Refinement of lentiviral vector for improved RNA processing and reduced rates of self inactivation repair

Background Lentiviral gene therapy vectors are now finding clinical application. In order to fully exploit their potential it is important that vectors are made as efficient and as safe as possible. Accordingly, we have modified a previously reported vector to improve RNA processing, minimise Human Immunodeficiency Virus Type-1 (HIV-1) sequence content and reduce repair of the self inactivating (SIN) deletion. Results HIV-1 sequence in the vector was reduced by substituting the polyadenylation signal with a heterologous signal. Mutation of splice donor sites was undertaken to prevent the majority of splicing within the vector genomic RNA. In addition, a number of other sequences within the vector were deleted. The combination of these modifications was able to significantly reduce the rates of both vector mobilisation and repair of the self inactivating deletion after two rounds of marker rescue. Conclusion RNA processing can be improved by mutation of the major and minor HIV-1 splice donor sites in the vector. In addition the rate of vector mobilisation and repair of SIN vectors can be successfully reduced by careful vector design that reduces homology between the 5' and 3' long terminal repeats (LTRs) to a minimum.Rachel M Koldej and Donald S Anso

Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

BACKGROUND: Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder. METHODS: The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis. RESULTS: Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment. CONCLUSION: Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA

Spatial proteomics identifies a spectrum of immune dysregulation in acquired bone marrow failure syndromes

Poor graft function (PGF), manifested by multilineage cytopenias and complete donor chimerism post-allogeneic stem cell transplantation (alloSCT), and acquired aplastic anaemia (AA) are immune-mediated acquired bone marrow (BM) failure syndromes with a similar clinical presentation. In this study, we used spatial proteomics to compare the immunobiology of the BM microenvironment and identify common mechanisms of immune dysregulation under these conditions. Archival BM trephines from patients exhibited downregulation of the immunoregulatory protein VISTA and the M2 macrophage marker and suppressor of T-cell activation ARG1 with increased expression of the immune checkpoint B7-H3 compared to normal controls. Increased CD163 and CD14 expression suggested monocyte/macrophage skewing, which, combined with dysregulation of STING and VISTA, is indicative of an environment of reduced immunoregulation resulting in the profound suppression of hematopoiesis in these two conditions. There were no changes in the immune microenvironment between paired diagnostic AA and secondary MDS/AML samples suggesting that leukaemic clones develop in the impaired immune microenvironment of AA without the need for further alterations. Of the eight proteins with dysregulated expression shared by diagnostic AA and PGF, the diagnostic AA samples had a greater fold change in expression than PGF, suggesting that these diseases represent a spectrum of immune dysregulation. Unexpectedly, analysis of samples from patients with good graft function post-alloSCT demonstrated significant changes in the immune microenvironment compared to normal controls, with downregulation of CD44, STING, VISTA, and ARG1, suggesting that recovery of multilineage haematopoiesis post-alloSCT does not reflect recovery of immune function and may prime patients for the development of PGF upon further inflammatory insult. The demonstrable similarities in the immunopathology of AA and PGF will allow the design of clinical interventions that include both patient cohorts to accelerate therapeutic discovery and translation

The development of HIV-1 derived gene transfer technology: optimisation of vector safety, processing and production.

Vectors derived from Human Immunodeficiency Virus type 1 (HIV-1) are being widely developed for gene therapy applications, principally because they are able to transduce both dividing and non-dividing cells and result in stable, long term gene expression. However, these vectors are difficult to produce in high titres and sufficient volumes for large scale experiments and clinical application. Therefore, an investigation into methods to improve the production of HIV-1 derived gene transfer vectors was undertaken. One factor that limits the production of recombinant virus is the amount of viral genomic RNA available for packaging into virions. Therefore, a transfer vector was modified with the aim of increasing the amount of genomic RNA produced. Substitution of the polyadenylation (pA) signal, mutation splice donor sites and removal of unnecessary sequences were all examined. pA signal readthrough was quantified to determine the effect of these modifications on the rate of pA signal readthrough. Insertional mutagenesis and vector mobilisation are recognised risk factors with all integrating vectors. Self inactivating (SIN) vectors, which contain a deletion of U3 sequences in the 3’ LTR, demonstrate a reduced rate of mobilisation. Transduction with these vectors results in a provirus containing no viral promoter elements, with transcription of the transgene being controlled from an internal promoter. However, LTR repair of SIN vectors occurs at an appreciable frequency. Therefore, the extent of this deletion was maximised and the effect on the frequency of the repair examined. The production of lentiviral gene therapy vectors by large-scale transient transfection is both time consuming and technically difficult. Therefore, methods to increase the scale of production without compromising virus titre were developed. This resulted in fewer transfections and less handling of the cells when making virus on a large scale (3-4 L). In order to process the virus on this scale in a single day (i.e. 8 hours), new concentration and purification methods were established. The protocol consisted of low speed centrifugation, 0.45 μm filtration, 750 kDa ultrafiltration, 0.8 μm filtration and ultracentrifugation. However, the use of ultracentrifugation means that this protocol is not amenable to further scale up. Therefore, the replacement of the ultracentrifugation step with anion exchange was investigated. A number of different resins and anion exchange devices were investigated, two of which show promise for large scale purification of HIV-1 derived gene transfer vectors. In an ideal world, HIV-1 derived gene transfer vectors would be produced using stable packaging cell lines engineered to produce the desired virus. However, previous attempts to produce such a cell line with the desired properties have had limited success and have generally used outdated helper systems. Therefore, in an attempt to combine the efficiency advantages of having a single helper plasmid with the safety advantages of expressing each protein separately, a single packaging construct that contained separate transcription units for each of the required proteins was produced. Transcription of cyotoxic proteins was controlled by inducible promoters. Initial results suggest that such a system is technically feasible but that further work is required to optimise the expression of helper functions.Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 200

Immunotherapeutics in Multiple Myeloma: How Can Translational Mouse Models Help?

Multiple myeloma (MM) is usually diagnosed in older adults at the time of immunosenescence, a collection of age-related changes in the immune system that contribute to increased susceptibility to infection and cancer. The MM tumor microenvironment and cumulative chemotherapies also add to defects in immunity over the course of disease. In this review we discuss how mouse models have furthered our understanding of the immune defects caused by MM and enabled immunotherapeutics to progress to clinical trials, but also question the validity of using immunodeficient models for these purposes. Immunocompetent models, in particular the 5T series and Vk⁎MYC models, are increasingly being utilized in preclinical studies and are adding to our knowledge of not only the adaptive immune system but also how the innate system might be enhanced in anti-MM activity. Finally we discuss the concept of immune profiling to target patients who might benefit the most from immunotherapeutics, and the use of humanized mice and 3D culture systems for personalized medicine