Development of antisense oligonucleotide combinatorial therapies for spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is a devastating genetically inherited neuromuscular disorder characterized by the progressive loss of α-motor neurons (MNs) in the anterior horn of the spinal cord, leading to muscle atrophy and weakness. Without treatment, SMA is the leading genetic cause of infant death. Although SMA is caused by homozygous mutations in survival motor neuron 1 (SMN1) gene, the disease severity is mainly determined by SMN2 copy number, an almost identical gene that produces ~10% correctly spliced full length SMN transcripts. Recently, three FDA- and EMA-approved therapies that either increase correctly spliced SMN2 transcripts (nusinersen - antisense oligonucleotide, ASO - and risdiplam - small molecule - ) or replace SMN1 gene (onasemnogen abeparvovec-xioi) have revolutionized the clinical outcome of SMA patients. However, clinical and scientific evidence emphasize the importance of a presymptomatic treatment in order achieve a significant therapeutic outcome. Moreover, there is a significant number of patients that do not respond to the SMN-enhancing treatments. Indeed, for severely affected SMA individuals carrying only two SMN2 copies even a presymptomatic therapy might be insufficient to fully counteract disease development. Therefore, SMN-independent compounds supporting SMN-dependent therapies represent a promising therapeutic approach. In this regard, genetic modifiers such as PLS3, NCALD or CHP1 have proved to act protective against SMA across species. Therefore, the aims of this work are the following: 1) To test a combinatorial therapy using SMN-ASO and Chp1-ASOs in SMA mice, 2) To develop an efficient MN differentiation protocol from healthy and SMA hiPSCs to use as a platform for compound screening (ASOs), 3) To test the long-term effect of the combinatorial therapy based on SMN-ASO and Ncald-ASO. In the first part of this work, we focused on the combinatorial treatment enhancing SMN protein levels and reducing CHP1 protein amount. CHP1, calcineurin-like EF-hand protein 1, is an interacting partner of PLS3, a strong modifier of SMA, and acts as a negative regulator of neurite outgrowth and endocytosis. Importantly, a significant amelioration of SMA disease hallmarks was observed in a severely affected SMA mouse model carrying a mutant Chp1 allele when combined with a suboptimal dose of SMN-ASO treatment. In this work, we aimed to pharmacologically reduce CHP1 levels in an ASO-based combinatorial therapy targeting SMN and Chp1 in SMA mice. Notably, Chp1 modulation represents a major challenge since its reduction to ~50% showed an amelioration of SMA pathology, while the downregulation below those levels is detrimental and prompts cerebellar ataxia characterized by Purkinje neuron loss. Hereby, efficacy and tolerability studies in neonatal wild type mice determined that a single injection of 30 µg Chp1-ASO4 in the CNS is a safe dosage that significantly reduced CHP1 levels to about 50% at postnatal day (PND)14 in the brain and the spinal cord. Unfortunately, neither electrophysiological predictors such as compound muscle action potential XVI | S U M M A R Y (CMAP) or motor unit number estimation (MUNE) nor morphological properties of the neuromuscular junctions (NMJ), the spinal cord or the tibialis anterior muscle were ameliorated in SMA mice treated with Chp1-ASO4 compared to CTRL-ASO at PND21. Unexpectedly, CHP1 levels were not reduced at 4- weeks post injection, indicating a rather short-term effect and stability of the ASO. Next, we re�administrated Chp1-ASO4 by i.c.v. bolus injection at PND28. However, no significant improvement of SMA hallmarks was observed at 2 month-of-age. Taken together, in contrast to the protective effect of the genetically-induced Chp1 reduction on SMA, combinatorial therapy based on Chp1- and SMN�ASOs failed to significantly ameliorate the SMA pathology in mice. The short-term stability of Chp1- ASOs compared to SMN-ASO suggests that further optimization of the ASO may be required to fully explore the combination of treatments. The second part of this work focused on the long-term combinatorial treatment targeting SMN upregulation and Ncald reduction. NCALD, neurocalcin delta, was identified as an SMA protective modifier in a SMA discordant family, where asymptomatic individuals exhibited a 4-5 fold NCALD reduction. NCALD is a calcium sensor protein that negatively regulates clathrin-mediated endocytosis and axonal outgrowth. NCALD reduction improves impaired endocytosis and ameliorates SMA pathology across species. Moreover, previous work carried out in our research group demonstrated that NCALD pharmacological reduction using Ncald-ASO in a low-dose SMN-ASO treated severe SMA mouse model, significantly improved electrophysiological and morphological pathology hallmarks at PND21. However, at 3 months of age SMA mice exhibited amelioration of motoric abilities solely. In the present work, we aimed to study the long-term effect of Ncald-ASO by re-injecting it via i.c.v. bolus administration at PND28. In parallel, we aimed to test the therapeutic effect of human NCALD-ASOs in MNs differentiated from patient-derived hiPSCs. First, 500µg Ncald-ASO administered via i.c.v. bolus injection at PND28 in wild type mice showed a good tolerability of the procedure and a significant reduction of NCALD in the brain and the spinal cord after two weeks. Importantly, Ncald-ASO re�injection prolonged the amelioration of electrophysiological parameters and rescued denervation defects. Moreover, human NCALD-ASO69 significantly downregulated NCALD levels in hiPSC derived MNs from healthy and SMA type I individuals. In addition, growth cone morphology and spontaneous neuronal activity were improved upon NCALD-ASO69 treatment. These data strongly supports the therapeutic role of NCALD in SMA and the importance of maintaining its reduction in order to achieve better clinical outcome

