter, more or less intertwined was also formed but was scarcely able to sustain a macroscopic gel on its own. The gels were analysed by TEM (Transmission Electronic Microscopy), cryo-TEM and SAXS (Small Angle X-ray Scattering). Molecular modelling was also used to highlight the possible conformations the hydrogelator can take. The gels displayed a weak and reversible transition near 20°C, close to room temperature, ascribed to the wormlike micelles 2D-sheets network. Heating over 30°C led to the loss of the gel macroscopic integrity, but gel fragments were still observed in suspension. A second transition near 50°C, ascribed to the network of helical fibres, finally dissolved completely these fragments. The gels showed thixotropic behaviour, recovering slowly their initial elastic modulus, in few hours, after injection through a needle. Stable gels were tested as scaffold for neural cell line culture, showing a reduced biocompatibility. This new gelator is a clear illustration of how controlling the pathway was critical for gel formation and how a new kind of self-assembly was obtained by shearing.

Enhancing Plasticity of the Central Nervous System: Drugs, Stem Cell Therapy, and Neuro-Implants.

Stroke represents the first cause of adult acquired disability. Spontaneous recovery, dependent on endogenous neurogenesis, allows for limited recovery in 50% of patients who remain functionally dependent despite physiotherapy. Here, we propose a review of novel drug therapies with strong potential in the clinic. We will also discuss new avenues of stem cell therapy in patients with a cerebral lesion. A promising future for the development of efficient drugs to enhance functional recovery after stroke seems evident. These drugs will have to prove their efficacy also in severely affected patients. The efficacy of stem cell engraftment has been demonstrated but will have to prove its potential in restoring tissue function for the massive brain lesions that are most debilitating. New answers may lay in biomaterials, a steadily growing field. Biomaterials should ideally resemble lesioned brain structures in architecture and must be proven to increase functional reconnections within host tissue before clinical testing.

Corticospinal Tract Tracing in the Marmoset with a Clinical Whole-Body 3T Scanner Using Manganese-Enhanced MRI.

Manganese-enhanced MRI (MEMRI) has been described as a powerful tool to depict the architecture of neuronal circuits. In this study we investigated the potential use of in vivo MRI detection of manganese for tracing neuronal projections from the primary motor cortex (M1) in healthy marmosets (Callithrix Jacchus). We determined the optimal dose of manganese chloride (MnCl2) among 800, 400, 40 and 8 nmol that led to manganese-induced hyperintensity furthest from the injection site, as specific to the corticospinal tract as possible, and that would not induce motor deficit. A commonly available 3T human clinical MRI scanner and human knee coil were used to follow hyperintensity in the corticospinal tract 24h after injection. A statistical parametric map of seven marmosets injected with the chosen dose, 8 nmol, showed the corticospinal tract and M1 connectivity with the basal ganglia, substantia nigra and thalamus. Safety was determined for the lowest dose that did not induce dexterity and grip strength deficit, and no behavioral effects could be seen in marmosets who received multiple injections of manganese one month apart. In conclusion, our study shows for the first time in marmosets, a reliable and reproducible way to perform longitudinal ME-MRI experiments to observe the integrity of the marmoset corticospinal tract on a clinical 3T MRI scanner.

Strength and fine dexterity recovery profiles after a primary motor cortex insult and effect of a neuronal cell graft.

The aim of this study was to set up (a) a large primary motor cortex (M1) lesion in rodent and (b) the conditions for evaluating a long-lasting motor deficit in order to propose a valid model to test neuronal replacement therapies aimed at improving motor deficit recovery. A mitochondrial toxin, malonate, was injected to induce extensive destruction of the forelimb M1 cortex. Three key motor functions that are usually evaluated following cerebral lesion in the clinic-strength, target reaching, and fine dexterity-were assessed in rats by 2 tests, a forelimb grip strength test and a skilled reaching task (staircase) for reaching and dexterity. The potential enhancement of postlesion recovery induced by a neuronal cell transplantation was then explored and confirmed by histological analyses. Both tests showed a severe functional impairment 2 days post lesion, however, reaching remained intact. Deficits in forelimb strength were long lasting (up to 3 months) but spontaneously recovered despite the extensive lesion size. This natural grip strength recovery could be enhanced by cell therapy. Histological analyses confirmed the presence of grafted cells 3 months postgraft and showed partial tissue reconstruction with some living neuronal cells in the graft. In contrast, fine dexterity never recovered in the staircase test even after grafting. These results suggest that cell replacement was only partially effective and that the forelimb M1 area may be a node of the sensorimotor network, where compensation from secondary pathways could account for strength recovery but recovery of forelimb fine dexterity requires extensive tissue reconstruction.

