Archaeosomes based on synthetic tetraether-like lipids as novel versatile gene delivery systems.

Novel cationic liposomes, termed "archaeosomes", based on mixtures of neutral/cationic bilayer-forming lipids and archaeobacterial synthetic tetraether-type bipolar lipids show efficient in vitro gene transfection properties and represent a new approach for modulating the lipidic membrane fluidity of the complexes they form with DNA.

Bone marrow transplantation for chronic granulocytic leukemia.

Thirty-seven patients with chronic granulocytic leukemia have been treated with supralethal chemoradiotherapy followed by transplantation of bone marrow from HLA-identical donors. All patients showed engraftment, and the Philadelphia chromosome (PH1) disappeared in each case. Four patients had syngeneic grafts before blast crisis and are still alive; 2 are in remission not maintained by therapy, and 2 others are receiving chemotherapy after having relapsed in the chronic phase. Thirty-three patients had allogeneic grafts; only 2 received the grafts during blast crisis, and neither is a long-term survivor. Of the 13 patients who had grafts in the accelerated phase, 6 died of complications related to the transplantation, and 1 died after a myeloblastic relapse. Thus 6 patients are in unmaintained remission with a median follow-up of 13 months. Eighteen patients received grafts in the chronic phase. All 10 survivors are in unmaintained remission with a median follow-up of 14 months; in this group, no patient has relapsed. The granulocytic hyperplasia of the chronic phase can be more effectively ablated than established blastic leukemia. The mortality rate of transplant-related complications must be weighted against the typical rate of progression of chronic granulocytic leukemia. Although a longer follow-up period is needed for full evaluation, bone marrow transplantation may now be offered to patients in the chronic phase in an attempt to achieve long-term survival or cure of more than one-half of these patients.

The design of cationic lipids for gene delivery.

Synthetic gene delivery vectors are gaining increasing importance in gene therapy as an alternative to recombinant viruses. Among the various types of non-viral vectors, cationic lipids are especially attractive as they can be prepared with relative ease and extensively characterised. Further, each of their constituent parts can be modified, thereby facilitating the elucidation of structure-activity relationships. In this forward-looking review, cationic lipid-mediated gene delivery will mainly be discussed in terms of the structure of the three basic constituent parts of any cationic lipid: the polar headgroup, hydrophobic moiety and linker. Particular emphasis will be placed on recent advances in the field as well as on our own original contributions. In addition to reviewing critical physicochemical features (such as headgroup hydration) of monovalent lipids, the use of headgroups with known nucleic-acid binding modes, such as linear and branched polyamines, aminoglycosides and guanidinium functions, will be comprehensively assessed. A particularly exciting innovation in linker design is the incorporation of environment-sensitive groups, the intracellular hydrolysis of which may lead to more controlled DNA delivery. Examples of pH-, redox- and enzyme-sensitive functional groups integrated into the linker are highlighted and the benefits of such degradable vectors can be evaluated in terms of transfection efficiency and cationic lipid-associated cytotoxicity. Finally, possible correlations between the length and type of hydrophobic moiety and transfection efficiency will be discussed. In conclusion it may be foreseen that in order to be successful, the future of cationic lipid-based gene delivery will probably require the development of sophisticated virus-like systems, which can be viewed as "programmed supramolecular systems" incorporating the various functions required to perform in a chronological order the different steps involved in gene transfection.

Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor.

The ex vivo approach to hepatic gene therapy involves several steps, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy.

Gene therapy using bone marrow transplantation.

As a result of new techniques in molecular biology, the basic defects in a variety of genetic diseases have been characterized and the corresponding normal genes cloned. Methods have been devised for transferring specific genes with high efficiency into various cell types that exploit the use of replication-defective retroviruses as vectors. Bone marrow may be the ideal target for these attempts to transfer genes, because it contains self-renewing stem cells and because it can readily be collected by standard techniques, manipulated ex vivo as required with comparative ease and then reinfused. In a murine system, recent studies have shown that genes can indeed be incorporated into haemopoietic stem cells that can then completely reconstitute the marrows of syngeneic recipients. Thus far, such genes after transfer have not been appropriately expressed in vivo, although appreciable expression has been documented in cell culture studies in vitro. It is reasonable to expect that improvements in the design of retroviral vectors should permit appropriate expression of a transferred gene. When this is achieved, the use of bone marrow transplantation for gene transfer will offer new options for therapy.

