The application of new protocols for gene therapy against monogenic diseases requires the development of safer therapeutic vectors, particularly in the case of diseases in which expression of the mutated gene is subject to fine regulation, as it is with CD40L (CD154). CD40L, the gene mutated in the X-linked hyper-immunoglobulin M syndrome (HIGM1), is tightly regulated to allow surface expression of its product only on T cells stimulated by antigen encounter. Previous studies in an HIGM1 animal model showed that transduction of progenitor cells corrected the syndrome but caused a thymic lymphoproliferative disease because of the unregulated expression of the transgene by constitutive vectors. To develop a tissue-specific, activation-inducible, lentiviral vector (LV) for gene therapy to counter HIGM1, we have constructed two self-inactivating LVs, pCD40L-eGFP and pCD40L-CD40L, regulated by a 1.3 kb fragment of the human CD40L proximal promoter. The expression of pCD40L-eGFP LV is restricted to cells in which mRNA transcripts of the endogenous CD40L gene can be detected. Moreover, the expression of the reporter gene in primary T lymphocytes depends on the activation state of the cells. Remarkably, primary HIGM1 lymphocytes transduced with pCD40L-CD40L LV expressed CD40L only after T-cell stimulation. Therefore, the CD40L-promoter-driven vectors are able to achieve a near-physiological expression pattern that follows very closely that of the endogenous CD40L gene.
Cannabinoids act as pleiotropic compounds exerting, among others, a broad-spectrum of neuroprotective effects. These effects have been investigated in the last years in different preclinical models of neurodegeneration, with the cannabinoid type-1 (CB1) and type-2 (CB2) receptors concentrating an important part of this research. However, the issue has also been extended to additional targets that are also active for cannabinoids, such as the orphan G-protein receptor 55 (GPR55). In the present study, we investigated the neuroprotective potential of VCE-006.1, a chromenopyrazole derivative with biased orthosteric and positive allosteric modulator activity at GPR55, in murine models of two neurodegenerative diseases. First, we proved that VCE-006.1 alone could induce ERK1/2 activation and calcium mobilization, as well as increase cAMP response but only in the presence of lysophosphatidyl inositol. Next, we investigated this compound administered chronically in two neurotoxin-based models of Parkinson’s disease (PD), as well as in some cell-based models. VCE-006.1 was active in reversing the motor defects caused by 6-hydroxydopamine (6-OHDA) in the pole and the cylinder rearing tests, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity detected in the substantia nigra. Similar cytoprotective effects were found in vitro in SH-SY5Y cells exposed to 6-OHDA. We also investigated VCE-006.1 in LPS-lesioned mice with similar beneficial effects, except against glial reactivity and associated inflammatory events, which remained unaltered, a fact confirmed in BV2 cells treated with LPS and VCE-006.1. We also analyzed GPR55 in these in vivo models with no changes in its gene expression, although GPR55 was down-regulated in BV2 cells treated with LPS, which may explain the lack of efficacy of VCE-006.1 in such an assay. Furthermore, we investigated VCE-006.1 in two genetic models of amyotrophic lateral sclerosis (ALS), mutant SOD1, or TDP-43 transgenic mice. Neither the neurological decline nor the deteriorated rotarod performance were prevented with this compound, and the same happened with the elevated microglial and astroglial reactivities, albeit modest spinal motor neuron preservation was achieved in both models. We also analyzed GPR55 in these in vivo models and found no changes in both TDP-43 transgenic and mSOD1 mice. Therefore, our findings support the view that targeting the GPR55 may afford neuroprotection in experimental PD, but not in ALS, thus stressing the specificities for the development of cannabinoid-based therapies in the different neurodegenerative disorders.
Primary immunodeficiencies (PID) are caused by mutations in genes that impair the development or activity of the immune system. Although bone marrow transplants achieve long time restoration in up to 90% of treated patients, morbidity and mortality are still high for some PID and adequate donors are not always available. Gene Therapy (GT) was envisioned as an alternative treatment for PID by inserting the correct gene into the patient's haematopoietic stem cells (HSCs). Up to date, GT for PID has succeeded in 40 of 44 patients treated in four clinical trials. However, five children enrolled in the SCID-X1 clinical trial developed leukaemia-like disease produced by aberrant expression of oncogenes. This phenomenon resulted fatal in one patient and represented a severe setback for gene therapy. Since then, vector development has been a priority in the GT field, by refining existing Murine laeukemia virus (MLV)-based vectors or by developing new ones. This review summarize existing methodologies for PID GT highlighting the importance of animal models in the PID GT success and focusing on new gene transfer vectors to achieve safe, efficient and stable gene modification.
Oxidative stress and inflammation in the brain are two key hallmarks of neurodegenerative diseases (NDs) such as Alzheimer’s, Parkinson’s, Huntington’s and multiple sclerosis. The axis NRF2-BACH1 has anti-inflammatory and anti-oxidant properties that could be exploited pharmacologically to obtain neuroprotective effects. Activation of NRF2 or inhibition of BACH1 are, individually, promising therapeutic approaches for NDs. Compounds with dual activity as NRF2 activators and BACH1 inhibitors, could therefore potentially provide a more robust antioxidant and anti-inflammatory effects, with an overall better neuroprotective outcome. The phytocannabinoid cannabidiol (CBD) inhibits BACH1 but lacks significant NRF2 activating properties. Based on this scaffold, we have developed a novel CBD derivative that is highly effective at both inhibiting BACH1 and activating NRF2. This new CBD derivative provides neuroprotection in cell models of relevance to Huntington’s disease, setting the basis for further developments in vivo.
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