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1.
Cell Mol Life Sci ; 65(16): 2594-612, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18597042

ABSTRACT

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.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Models, Molecular , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Cross-Linking Reagents/metabolism , Disulfides/metabolism , Humans , Molecular Sequence Data , Mutation/genetics , Protein Structure, Secondary , Protein Structure, Tertiary
2.
Chem Commun (Camb) ; (20): 2054-6, 2007 May 28.
Article in English | MEDLINE | ID: mdl-17713076

ABSTRACT

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.


Subject(s)
Glyceryl Ethers/chemistry , Transfection/methods , Cations/chemistry , Cell Line, Tumor , DNA/genetics , Glyceryl Ethers/chemical synthesis , Humans , Liposomes , Molecular Structure , Plasmids/genetics
3.
Cell Mol Life Sci ; 62(18): 2112-23, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16132229

ABSTRACT

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.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Amino Acid Sequence , Amino Acids, Aromatic/chemistry , Amino Acids, Aromatic/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Dimerization , Humans , Molecular Sequence Data , Mutation , Nucleotides/metabolism , Protein Structure, Tertiary , Sequence Analysis, Protein
4.
Curr Pharm Des ; 11(3): 375-94, 2005.
Article in English | MEDLINE | ID: mdl-15723632

ABSTRACT

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.


Subject(s)
Cations , Drug Design , Gene Transfer Techniques/trends , Lipids , Cations/chemistry , Genetic Vectors/chemical synthesis , Genetic Vectors/pharmacokinetics , Humans , Lipids/chemistry
5.
Cell Mol Life Sci ; 61(2): 230-42, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14745501

ABSTRACT

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.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Nucleotides/metabolism , Amino Acid Sequence , Binding Sites , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Dimerization , Humans , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, Protein
6.
J Gene Med ; 3(5): 478-87, 2001.
Article in English | MEDLINE | ID: mdl-11601761

ABSTRACT

BACKGROUND: Colloidal stability of lipid/DNA aggregates is a major requirement for cationic lipid-mediated transfection which is particularly difficult to fulfil at the high DNA concentrations used for in vivo gene delivery. Thus, we have investigated the potential of poly(ethyleneglycol) (PEG) conjugates for steric stabilization of lipoplexes formed by bis(guanidinium)-tren-cholesterol/dioleoyl phosphatidylethanolamine (BGTC/DOPE) liposomes, a class of cationic liposomes we have developed over the past few years. METHODS AND RESULTS: We demonstrate that adequate lipophilic PEG derivatives can stabilize BGTC/DOPE lipoplexes formed at high DNA concentration. We also report the results of cryotransmission electron microscopy studies indicating that PEG-stabilized lipoplexes form DNA-coated structures which assemble into clusters exhibiting various complex morphologies. Finally, we report data from in vivo transfection experiments suggesting that PEG-mediated colloidal stabilization of concentrated lipoplex solutions may allow enhanced transfection of the mouse airways via intranasal administration. CONCLUSION: Our results represent an important step towards the design of multimodular BGTC-based systems for improved in vivo gene transfection.


Subject(s)
Chloramphenicol/analogs & derivatives , Cholesterol/analogs & derivatives , Cholesterol/genetics , Glycerophospholipids/genetics , Lung/metabolism , Phosphatidylethanolamines , Transfection , Animals , Cell Survival , Chloramphenicol/metabolism , Cholesterol/chemistry , Cholesterol/metabolism , DNA/chemistry , DNA/ultrastructure , Female , Gene Transfer Techniques , Genetic Vectors , Glycerophospholipids/chemistry , Glycerophospholipids/metabolism , Guanidines/chemistry , Guanidines/metabolism , Mice , Mice, Inbred BALB C , Polyethylene Glycols/chemistry , Tumor Cells, Cultured
7.
Glycobiology ; 11(9): 741-50, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11555618

ABSTRACT

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.


