Targets in Gene Therapy

This book aims at providing an up-to-date report to cover key aspects of existing problems in the emerging field of targets in gene therapy. With the contributions in various disciplines of gene therapy, the book brings together major approaches: Target Strategy in Gene Therapy, Gene Therapy of Cancer and Gene Therapy of Other Diseases. This source enables clinicians and researchers to select and effectively utilize new translational approaches in gene therapy and analyze the developments in target strategy in gene therapy

In silico site-directed mutagenesis of Acinetobacter Haemolyticus Lipase KV1 for improved alkaline stability

The interest on alkaline-stable lipases by the scientific community is increasing due to its great potential use. As most industrial processes are performed under highly basic conditions, alkaline-stable lipases become hugely valued biocatalysts. In this study, three aspartic acid residues at positions 51, 122 and 247 in the outer loop of LipKV1 from Acinetobacter haemolyticus was computationally mutated into lysine using the SWISS-MODEL program, followed by energy minimization of the protein models. PROCHECK, ERRAT and Verify3D refined models of LipKV1 and Mut-LipKV1 indicated that the Mut-LipKV1 protein conformation is in a good condition. The study found that the overall electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0 −10.0). Molecular dynamics (MD) simulation of Lip-KV1 and Mut-LipKV1 protein models under different alkaline pHs using GROMACS version 2018.6 revealed that Mut-LipKV1 was more stable at the high pH 9.0 (RMSD ~0.3 nm, RMSF ~0.05 – 0.2 nm), compared the optimal pH 8.0 of LipKV1 (RMSD 0.3 nm, RMSF 0.05 – 0.20 nm). Molecular docking using AutoDock Vina with tributyrin as the substrate identified detailed changes that occurred post mutation. The highest binding affinity (−4.1 kcal/mol) with Mut-LipKV1 which occurred at pH 9.0 was from a single hydrogen bond with His289. MD simulations showed that configurations which formed between Mut-LipKV1-tributyrin (RMSD 0.3 nm; RMSF 0.05 − 0.3 nm) and the LipKV1-tributyrin complexes (RMSD 0.35 nm; RMSF 0.05 − 0.4 nm) were comparably stable at pH 8.0. Furthermore, MM-PBSA calculation validated that the Mut-LipKV1-tributyrin complex at pH 8.0 (-44.01 kcal/mol) showed comparable binding free energy to LipKV1-tributyrin complex (−43.83 kcal/mol). Whereas the lowest binding free energy for Mut-LipKV1-tributyrin complex was simulated at pH 12.0 (−44.04 kcal/mol). Thus, adaptive strategy of replacing the outer loop surface aspartic acid to lysine in LipKV1 successfully broadened pH stability of Mut-LipKV1 towards higher pH, raising it from pH 8.0 − 11.0 to pH 8.0 − 12.0 in the mutant lipase. In a nutshell, this research offered a considerable insight for further improving the alkaline tolerance of lipases

Clinical and genetic characterisation of hereditary motor neuropathies

PhD ThesisInherited peripheral neuropathies or Charcot-Marie-Tooth disease (CMT) are common neuromuscular conditions, characterised by distal motor atrophy and weakness with variable range of sensory impairment and classified according to demyelinating (CMT1) or axonal (CMT2) pathology. The number of genes causing CMT has rapidly increased due to improved genetic testing technology, even though gene identification has remained challenging in some subgroups of CMT. Hereditary motor neuropathies (HMN) encompass heterogeneous groups of disorders caused by motor axon and neuron pathology. The distal hereditary motor neuropathies (dHMN) are rare length-dependent conditions, which show significant clinical and genetic overlap with motor neuron diseases. Several (>30) causative genes have been identified for ~20% of dHMN patients, which predicts extreme genetic heterogeneity in this group. My study was designed to investigate the prevalence, clinical presentation, molecular cause and phenotype-genotype correlations of hereditary motor neuropathies in a large cohort of patients. I aimed to identify novel disease genes and reassessed mutation detection rate in dHMN. Furthermore, I studied common pathomechanisms and targets for therapy approaches in hereditary motor neuropathies. Detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in 105 patients with clinical symptoms of distal hereditary motor neuropathy (dHMN, 64 patients), axonal motor neuropathy (motor CMT2, 16 patients) or complex neurological disease predominantly affecting the motor nerves (dHMN plus, 25 patients). I calculated the dHMN prevalence 2.14 affected individuals per 100.000 inhabitants (95% CI: 1.62-2.66) in the North of England. Causative mutations were identified in overall 47.9% in the motor neuropathy patient cohort. In the dHMN group the diagnostic rate was 42.5%, significantly higher than the previously reported 20%. The significant increase in the mutation detection rate could be attributed to the development of next generation techniques. Many of the genes were shared between dHMN and motor CMT2, indicating identical disease mechanisms. I examined the phenotypic variability and the correlations with the identified genetic background. We described the novel phenotype of non-progressive motor neuropathy with fatigable weakness due to presynaptic neuromuscular transmission defect caused by synaptotagmin 2 mutations. I indentified further novel genes involved in intracellular signal transduction and ii transcriptional regulatory cascades, which might indicate common pathways and highlight further targets in the therapy of motor neuropathies. We detected a potentially treatable defect of neuromuscular transmission in some genetic forms, which raise the possibility that neuromuscular junction defects can cause or accompany motor neuropathy. The preliminary results suggested the potential treatability of the neuromuscular transmission defect, although long term effects will still need to be evaluated. In summary, detailed clinical characterisation and segregation analysis improved the detection rate in our cohort and highlighted that clinical expertise are still essential in confirming the diagnosis of inherited motor neuropathies. Increasing knowledge on disease pathways will not only help to identify new genes with shared pathomechanisms but will provide a basis for novel therapy approaches.Medical Research Council Centre for Translational Research in Neuromuscular Diseases for funding my PhD research

In silico analysis of a putative dehalogenase from the genome of halophilic bacterium Halomonas smyrnensis AAD6T

Microbial-assisted removal of natural or synthetic pollutants is the prevailing green, low-cost technology to treat polluted environments. However, the challenge with enzyme-assisted bioremediation is the laborious nature of dehalogenase-producing microorganisms’ bioprospecting. This bottleneck could be circumvented by in-silico analysis of certain microorganisms’ whole-genome sequences to predict their protein functions and enzyme versatility for improved biotechnological applications. Herein, this study performed structural analysis on a dehalogenase (DehHsAAD6) from the genome of Halomonas smyrnensis AAD6 by molecular docking and molecular dynamic (MD) simulations. Other bioinformatics tools were also employed to identify substrate preference (haloacids and haloacetates) of the DehHsAAD6. The DehHsAAD6 preferentially degraded haloacids and haloacetates ( 3.2–4.8 kcal/ mol) and which formed three hydrogen bonds with Tyr12, Lys46, and Asp182. MD simulations data revealed the higher stability of DehHsAAD6-haloacid- (RMSD 0.22–0.3 nm) and DehHsAAD6-haloacetates (RMSF 0.05–0.14nm) complexes, with the DehHsAAD6-L-2CP complex being the most stable. The detail of molecular docking calculations ranked complexes with the lowest binding free energies as: DehHsAAD6-L-2CP complex ( 4.8 kcal/mol) ¼ DehHsAAD6-MCA ( 4.8 kcal/mol) < DehHsAAD6-TCA ( 4.5 kcal/mol) < DehHsAAD6-2,3-DCP ( 4.1 kcal/mol) < DehHsAAD6-D-2CP ( 3.9 kcal/mol) < DehHsAAD6-2,2-DCP ( 3.5 kcal/mol) < DehHsAAD6-3CP ( 3.2 kcal/mol). In a nutshell, the study findings offer valuable perceptions into the elucidation of possible reaction mechanisms of dehalogenases for extended substrate specificity and higher catalytic activity

Understanding malignant hyperthermia : bioinformatic approaches to identify pathogenic genetic variants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

Technological advances and decreasing costs in genome sequencing have greatly sped up the rate of identification of the genetic causes of inherited disease. One such human disorder in which genome sequencing is being applied in hope that the genetic causes will be identified is malignant hyperthermia (MH). MH is an autosomal dominant pharmacogenetic disorder which has long perplexed researchers due to its phenotypic and genetic complexity. Individuals susceptible to MH are at risk of a sometimes deadly hypermetabolic episode of skeletal muscle triggered by potent volatile general anaesthetic agents. Although the genetic origin of the disorder has been determined in over half of all MH families, there are many families for which the origin has not yet been elucidated. This research aimed to identify genetic variants that may be pathogenic for MH-susceptibility in six New Zealand families for which the genetic cause has not yet been identified. Targeted next-generation sequencing of the genome was undertaken on a number of individuals from each family. Bioinformatic approaches were developed and applied to identify candidate genetic variants. Segregation analysis was carried out for some of the identified candidate variants, which failed to establish an association with MH-susceptibility, although a number of variants were ruled out as being pathogenic for MH-susceptibility. Additionally, a common polymorphism that has been previously postulated to have a modifying effect on MH-susceptibility was identified within a large MH family. A genotype/phenotype association study was carried, however the study did not find an association. Overall, this work has reinforced that MH does not have a simple, easily identifiable genetic origin, suggesting there is one or more missing elements to the current understanding of MH. Importantly, it has established an effective MH-specific bioinformatic protocol that can be applied to other sequencing data in the future