Marxist Nation Building in the Democratic Republic of Vietnam

With the surrender of the Japanese in 1945, Ho Chi Minh and his compatriots faced the problem of building an industrialized Marxist nation from an extremely localistic peasant society

Geometries of third-row transition-metal complexes from density-functional theory

A set of 41 metal-ligand bond distances in 25 third-row transition-metal complexes, for which precise structural data are known in the gas phase, is used to assess optimized and zero-point averaged geometries obtained from DFT computations with various exchange-correlation functionals and basis sets. For a given functional (except LSDA) Stuttgart-type quasi-relativistic effective core potentials and an all-electron scalar relativistic approach (ZORA) tend to produce very similar geometries. In contrast to the lighter congeners, LSDA affords reasonably accurate geometries of 5d-metal complexes, as it is among the functionals with the lowest mean and standard deviations from experiment. For this set the ranking of some other popular density functionals, ordered according to decreasing standard deviation, is BLYP > VSXC > BP86 approximate to BPW91 approximate to TPSS approximate to B3LYP approximate to PBE > TPSSh > B3PW91 approximate to B3P86 approximate to PBE hybrid. In this case hybrid functionals are superior to their nonhybrid variants. In addition, we have reinvestigated the previous test sets for 3d- (Buhl M.; Kabrede, H. J. Chem. Theory Comput. 2006, 2, 1282-1290) and 4d- (Waller, M. P.; Buhl, M. J. Comput. Chem. 2007,28,1531-1537) transition-metal complexes using all-electron scalar relativistic DFT calculations in addition to the published nonrelativistic and ECP results. For this combined test set comprising first-, second-, and third-row metal complexes, B3P86 and PBE hybrid are indicated to perform best. A remarkably consistent standard deviation of around 2 pm in metal-ligand bond distances is achieved over the entire set of d-block elements.PostprintPeer reviewe

Design of a highly active Pd catalyst with P,N hemilabile ligands for alkoxycarbonylation of alkynes and allenes : a density functional theory study

Authors thanks EaStCHEM and the School of Chemistry for support.In palladium-catalysed methoxycarbonylation of technical propyne, the presence of propadiene poisons the hemilabile Pd(P,N) catalyst. According to density functional theory calculations (B3PW91-D3/PCM level), a highly stable π-allyl intermediate is the reason for this catalyst poisoning. Predicted regioselectivities suggest that at least 11% of propadiene should yield this allyl intermediate, where the reaction gets stalled under the turnover conditions due to an insurmountable methanolysis barrier of 25.8 kcal mol-1. Results obtained for different ligands and substrates are consistent with the available experimental data. A new ligand, (6-Cl-3-Me-Py)PPh2, is proposed, which is predicted to efficiently control the branched/linear selectivity, avoiding rapid poisoning (with only 0.2% of propadiene being trapped as Pd allyl complex), and to tremendously increase the catalytic activity by decreasing the overall barrier to 9.1 kcal mol-1.PostprintPeer reviewe

Computational modelling of Pd-catalysed alkoxycarbonylation of alkenes and alkynes

The work was supported by the School of Chemistry and EaStCHEM.This perspective highlights the computational modelling of alkene and alkyne alkoxycarbonylation at palladium catalysts. We cover studies on Pd-catalysed alkoxycarbonylation of alkenes with bidentate diphosphine ligands, which reveal a hydride pathway is operating with an intermolecular alcoholysis step, where explicit solvation is mandatory to estimate the overall barriers correctly and model alcoholysis/copolymerisation selectivities. Subsequently, we discuss Pd-catalysed alkyne alkoxycarbonylation with P,N-chelating ligands, where an in situ base mechanism is operating involving ketene-type intermediates. We also discuss catalyst poisoning due to allene and designing a potential new catalyst tolerant towards allene poisoning.Publisher PDFPeer reviewe

Hydricity Of 3d transition metal complexes from density functional theory : a benchmarking study

A range of modern density functional theory (DFT) functionals have been benchmarked against experimentally determined metal hydride bond strengths for three first-row TM hydride complexes. Geometries were found to be produced sufficiently accurately with RI-BP86-D3(PCM)/def2-SVP and further single-point calculations with PBE0-D3(PCM)/def2-TZVP were found to reproduce the experimental hydricity accurately, with a mean absolute deviation of 1.4 kcal/mol for the complexes studied.Publisher PDFPeer reviewe

Laccase redox potentials: pH dependence and mutants, a QM/MM study

The authors are grateful for funding by the Engineering and Physical Sciences Research Council, grant “Clean catalysis for sustainable development” (Ref. EP/J018139/1).We have studied the T. versicolor laccase T1 site redox potential (RP) at the M06/6-311++G**/SDD(Cu) level of theory, employing QM/MM optimised geometries (RI-BP86/def2-SVP/def2-TZVP(Cu):CHARMM) of the whole protein system with electronic embedding. The oxidation state of the trinuclear cluster was found to affect the T1 site RP by about 0.2-0.3 V, depending on the protein protonation state. The computed laccase RP can be drastically lowered upon introduction of a protonation state corresponding to a neutral environment, by up to -1.37 V, which is likely an overestimation of the effect in vivo. The gradual change of the protonation state by single points without optimisation or equilibration results in a change that is even larger, namely up to about -2.6 V. Thus, the preferred protein conformation supports a high redox potential, compensating for the RP-lowering effect of surface charges. The predicted change in RP on going to the F463M mutant, ca. -0.1 V, is consistent with observations for a related laccase. Based on our results, we also propose and test a D206N mutant, but find it to be locked in a conformation with slightly lower RP.PostprintPeer reviewe

Second-Order Latent Growth Models with Shifting Indicators

Second-order latent growth models assess longitudinal change in a latent construct, typically employing identical manifest variables as indicators across time. However, the same indicators may be unavailable and/or inappropriate for all time points. This article details methods for second-order growth models in which constructs’ indicators shift over time

[UO2(NH3)5]Br2 ∙ NH3 : synthesis, crystal structure, and speciation in liquid ammonia solution by first-principles molecular dynamics simulations

Pentaammine dioxido uranium(VI) dibromide ammonia (1/1), [UO2(NH3)5]Br2·NH3, was synthesized in the form of yellow crystals by the reaction of uranyl bromide, UO2Br2, with dry liquid ammonia. The compound crystallizes orthorhombic in space group Cmcm and is isotypic to [UO2(NH3)5]Cl2·NH3 with a = 13.2499(2), b = 10.5536(1), c = 8.9126(1) Å, V = 1246.29(3) Å3 and Z = 4 at 123 K. The UO22+ cation is coordinated by five ammine ligands and the coordination polyhedron can be best described as pentagonal bipyramid. Car–Parrinello molecular dynamics simulations are reported for [UO2(NH3)5]2+ in the gas phase and in liquid NH3 solution (using the BLYP density functional). According to free-energy simulations, solvation by ammonia has only a small effect on the uranyl–NH3 bond strength.PostprintPeer reviewe