Subject(s)
Glycoside Hydrolases/metabolism , Iduronidase/metabolism , Mucopolysaccharidosis I/enzymology , Mutation , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Base Sequence , Binding Sites , Blotting, Western , CHO Cells , Cricetinae , DNA Primers , Epitope Mapping , Glycoside Hydrolases/genetics , Glycoside Hydrolases/immunology , Humans , Iduronidase/genetics , Iduronidase/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Subcellular Fractions/enzymology
8.
Biochem Biophys Res Commun ; 281(2): 536-43, 2001 Feb 23.
Article in English | MEDLINE | ID: mdl-11181080

ABSTRACT

We have previously shown that cationic cholesterol derivatives bearing guanidinium groups were efficient vectors for gene transfer. To further evaluate the potentiality of this novel class of cationic lipids, we undertook to study the transfection efficiency of guanidinium-based lipids with other hydrophobic moieties. Specifically, we synthesized a reagent where two guanidinium groups are linked to a diacetylene lipid which may provide the lipoplexes with favorable structural features. We report here that the cationic lipid bisguanidinium-diacetylene (BGDA) is highly efficient for in vitro gene transfection when formulated with dioleoylphosphatidyl ethanolamine (DOPE). We also show that liposomes composed of BGDA, DOPE, and a neutral diacetylene colipid, hydroxyethylenediacetylene (HEDA), are efficient for transfection. Thus, diacetylene-based lipids provide a novel scaffold for gene transfection and will be particularly useful for gaining new insights into the structure-activity relationships of the lipid/DNA complexes as they offer a means to study the effects of polymerizable domains.


Subject(s)
DNA/genetics , Gene Transfer Techniques , Lipids/administration & dosage , Acetylene/chemistry , Culture Media, Serum-Free/pharmacology , DNA/administration & dosage , DNA/chemistry , Guanidines/chemistry , HeLa Cells , Humans , Lipids/chemistry , Liposomes , Luciferases/drug effects , Luciferases/genetics , Luciferases/metabolism , Plasmids/administration & dosage , Plasmids/genetics , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection/methods
9.
Glycobiology ; 10(11): 1217-24, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11087714

ABSTRACT

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.


Subject(s)
Glucosylceramidase/genetics , Animals , Base Sequence , Catalytic Domain/genetics , Cell Line , DNA, Complementary/genetics , Gene Expression , Glucosylceramidase/chemistry , Glucosylceramidase/metabolism , Glycosylation , Humans , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Subcellular Fractions/enzymology , Transduction, Genetic
10.
Hum Mol Genet ; 9(6): 967-77, 2000 Apr 12.
Article in English | MEDLINE | ID: mdl-10767320

ABSTRACT

Lysosomal storage diseases are due to inherited deficiencies in various enzymes involved in basic metabolic processes. As with other genetic diseases, accurate structure data for these enzymatic proteins should help in better understanding the molecular effects of mutations identified in patients with the corresponding lysosomal diseases; however, no such three-dimensional (3D) structure data are available for many lysosomal enzymes. Thus, we herein intend to illustrate for an audience of molecular geneticists how structure information can nonetheless be obtained via a bioinformatics approach in the case of five human lysosomal glycoside hydrolases. Indeed, using the two-dimensional hydrophobic cluster analysis method to decipher the sequence information available in data banks for the large group of glycoside hydrolases (clan GH-A) to which these human lysosomal enzymes belong, we could deduce structure predictions for their catalytic domains and propose explanations for the molecular effects of mutations described in patients. In addition, in the case of human beta-glucuronidase for which experimental 3D data have been reported, we also show here that bioinformatics methods relying on the available 3D structure information can be used to obtain further insights into the effects of various mutations described in patients with Sly disease. In a broader perspective, our work stresses that, in the context of a rapid increase in protein sequence information through genome sequencing, bioinformatics approaches might be highly useful for generating structure-function predictions based on sequence-structure interrelationships.


