Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Egg envelope synthesis and chorion modification after oviposition in the dragonfly Libellula depressa (Odonata, Libellulidae).

Libellula depressa (Odonata, Libellulidae) is an exophytic dragonfly ovidepositing eggs in clutches on the surface of floating plants and algae. The present work investigates, at ultrastructural level, the gradual differentiation of the egg envelopes and the chorionic changes after egg deposition in water. The ovary of the mature female of L. depressa is composed of numerous strings of panoistic ovarioles, where the eggshell formation takes place gradually throughout the activity of the follicle cells. The present data show that the egg envelopes are constituted of a very thick electrondense vitelline envelope, a thin endochorion and an extremely thick exochorion composed of a fibrillar matrix resting on a thin electrondense layer. After deposition in water, L. depressa eggs, initially white and almost transparent, gradually become brown spots in a semitransparent jelly coat, rich of incorporated debris. The jelly coat enveloping the eggs of L. depressa derives exclusively from the exochorion, constituted of a fibrillar matrix, which swell at contact with water. The jelly-like coat performs an adhesive function and presumably a protective role during egg segmentation and ensuing larval hatching.

Lentiviral transduction of human postnatal skeletal (stromal, mesenchymal) stem cells: in vivo transplantation and gene silencing.

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

Two ribosomal DNA-binding factors interact with a cluster of motifs on the 5' external transcribed spacer, upstream from the primary pre-rRNA processing site in a higher plant.

In radish the primary processing site in pre-rRNA has been mapped to a TTTTCGCGC sequence (motif P) in the 5' external transcribed spacer (5' ETS) of the ribosomal DNA (rDNA) [Delcasso-Tremousaygue, D., Grellet, F., Panabières, F., Ananiev, E. & Delseny, M. (1988) Eur. J. Biochem. 172, 767-776]. The processing site is just downstream of four similar motifs named A1, A2, A3 and B. The five motifs constitute cluster A123BP. We have described previously that in radish extracts a nuclear protein, nuclear factor B (NF B) specifically binds to motif B [Echeverría, M., Penon, P. & Delseny, M. (1994) Mol. Gen. Genet. 243, 442-452]. Here, by means of electrophoretic-mobility-shift assays, we describe an rDNA-binding activity, nuclear factor D (NF D), that interacts with the A123BP cluster. Using various rDNA probes and competitors we show that NF D binds specifically to the A123 clustered motifs but not to similar B or P motifs. We used sequence-specific DNA-affinity chromatography to separate NF D from NF B. DNase I footprinting was used to map the binding site of NF D on the A123BP cluster and we compared it with that of NF B on the same probe. The footprint of NF D extends from the A1 motif to the 5' end of the NF B-binding site and includes motifs A2 and A3 on each strand. The footprinting of NF B is restricted to motif B and adjacent nucleotides. Thus the NF D-binding and NF B-binding sites are distinct but overlap. These two factors bind with a high specificity to the A123BP cluster in the radish 5' ETS. The possibility that these factors regulate rDNA transcription elongation at the level of the primary pre-rRNA processing site in crucifers is discussed.