Investigation of the competition between cell/surface and cell/cell interactions during neuronal cell culture on a micro-engineered surface.

To investigate the respective roles of topography and cell/cell interactions in the development of a guided neuronal network on an engineered surface, micropatterned PDMS substrates were generated with different microgrooves geometry and investigated for the influence of cell density on neurite outgrowth and alignment. Through this systematic investigation, using a human neuronal stem cell line, the rules of neuronal network development and guiding could be learned. The results show that when cell density is increased the influence on neuritic outgrowth and alignment is very different for the various grooves geometries. The data emphasized the competition, in neurite development, between physical cues brought by surface topographical features and cell to cell communications. These results can be of particular interest for designing functional neuronal networks with a controlled architecture.

[Micro/nano-engineering to control growth of neuronal cells and tissue engineering applied to the central nervous system]. / Micro/nano-ingénierie pour le contrôle de la croissance de cellules neuronales et ingénierie tissulaire appliquée au système nerveux central.

Central nervous system pathologies are often characterized by the loss of cell populations. A promising therapy now being developed consists in using bioactive materials, associating grafted cells to biopolymers which provide a scaffold for the in vitro building of new tissues, to be implanted in vivo. In the present article, the state of the art of this field, at crossroads between microtechnology and neuroscience, is described in detail; thereafter our own approach and results about interactions between adult human neural stem cells and microstructured polymers are summarized and discussed. In a second part, some central nervous system repair strategies, based on cerebral tissue engineering, are presented. We will report the main results of our studies to work out and characterize in vivo a cerebral bioprosthesis.

Elucidation of the role of carbon nanotube patterns on the development of cultured neuronal cells.

Carbon nanotubes (CNTs) promise various novel neural biomedical applications for interfacing neurons with electronic devices or to design appropriate biomaterials for tissue regeneration. In this study, we use a new methodology to pattern SiO(2) cell culture surfaces with double-walled carbon nanotubes (DWNTs). In contrast to homogeneous surfaces, patterned surfaces allow us to investigate new phenomena about the interactions between neural cells and CNTs. Our results demonstrate that thin layers of DWNTs can serve as effective substrates for neural cell culture. Growing neurons sense the physical and chemical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. Cells exhibit comparable adhesion and differentiation scores on homogeneous CNT layers and on a homogeneous control SiO(2) surface. Conversely, on patterned surfaces, it is found that cells preferentially grow on CNT patterns and that neurites are guided by micrometric CNT patterns. To further elucidate this observation, we investigate the interactions between CNTs and proteins that are contained in the cell culture medium by using quartz crystal microbalance measurements. Finally, we show that protein adsorption is enhanced on CNT features and that this effect is thickness dependent. CNTs seem to act as a sponge for culture medium elements, possibly explaining the selectivity in cell growth localization and differentiation.

Engineering of adult human neural stem cells differentiation through surface micropatterning.

Interaction between differentiating neural stem cells and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. To restore damage brain area by tissue engineering, the biomaterial scaffold has to mimic this microenvironment to allow organized tissue regeneration. To establish the validity of using microgrooved surfaces in order to simultaneously provide to primary adult human neural stem cells a permissive growth environment and a guide for neurite outgrowth in a pre-established direction, we have studied the long-term culture of adult human neural stem cells from patient biopsies on microgrooved polymers. By exploiting polymer moulding techniques, we engineered non-cytotoxic deep microstructured surfaces of polydimethylsiloxane (PDMS) exhibiting microchannels of various widths. Our results demonstrate that precoated micropatterned PDMS surfaces can serve as effective neurite guidance surfaces for human neural stem cells. Immunocytochemistry analysis show that channel width can impact strongly development and differentiation. In particular we found an optimal microchannel width, that conciliates a high differentiation rate with a pronounced alignment of neurites along the edges of the microchannels. The impact of the microstructures on neurite orientation turned out to be strongly influenced by cell density, attesting that cell/surface interactions at the origin of the alignment effect, are in competition with cell/cell interactions tending to promote interconnected networks of cells. Considering all these effects, we have been able to design appropriate structures allowing to obtain neuron development and differentiation rate comparable to a plane unpatterned surface, with an efficient neurite guidance and a long-term cell viability.

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo.

BACKGROUND: To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.

Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein.

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.

Proteolipidic vectors for gene transfer to the lung.

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.