Subject(s)
Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Mutation , Crystallography, X-Ray , Humans , Medical Informatics , Protein Conformation
11.
Proc Natl Acad Sci U S A ; 96(6): 2621-6, 1999 Mar 16.
Article in English | MEDLINE | ID: mdl-10077560

ABSTRACT

We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection in vitro and in vivo. In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 A and 68 A in BGTC/DOPE-DNA and BGTC-DNA aggregates, respectively. In addition, DNA lipoplexes with similar lamellar patterns were detected inside transfected HeLa cells by conventional transmission electron microscopy. These results suggest that DNA condensation by multivalent guanidinium-cholesterol cationic lipids involves the formation of highly ordered multilamellar domains, the DNA molecules being intercalated between the lipid bilayers. These results also invite further investigation of the intracellular fate of the internalized lipid/DNA structures during their trafficking toward the cell nucleus. The identification of the basic features of active complexes should indeed help in the design of improved guanidinium-based vectors.


Subject(s)
Cholesterol/chemistry , DNA/chemistry , Gene Transfer Techniques , Genetic Vectors/chemistry , Guanidine/chemistry , DNA/ultrastructure , Genetic Vectors/ultrastructure , HeLa Cells , Humans , Microscopy, Electron , X-Ray Diffraction
12.
Adv Drug Deliv Rev ; 30(1-3): 5-11, 1998 Mar 02.
Article in English | MEDLINE | ID: mdl-10837597

ABSTRACT

Although most research in the field of somatic gene therapy has investigated the use of recombinant viruses for transferring genes into somatic target cells, various methods for nonviral gene delivery have also been proposed. Both types of gene delivery systems have advantages and drawbacks. Schematically, viral vectors are particularly efficient for gene delivery, whereas nonviral systems are free of the difficulties associated with the use of recombinant viruses but need to be further optimized to reach their full potential. In order to bridge the gap between viral vectors and synthetic reagents, we discuss here some specific features of the viral vector systems of today that could advantageously be taken into account for the design of improved nonviral gene delivery systems. Indeed, although nonviral systems differ fundamentally from viral systems, one possible approach towards enhanced artificial reagents aims at developing 'artificial viruses' that mimic the highly efficient processes of viral infection.

13.
Mol Biol Evol ; 14(4): 372-80, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9100367

ABSTRACT

The species-specific pattern of cystic fibrosis transmembrane conductance regulator (CFTR) expression was investigated in order to identify species closely related to man which can be used as potential cystic fibrosis (CF) animal models. To this purpose, the nucleotide sequences of the CFTR promoter region of eight mammalian species representing four different orders (Primates, Artiodactyla, Lagomorpha and Rodentia) were analyzed. Distance matrices and unrooted trees of the CFTR promoter region sequences yielded two deeply separated groups, one including man (Homo sapiens), nonhuman primates (Hylobates lar, Macaca fascicularis, Saimiri sciureus), cow (Bos taurus), and rabbit (Oryctolagus cuniculus) and the other including the rodents (Rattus norvegicus, Mus musculus). Divergences between rodent and nonrodent groups have been observed in putative cis transcriptional regulatory elements and can be involved in the differences of pattern of expression between these two groups. Comparison of the available CFTR cDNA sequences enabled us to root the tree with a noneutherian outgroup and to perform a phylogenetic analysis. This analysis did not detect any base composition bias and supported polyphyletic Glires. Although a long-branch attraction artifact cannot be completely excluded, these findings converge toward the recent statement (Graur, Duret, and Gouy 1996) that Lagomorpha is more closely related to Primates than to Rodentia. In addition, the phenylalanine residue in exon 10 involved in the most common CF mutation in man is conserved in rabbit. These phylogenetic analyses as well as anatomical and developmental data suggest that, once rabbit embryonic stem cells become available, the rabbit will provide a suitable tool for both gene transfer and pharmacological investigations and could lead to a better CF model than the current murine models.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Phylogeny , Animals , Disease Models, Animal , Humans , Molecular Sequence Data , Rabbits , Rats , Species Specificity
14.
Proc Natl Acad Sci U S A ; 94(5): 1651-6, 1997 Mar 04.
Article in English | MEDLINE | ID: mdl-9050833

ABSTRACT

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.


