Clare E. Thomas

Bio: Clare E. Thomas is an academic researcher from University of Manchester. The author has contributed to research in topics: Immune system & Transgene. The author has an hindex of 12, co-authored 18 publications receiving 3749 citations. Previous affiliations of Clare E. Thomas include Stanford University & University of California, Los Angeles.

Progress and problems with the use of viral vectors for gene therapy

Abstract: Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors.

Abstract: Transduction of the liver with single-stranded adeno-associated virus serotype 2 (AAV2) vectors is inefficient; less than 10% of hepatocytes are permissive for stable transduction, and transgene expression is characterized by a lag phase of up to 6 weeks. AAV2-based vector genomes packaged inside AAV6 or AAV8 capsids can transduce the liver with higher efficiency, but the molecular mechanisms underlying this phenomenon have not been determined. We now show that the primary barrier to transduction of the liver with vectors based on AAV2 capsids is uncoating of vector genomes in the nucleus. The majority of AAV2 genomes persist as encapsidated single-stranded molecules within the nucleus for as long as 6 weeks after vector administration. Double-stranded vector genomes packaged inside AAV2 capsids are at least 50-fold more active than single-stranded counterparts, but these vectors also exhibit a lag phase before maximal gene expression. Vector genomes packaged inside AAV6 or AAV8 capsids do not persist as encapsidated molecules and are more biologically active than vector genomes packaged inside AAV2 capsids. Our data suggest that the rate of uncoating of vector genomes determines the ability of complementary plus and minus single-stranded genomes to anneal together and convert to stable, biologically active double-stranded molecular forms.

Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases.

Abstract: Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8+ cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders.

Astrocytes: biology and pathology

Abstract: Astrocytes are specialized glial cells that outnumber neurons by over fivefold. They contiguously tile the entire central nervous system (CNS) and exert many essential complex functions in the healthy CNS. Astrocytes respond to all forms of CNS insults through a process referred to as reactive astrogliosis, which has become a pathological hallmark of CNS structural lesions. Substantial progress has been made recently in determining functions and mechanisms of reactive astrogliosis and in identifying roles of astrocytes in CNS disorders and pathologies. A vast molecular arsenal at the disposal of reactive astrocytes is being defined. Transgenic mouse models are dissecting specific aspects of reactive astrocytosis and glial scar formation in vivo. Astrocyte involvement in specific clinicopathological entities is being defined. It is now clear that reactive astrogliosis is not a simple all-or-none phenomenon but is a finely gradated continuum of changes that occur in context-dependent manners regulated by specific signaling events. These changes range from reversible alterations in gene expression and cell hypertrophy with preservation of cellular domains and tissue structure, to long-lasting scar formation with rearrangement of tissue structure. Increasing evidence points towards the potential of reactive astrogliosis to play either primary or contributing roles in CNS disorders via loss of normal astrocyte functions or gain of abnormal effects. This article reviews (1) astrocyte functions in healthy CNS, (2) mechanisms and functions of reactive astrogliosis and glial scar formation, and (3) ways in which reactive astrocytes may cause or contribute to specific CNS disorders and lesions.

Non-viral vectors for gene-based therapy

Abstract: Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches

Progress and problems with the use of viral vectors for gene therapy

Abstract: Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

Abstract: We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.

MicroRNA: Biogenesis, Function and Role in Cancer.

Abstract: MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. The conventional biogenesis pathway consists of two cleavage events, one nuclear and one cytoplasmic. However, alternative biogenesis pathways exist that differ in the number of cleavage events and enzymes responsible. How microRNA precursors are sorted to the different pathways is unclear but appears to be determined by the site of origin of the microRNA, its sequence and thermodynamic stability. The regulatory functions of microRNAs are accomplished through the RNA-induced silencing complex (RISC). MicroRNA assembles into RISC, activating the complex to target messenger RNA (mRNA) specified by the microRNA. Various RISC assembly models have been proposed and research continues to explore the mechanism(s) of RISC loading and activation. The degree and nature of the complementarity between the microRNA and target determine the gene silencing mechanism, slicer-dependent mRNA degradation or slicer-independent translation inhibition. Recent evidence indicates that P-bodies are essential for microRNA-mediated gene silencing and that RISC assembly and silencing occurs primarily within P-bodies. The P-body model outlines microRNA sorting and shuttling between specialized P-body compartments that house enzymes required for slicer –dependent and –independent silencing, addressing the reversibility of these silencing mechanisms. Detailed knowledge of the microRNA pathways is essential for understanding their physiological role and the implications associated with dysfunction and dysregulation.