Olivier Danos

Nucleotide sequence of the AIDS virus, LAV

The complete 9193-nucleotide sequence of the probable causative agent of AIDS, lymphadenopathy-associated virus (LAV), has been determined. The deduced genetic structure is unique: it shows, in addition to the retroviral gag, pol, and env genes, two novel open reading frames we call Q and F. Remarkably, Q is located between pol and env and F is half-encoded by the U3 element of the LTR. These data place LAV apart from the previously characterized family of human T cell leukemia/lymphoma viruses.

Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus

We have determined the complete 9202 nucleotide sequence of the visna lentivirus. The deduced genetic organization most closely resembles that of the AIDS retrovirus in that there is a novel central region separating pol and env. Moreover, there is a close phylogenetic relationship between the conserved reverse transcriptase and endonuclease/integrase domains of the visna and AIDS viruses. These findings support the inclusion of the AIDS virus in the retroviral subfamily Lentivirinae.

Rescue of Dystrophic Muscle Through U7 snRNA-Mediated Exon Skipping

Most mutations in the dystrophin gene create a frameshift or a stop in the mRNA and are associated with severe Duchenne muscular dystrophy. Exon skipping that naturally occurs at low frequency sometimes eliminates the mutation and leads to the production of a rescued protein. We have achieved persistent exon skipping that removes the mutated exon on the dystrophin messenger mRNA of the mdx mouse, by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. We report the sustained production of functional dystrophin at physiological levels in entire groups of muscles and the correction of the muscular dystrophy.

Polyethylenimine-mediated gene delivery: a mechanistic study

Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes.

Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome: Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products

The complete nucleotide sequence and genomic organization of human papillomavirus type 18, associated with cervical cancer, has been established. A detailed comparative analysis was undertaken leading to the identification of a number of features specific for genital papillomaviruses and the construction of a phylogenetic tree. Genital papillomaviruses differ from other human and animal papillomaviruses as they possess a longer E1 open reading frame (ORF) and have a characteristic control region. Phylogenetically, HPV 18 is located between the benign genital viruses, HPV 6 and HPV 11, and the malignant isolates, HPV 16 and HPV 33, and may represent an evolutionary intermediate among oncogenic papillomaviruses. Viral gene products known to be involved in cellular transformation are those of ORFs E5, E6 and E7. Significant sequence variation was found between the E6 to E7 regions of different integrated forms of HPV 18. On re-examination of the E6 primary structures we noticed that the gene has evolved by successive duplications of a unit encoding 33 amino acids, which include a Cys-X-X-Cys motif. Furthermore, the E7 gene product has apparently evolved in the same manner and is related to E6. Both gene products bear a striking resemblance to the transcriptional factor IIIA of Xenopus laevis, the prototype of a new class of nucleic acid binding proteins.

Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation.

ABSTRACT The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus. Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A1, brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form. MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.

Canine Adenovirus Vectors: an Alternative for Adenovirus-Mediated Gene Transfer

ABSTRACT Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.

Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells

Gene delivery has shown potential in a wide variety of applications, including basic research, therapies for genetic and acquired diseases, and vaccination. Most available nonviral systems have serious drawbacks such as the inability to control and scale the production process in a reproducible manner. Here, we demonstrate a biotechnologically feasible approach for gene delivery, using synthetic cationic amphipathic peptides containing a variable number of histidine residues. Gene transfer to different cell lines in vitro was achieved with an efficiency comparable to commercially available reagents. We provide evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Endosomal acidification is also required. Interestingly, even when complexed to DNA these peptides maintain a high level of antibacterial activity, opening the possibility of treating the genetic defect and the bacterial infections associated with cystic fibrosis with a single compound. Thus, this family of peptides represents a new class of agents that may have broad utility for gene transfer and gene therapy applications.

FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia

Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34(+) cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41(+)CD42(-) progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41(+)CD42(-) cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41(+)CD42(-) cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1(+) or FLI1(-) CD41(+)CD42(-) cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.

Intranasal gene delivery with a polyethylenimine: PEG conjugate

Polyethylenimines (PEIs) are among the most efficient synthetic DNA carriers. High levels of reporter gene expression can be obtained with these agents on a variety of cells. Nevertheless, the gap between their efficiency and that required for therapeutic approaches is still important. With the aim to improve the in vivo transfection properties of PEIs, we have synthesized a conjugate consisting of the linear polymer of 22 kDa covalently modified with polyethyleneglycol (PEG) residues. The resulting conjugate was able to complex DNA and allowed the preparation of highly concentrated polyplexes, in contrast to non-modified PEIs. Administration by nasal instillation of PEI-PEG/DNA complexes in mice resulted in significant levels of transgene expression. Luciferase activity was greatest 24 h after delivery and decreased thereafter. Our results show that the grafting of PEGs can improve some of the properties of PEIs.