Subject(s)
Cholesterol/analogs & derivatives , Gene Transfer Techniques , Genetic Vectors , Guanidines , Nasal Polyps/metabolism , Trachea/metabolism , Adenoviridae/genetics , Animals , Cells, Cultured , Cystic Fibrosis/therapy , Epithelium/metabolism , Escherichia coli/enzymology , Genes, Reporter/genetics , Genetic Therapy , Humans , Liposomes/administration & dosage , Luciferases/genetics , Luciferases/metabolism , Male , Mice , Phosphatidylethanolamines , Plasmids , Transfection , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
15.
Glycobiology ; 7(2): 277-84, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9134434

ABSTRACT

The clan GH-A is a group of more than 200 proteins representing nine established families of glycosyl hydrolases that act on a large variety of substrates. This clan includes five enzymes implicated in lysosomal storage diseases: beta-glucuronidase (Sly disease), beta-glucocerebrosidase (Gaucher disease), beta-galactosidase (Landing disease and Morquito type B disease), beta-mannosidase (mannosidosis) and alpha-L-iduronidase (Hurler-Scheie disease). Examination of known 3D structures from some families of the clan allowed us to deduce structural and functional features shared by these proteins. We then used the hydrophobic cluster analysis method to study the protein sequences of the entire clan. Our results reveal that, despite low levels of sequence identity, all the proteins of the clan (including the aforementioned lysosomal enzymes) likely share a similar catalytic domain consisting of an (alpha/beta)8 barrel with conserved functional amino acids located at the C-terminal ends of six of the eight strands constituting the beta-barrel. Interestingly, several mutations reported to be responsible for lysosomal storage diseases are located within these conserved regions of the lysosomal enzyme catalytic domains.


Subject(s)
Glycoside Hydrolases/chemistry , Lysosomes/enzymology , Protein Structure, Secondary , Protein Structure, Tertiary , Amino Acid Sequence , Animals , Binding Sites , Cluster Analysis , Conserved Sequence , Glycoside Hydrolases/classification , Humans , Molecular Sequence Data , Sequence Analysis/methods , Sequence Homology, Amino Acid
16.
Transfus Clin Biol ; 4(3): 267-73, 1997.
Article in French | MEDLINE | ID: mdl-9264784

ABSTRACT

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.


Subject(s)
Hematopoietic Stem Cells/cytology , Thymus Gland/cytology , Transgenes , Animals , Antigens, CD34/analysis , Cell Transformation, Viral , Fetal Blood/cytology , Gene Transfer Techniques , Genetic Markers , Hematopoietic Stem Cells/chemistry , Humans , Mice , Mice, SCID , Retroviridae , Thymus Gland/embryology
17.
Proc Natl Acad Sci U S A ; 93(18): 9682-6, 1996 Sep 03.
Article in English | MEDLINE | ID: mdl-8790391

ABSTRACT

Two cationic lipids, bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.


Subject(s)
Cholesterol/analogs & derivatives , Guanidines/chemical synthesis , Transfection/methods , Animals , Cell Line , Cholesterol/chemical synthesis , Dogs , Genes, Reporter , Haplorhini , HeLa Cells , Humans , Liposomes , Luciferases/genetics , Mice , Rats
18.
Blood ; 88(1): 107-13, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8704164

ABSTRACT

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T-cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.


Subject(s)
CD2 Antigens/genetics , Fetal Blood/cytology , Fetal Tissue Transplantation , Genetic Markers , Genetic Vectors/genetics , Hematopoietic Stem Cell Transplantation , Retroviridae/genetics , T-Lymphocytes/cytology , Thymus Gland/transplantation , Transplantation, Heterologous , Animals , Antigens, CD34/analysis , CD2 Antigens/biosynthesis , Cell Differentiation , Cell Lineage , Chimera , Coculture Techniques , Flow Cytometry , Genetic Therapy/methods , Graft Survival , Humans , Immunophenotyping , Infant, Newborn , Mice , Mice, SCID , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Thymus Gland/cytology , Virus Integration
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