Functional characterization of the Mediator subunit MED25

In this study a structure–function analysis has been employed to analyze transcriptional regulation through the Mediator subunit MED25. A relationship could be established between predicted structural domains and functional characteristics of this protein. Most critically the region responsible for interaction of MED25 with the Mediator was identified. Immunoprecipitation experiments demonstrated that the so–called VWA domain (von–Willebrand A domain, amino acids 1–290) is both sufficient and required for this contact. Site–directed mutagenesis indicates that this binding reaction involves the non–conserved loop SR2, which is protruding from this domain. Based on the results of this analysis a model was proposed, in which the primary contact is established by ionic forces and is further stabilized by hydrophobic interactions. The previously identified ACID domain was reported to bind to VP16. Targeted mutagenesis of four different motifs in this region impaired not only transcriptional activation through MED25 but also led to reduced binding to VP16. In particluar a lysine–rich motif is also present in two domains of PTOV1, a close homolog of MED25. Noteworthy, K518 is not conserved in the PTOV1_B domain, which in contrast to PTOV1_A and the ACID domain of MED25 does not bind to VP16. This led to the hypothesis that K518 is critically involved in the binding of VP16 to MED25. Furthermore it could be demonstrated that MED25 contains an intrinsic transcriptional activation capacity, which is localized in the region 290–715. This indicates additional recruitment of other factors to promoters through this region. Together with the Mediator binding VWA–domain and the VP16–interaction domain this region might facilitate transcriptional activation. A genome–wide screen showed downregulation of c–Jun and FosB following overexpression of MED25. Interestingly, expression of GSK3β, a downstream target of which is cyclin D1, seems to be stimulated by MED25. Together with the finding that overexpression of MED25 leads to activation of a p21 reporter, this raises the possibility that MED25 is involved in cell cycle control. An overlap has been discovered by comparison of MED25 target genes and genes identified previously as target for the viral activator EBNA2. The close homology between the activation domains of EBNA2 and VP16 implies a common mechanism of transcriptional activation by these two viral proteins through MED25. The involvement of MED25 in gene activation by viral activators might indicate a role for this Mediator subunit in viral transcription

Genetics of atypical haemolytic uraemic syndrome

PhD ThesisAtypical haemolytic uraemic syndrome (aHUS) is a life threatening renal disease, caused by deregulation of the alternative complement pathway. Several genes within this pathway are associated with aHUS. At the outset of this project, the genetic cause had been identified in 45% of familial cases in the Newcastle aHUS cohort. The aim of this project was to identify the genetic cause of disease in the remaining 55%. Complement Factor H (CFH) and Complement Factor H-related (CFHRs) are found on chromosome 1. This area contains several low copy repeats, the result of genomic duplications that occurred early in evolution. This causes genomic instability, which can lead to gene conversions or rearrangements. Sanger sequencing will not always detect these abnormalities, therefore patients were also screened using multiplex ligation-dependent probe amplification and western blotting. A novel hybrid CFH/CFHR3 gene was described, which arose by microhomology-mediated end joining. Functional analysis demonstrated that it was defective at regulating complement at the cell surface, which was predicted to predispose this patient to disease. Review of all patients in the Newcastle aHUS cohort with CFH abnormalities, identified a third of patients had a genomic rearrangement between CFH and CFHRs. The relative frequency of genomic rearrangements emphasised the importance of undertaking copy number analysis in aHUS diagnostic testing, because often they are not detected by Sanger sequencing. Whole exome sequencing was then undertaken in Newcastle familial cohort with an unknown genetic aetiology. Pathogenic sequence variants were identified in genes, known to be associated with thrombotic microangiopathies. Sequence variants that were predicted to be pathogenic, were found in three genes not previously associated with disease. Two of these genes were located outside of the complement system, indicating that complement-directed therapies may be contraindicated. In this project, a genetic cause of disease was found in 54% of familial cases tested