Gene therapy using bone marrow transplantation: a 1990 update.

The use of recombinant retroviruses for gene transfer into the haematopoietic tissue in vivo is still a new area of research. The initial results of in vitro studies were very exciting. In contrast, the early in vivo studies in mice were somewhat disappointing because of the transient and low levels of expression of the transferred gene. Recently, however, better results have been obtained in the murine system in vivo. New packaging cell lines have been constructed, which are safer and still efficient. Better vectors have been designed. Thus, significant levels of expression of the transgene have been achieved in murine long-term transplant recipients. However, the results obtained so far in large animal studies are still disappointing. It should be emphasized that further progress must be based on a simple overall strategy involving better understanding of the functioning of the gene to be transferred by gene expression studies, design of vectors carrying a fully active and correctly regulated minilocus and better knowledge of the biological properties of the target cells, the haematopoietic stem cells.

[Transgenic human thymopoiesis from retrovirally transduced umbilical cord blood hematopoietic stem cells: experimental studies in the SCID-hu mouse]. / Thymopoïèse humaine transgénique à partir des cellules souches hématopoïétiques du sang de cordon ombilical génétiquement modifiées par voie rétrovirale: étude expérimentale dans la souris SCID-hu.

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.

Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.

We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.

Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.

[General principles of the production and use of retroviral vectors]. / Principes généraux de la production et de l'utilisation des vecteurs rétroviraux.

This paper describes the various strategies for somatic gene therapy. There are two main scenarios: the ex vivo approach and the in vivo approach. As recombinant retroviruses were used in the first clinical trials of gene therapy, their main characteristics (structure, efficient production of helper-free viral stocks by using packaging cells, transduction procedures) are discussed as well as the related safety issues. Finally, retroviral vectors can also be used as a new tool to investigate various clinical situations as they allow to mark cells by stably integrating in the cellular genome.

[Gene therapy in cystic fibrosis: molecular and cellular aspects]. / Thérapie génique de la mucoviscidose: aspects moléculaires et cellulaires.

Gene therapy has become a potential treatment for Cystic Fibrosis (CF) when the CFTR gene responsible for the disease was isolated in 1989. As premature death is still the norm, the demand is now for a dramatically improved therapy. The pulmonary manifestations of CF being life-limiting, many gene therapy studies have focused on gene transfer via various vectors into airway epithelial cells, namely in murine models of CF. These preclinical studies led to the first clinical trials, which showed that many hurdles still do exist and that more efficient vector systems need to be developed. Both a remarkable scientific effort and a close collaboration between scientists and clinicians are highly necessary in order to move from the present stage of "cautious optimism" toward the ultimate goal: a cure for CF.

[Transfer of genes in hematopoietic tissue: from research to applications by autografts]. / Transfert de gènes dans le tissu hématopoïétique: de la recherche aux applications par autogreffes.

Gene therapy is a new field of biomedical research just entering clinical practice. Recombinant retroviruses are at present the best vectors for gene transfer into a permanently dividing tissue like the hematopoietic tissue. The ex vivo strategy consists of an autologous transplantation of genetically modified cells. Although the hematopoietic tissue can be manipulated with comparative ease, its development and its structure are rather complex. Therefore, the different sources of hematopoietic tissue and the diverse cell types represent a wide array of targets for gene transfer. In vivo studies in mice have shown that the hematopoietic stem cells can be efficiently transduced by retroviral vectors carrying therapeutic genes. In contrast, preclinical studies in large outbred animals indicate that only a small proportion of stem cells can be transduced, although much better results can be obtained with primitive progenitors detectable in vitro. However, some clinical trials have already shown interesting and useful results.

Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.