Efficient gene expression from integration-deficient lentiviral vectors in the spinal cord.

Gene transfer to spinal cord cells may be crucial for therapy in spinal muscular atrophy, amyotrophic lateral sclerosis and spinal cord injury. Lentiviral vectors are efficient for transduction of a variety of cells, but like all integrating vectors they pose a risk of insertional mutagenesis. Integration-deficient lentiviral vectors (IDLVs) remain episomal but retain the transduction efficiency of standard integrating lentiviral vectors, particularly when the episomes are not diluted out through repeated cell division. We have now applied IDLVs for transduction of spinal cord in vitro, in explants and in vivo. Our results demonstrate similar efficiency of eGFP expression from integrating lentiviral vectors and IDLVs in most cell types analyzed, including motor neurons, interneurons, dorsal root ganglia (DRG) neurons and astroglia. IDLV-mediated expression of pro-glial-cell-derived neurotrophic factor (Gdnf) rescues motor neuron cultures from death caused by removal of exogenous trophic support. IDLVs also mediate efficient RNA interference in DRG neuron cultures. After intraparenchymal injection in the rat and mouse cervical and lumbar regions in vivo, transduction is mainly neuronal, with both motor neurons and interneurons being efficiently targeted. These results suggest that IDLVs could be efficient and safer tools for spinal cord transduction in future therapeutic strategies.

Nonviral gene delivery to the central nervous system based on a novel integrin-targeting multifunctional protein.

Successful introduction of therapeutic genes into the central nervous system (CNS) requires the further development of efficient transfer vehicles that avoid viral vector-dependent adverse reactions while maintaining high transfection efficiency. The multifunctional protein 249AL was recently constructed for in vitro gene delivery. Here, we explore the capability of this vector for in vivo gene delivery to the postnatal rat CNS. Significant transgene expression was observed both in the excitotoxically injured and noninjured brain after intracortical injection of the DNA-contaning-249AL vector. In the injured brain, a widespread expression occurred in the entire lesioned area and retrograde transport of the vector toward distant thalamic nuclei and transgene expression were observed. Neurons, astrocytes, microglia, and endothelial cells expressed the transgene. No recruitment of leukocytes, demyelination, interleukin-1beta expression, or increase in astrocyte/microglial activation was observed at 6 days postinjection. In conclusion, the 249AL vector shows promising properties for gene therapy intervention in the CNS, including the targeting of different cell populations.

Riluzole promotes survival of rat motoneurons in vitro by stimulating trophic activity produced by spinal astrocyte monolayers.

In the present study we have assessed whether riluzole stimulates the production of trophic activities for motoneurons by spinal astrocyte cultures. Astrocyte monolayers prepared from new-born rats were exposed to vehicle or riluzole (1-10 microM) for 30-36 h, then washed and further incubated without riluzole for 24 h in L15 medium to obtain the astrocyte conditioned media (ACM). Motoneuron-enriched cultures were used to test the ability of the ACM to support motoneuron viability. Astrocyte monolayers exposed to 1 microM riluzole did not show changes in morphology or in DNA or protein synthesis. However, the conditioned medium obtained from astrocyte monolayers after this treatment increased motoneuron survival compared to that from vehicle-treated cultures. A similar effect was found when astrocytes were exposed to a higher riluzole concentration (10 microM) but with greater dilutions of the conditioned medium. This trophic activity was abolished by boiling or after treatment with trypsin. These findings strongly suggest the existence of a new trophic mechanism, through which riluzole may exert motoneuron protection.

N-acetylaspartylglutamate acetoxymethyl triester (NAAG.AM) as a tool for loading the neuropeptides NAAG and succinimidyl-NAAG into intact cells: effect on [3H]-dopamine exocytosis.

Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8% of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCl depolarization reaching an inhibition of 49% with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.

N-acetylaspartylglutamate acetoxymethyl triester (NAAG.AM) as a tool for loading the neuropeptides NAAG and succinimidyl-NAAG into intact cells: effect on [3H]-dopamine exocytosis

Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8 percent of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCI depolarization reaching an inhibition of 49 percent with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.