Characterisation Of Adeno-Associated Virus DNA contaminants and Development of a P5 promoter replacement, high purity Adeno-Associated Virus production system

Gene therapy is the transfer of nucleic acids for therapeutic benefit, a process potentially achieved through several delivery methods. One of the most common gene therapy delivery modalities is adeno associated virus (AAV). The availability of a system to produce large quantities of AAV, and of diverse AAV serotypes, that can infect different cell types makes AAV very attractive for widespread clinical implementation. Viruses like AAV that are manipulated to become highly useful delivery tools rely on complex biological systems for production. It is imperative that these systems are well characterised and designed to be highly safe and efficacious. This PhD thesis investigated nucleic acid contamination of AAV preparations, looking at the production system as a source, and the problems that this may pose for AAV infected cells. Specific regions of DNA from AAV producer plasmids were incorporated into virions more abundantly. These were found adjacent to the outside of the inverted terminal repeats (ITRs) that flank the expression cassette, and on the plasmid containing the replication and capsid genes, directly upstream of the P5 promoter which drives expression of the replication proteins, REP78 and REP68. Contaminant sequences were found to be transferred into transduced cells during AAV infection, persistent within cells and transcriptionally active. Investigation of P5 upstream contaminants showed potential for protein to be produced persistently from contaminant sequences both in vitro and in vivo. Finally, precise sequence regions causing DNA incorporation upstream of the P5 promoter were examined. This information was used to produce alternative production 3 reagents that, in their first iteration, provided proof of principle of P5-related contaminant removal from the final AAV product, and in additional iterations resulted in a modified P5 promoter (P5-HS). This promoter limited upstream contamination thereby resulting in higher purity AAV, whilst retaining vector yields equivalent to the original AAV production system

Exploring Anti-FVIII Antibodies in Haemophilia A - Role in In Vitro Haemostasis and Clinical Disease

Haemophilia A (HA) is caused by defective synthesis of coagulation factor VIII(FVIII), which has serious effects on haemostasis; joints being the most common site of bleeding. The development of FVIII replacements has improved the situation for patients with haemophilia such that chronic arthropathy can be prevented, and life expectancy and the quality of life have increased. However, approximately 20-30% of patients suffering from severe HA develop neutralizing antibodies (inhibitors) against FVIII. Alternative treatment, using by-passing agents, is available for patients exhibiting inhibitors, although these can only be used for the short-term treatment of acute haemorrhage and as prophylaxis during surgery. Furthermore, the clinical response to by-passing products is unpredictable. Two of the studies included in this thesis evaluated the response to by-passing therapy in plasma from patients with HA. The variation in thrombin production within families was found to be significantly lower than the variation between families, indicating that a familial predisposition may influence thrombin formation in response to by-passing agents (Paper I). Moreover, FVIII clotting factors were found to potentiate the in vitro effect of by-passing agents on thrombin formation in plasma from patients with HA exhibiting inhibitors, indicating that further assessment of this treatment strategy in a clinical context is warranted (Paper II). Not all anti-FVIII antibodies have neutralizing capacity. In the studies presented in Papers III & V, non-neutralizing anti-FVIII antibodies(NNAs) were investigated in two different cohorts, using an enzyme-linked immunosorbent assay (ELISA). NNAs were detected in 18.9% of siblings with HA, and in 12.8% of unrelated HA subjects followed for four years. The antibody response was assayed using three different rFVIII products. The antibody response was found to be heterogeneous, to vary considerably between individuals (Papers III and V), and also over time (Paper V). None of the patients in the cohort with NNAs observed longitudinally developed inhibitors (Paper V). However, in one patient with moderate HA, the detection of Bethesda-negative anti-FVIII antibodies coincided with a change in bleeding phenotype four years prior to FVIII inhibitor development. This finding suggests that immunoassays may be a useful complement in evaluating the immune response to FVIII (Paper IV). The potential clinical impact of NNAs was evaluated in the long term study (Paper V), showing no association between age, F8 mutation, or the influence of immune system challenges on NNA development. Interestingly, patients with NNAs had significantly fewer bleeding episodes than NNA-negative patients (p=0.048), raising questions about the possibility of yet undefined types of anti-FVIII antibodies with protective or potentiating effects on FVIII

Epidemiology of hepatitis C virus in Iran

In Iran, the prevalence of hepatitis C virus (HCV) infection is relatively low according to the populationb a s e d e p i d e m i o l o g i c a l s t u d i e s . H o w e ve r, t h e epidemiology of HCV is changing and the rate of HCV infection is increasing due to the growth in the number of injecting drug users in the society. In addition, a shift has occurred in the distribution pattern of HCV genotypes among HCV-infected patients in Iran. Genotype 1a is the most prevalent genotype in Iran, but in recent years, an increase in the frequency of 3a and a decrease in 1a and 1b have been reported. These variations in the epidemiology of HCV reflect differences in the routes of transmission, status of public health, lifestyles, and risk factors in different groups and geographic regions of Iran. Health policy makers should consider these differences to establish better strategies for control and prevention of HCV infection. Therefore, this review was conducted to present a clear view regarding the current epidemiology of HCV infection in Iran. Key words: Hepatitis C virus; Blood donors; Injecting drug users; Hemodialysis; Hemophilia; Thalassemia; Genotypes; Occult hepatitis C virus; Epidemiology; Ira

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

Epidemiology of hepatitis C virus in Iran

In Iran, the prevalence of hepatitis C virus (HCV) infection is relatively low according to the populationb a s e d e p i d e m i o l o g i c a l s t u d i e s . H o w e ve r, t h e epidemiology of HCV is changing and the rate of HCV infection is increasing due to the growth in the number of injecting drug users in the society. In addition, a shift has occurred in the distribution pattern of HCV genotypes among HCV-infected patients in Iran. Genotype 1a is the most prevalent genotype in Iran, but in recent years, an increase in the frequency of 3a and a decrease in 1a and 1b have been reported. These variations in the epidemiology of HCV reflect differences in the routes of transmission, status of public health, lifestyles, and risk factors in different groups and geographic regions of Iran. Health policy makers should consider these differences to establish better strategies for control and prevention of HCV infection. Therefore, this review was conducted to present a clear view regarding the current epidemiology of HCV infection in Iran. Key words: Hepatitis C virus; Blood donors; Injecting drug users; Hemodialysis; Hemophilia; Thalassemia; Genotypes; Occult hepatitis C virus; Epidemiology; Ira

Drug development progress in duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease