Optimized pharmacological control over the AAV-Gene-Switch vector for regulable gene therapy.

Gene therapy in its current design is an irreversible process. It cannot be stopped in case of unwanted side effects, nor can expression levels of therapeutics be adjusted to individual patient's needs. Thus, the Gene-Switch (GS) system for pharmacologically regulable neurotrophic factor expression was established for treatment of parkinsonian patients. Mifepristone, the synthetic steroid used to control transgene expression of the GS vector, is an approved clinical drug. However, pharmacokinetics and -dynamics of mifepristone vary considerably between different experimental animal species and depend on age and gender. In humans, but not in any other species, mifepristone binds to a high-affinity plasma carrier protein. We now demonstrate that the formulation of mifepristone can have robust impact on its ability to activate the GS system. Furthermore, we show that a pharmacological booster, ritonavir (Rtv), robustly enhances the pharmacological effect of mifepristone, and allows it to overcome gender- and species-specific pharmacokinetic and -dynamic issues. Most importantly, we demonstrate that the GS vector can be efficiently controlled by mifepristone in the presence of its human plasma carrier protein, α1-acid glycoprotein, in a "humanized" rat model. Thus, we have substantially improved the applicability of the GS vector toward therapeutic use in patients

Developing therapeutically more efficient Neurturin variants for treatment of Parkinson's disease

In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

Comparative Analysis of the Effects of Neurotrophic Factors CDNF and GDNF in a Nonhuman Primate Model of Parkinson's Disease

Cerebral dopamine neurotrophic factor (CDNF) belongs to a newly discovered family of evolutionarily conserved neurotrophic factors. We demonstrate for the first time a therapeutic effect of CDNF in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of Parkinson's disease in marmoset monkeys. Furthermore, we tested the impact of high chronic doses of human recombinant CDNF on unlesionedmonkeys and analyzed the amino acid sequence ofmarmoset CDNF. The severity of 6-OHDA lesions and treatment effects weremonitored in vivo using 123I-FP-CIT (DaTSCAN) SPECT. Quantitative analysis of 123I-FP-CIT SPECT showed a significant increase of dopamine transporter binding activity in lesioned animals treated with CDNF. Glial cell line-derived neurotrophic factor (GDNF), a well-characterized and potent neurotrophic factor for dopamine neurons, served as a control in a parallel comparison with CDNF. By contrast with CDNF, only single animals responded to the treatment with GDNF, but no statistical difference was observed in the GDNF group. However, increased numbers of tyrosine hydroxylase immunoreactive neurons, observed within the lesioned caudate nucleus of GDNF-treated animals, indicate a strong bioactive potential of GDNF.Peer reviewe

Excretion Rate and Distribution Volumes in Common Marmoset Monkeys After Slow and Fast Injection of Gadobutrol

Common marmoset monkeys (Callithrix jacchus) are increasingly used in models of neurodegenerative diseases. Serial measurements of gadolinium excretion were performed using the dual flip angle method to map R1 on three animals in the wrist coil of a 3T whole-body system. R1 values were analyzed in a ROI in the straight sinus and fitted with a single compartment model. Slow injection of 0.3 mmol Gadovist per kg bodyweight (2 min duration) resulted in faster equilibration and excretion, as well as smaller distribution volumes than fast injection (15 sec). Slow injection and an equilibration delay of 15 minutes are recommended

Pharmacokinetics of the MRI contrast agent gadobutrol in common marmoset monkeys (Callithrix jacchus)

Background: This study determined the pharmacokinetics of the contrast agent gadobutrol in marmosets by quantitative MRI to derive guidelines for neuroimaging protocols. Methods: Local concentrations of gadobutrol were determined from consecutive gradient echo-based mapping of the relaxation rate R1 on a clinical 3T MRI scanner. Half-time of renal elimination was measured after injection of a triple dose of gadobutrol (0.3 mmol/kg) into the saphenous vein. A first-order single-compartment model was fitted to the measured R1 values and verified by blood analysis. Results: Slow injection (1.5 minutes) resulted in an elimination half-time of 26±4 minutes. After bolus injection (15 seconds), elimination was much slower (62±8 minutes) with 45% larger distribution volumes. Importantly, more gadobutrol entered the cerebrospinal fluid. Conclusions: Slow injection and a latency of about 20 minutes are recommended to avoid extravasation. Application of a triple dose of gadobutrol compensates for the fast elimination in healthy marmosets

Oligodendroglia in cortical multiple sclerosis lesions decrease with disease progression, but regenerate after repeated experimental demyelination

Cerebral cortex shows a high endogenous propensity for remyelination. Yet, widespread subpial cortical demyelination (SCD) is a common feature in progressive multiple sclerosis (MS) and can already be found in early MS. In the present study, we compared oligodendroglial loss in SCD in early and chronic MS. Furthermore, we addressed in an experimental model whether repeated episodes of inflammatory SCD could alter oligodendroglial repopulation and subsequently lead to persistently demyelinated cortical lesions. NogoA(+) mature oligodendrocytes and Olig2(+) oligodendrocyte precursor cells were examined in SCD in patients with early and chronic MS, normal-appearing MS cortex, and control cortex as well as in the rat model of repeated targeted cortical experimental autoimmune encephalomyelitis (EAE). NogoA(+) and Olig2(+) cells were significantly reduced in SCD in patients with chronic, but not early MS. Repeated induction of SCD in rats resulted only in a transient loss of NogoA(+), but not Olig2(+) cells during the demyelination phase. This phase was followed by complete oligodendroglial repopulation and remyelination, even after four episodes of demyelination. Our data indicate efficient oligodendroglial repopulation in subpial cortical lesions in rats after repeated SCD that was similar to early, but not chronic MS cases. Accordingly, four cycles of experimental de- and remyelination were not sufficient to induce sustained remyelination failure as found in chronic cortical MS lesions. This suggests that alternative mechanisms contribute to oligodendrocyte depletion in chronic cortical demyelination in MS

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.status: publishe