Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography

Use of lentiviral vectors (LVs) in clinical Cell and Gene Therapy applications is growing. However, functional product loss during capture chromatography, typically anion-exchange (AIEX), remains a significant unresolved challenge for the design of economic processes. Despite AIEX's extensive use, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of LV adsorption and other types of vector delivery systems. This work demonstrates HIV-1-LV recovery over quaternary-amine membrane adsorbents is a function of time in the adsorbed state. Kinetic data for product loss in the column bound state was generated. Fitting a second order-like rate model, we observed a rapid drop in functional recovery due to increased irreversible binding for vectors encoding two separate transgenes ( t Y 1 / 2 ${t}_{{Y}_{1/2}}$  = 12.7 and 18.7 min). Upon gradient elution, a two-peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterizing the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak. This work highlights time spent in the adsorbed state as a critical factor impacting LV product loss and the need for consideration in LV AIEX process development workflows

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance

Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins

The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein–protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery

Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos

Characterization of the murine formylated peptide chemotactic receptor

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN017869 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Legal reasoning and constructive interpretation

Award date: February 1995Supervisor: M. La TorrePDF of thesis uploaded from the Library digitised archive of EUI PhD theses completed between 2013 and 201

Characterization of the murine n-formyl peptide chemotactic receptor

The N-formyl peptide receptor (FPR) present on neutrophils is of importance in providing the host with a detection mechanism of broad specificity for invading microorganisms and damaged tissue. The aim of this project was to develop an accurate physiological model for the study of FPR. Since neutrophils isolated from blood leukocytes are heterogeneous, short lived and terminally differentiated they do not make good models for the study of FPR. Hence, the need for in vitro model systems. The current model used, the human leukaemic cell line (HL-60), does not produce fully mature neutrophils. In contrast, the murine pluripotent stem cell line (FDCP), can be fully differentiated to mature neutrophils. This cell line was therefore chosen for the characterization and development of a model system for the FPR. A detailed study of cytokine-mediated differentiation was undertaken. Differentiated FDCP cells, expressed FPR and showed cell adhesion and degranulation in response to N-formyl peptides. The kinetics of the expressed murine FPR and the efficacy of a number of synthetic N-formyl peptides was established. The peptide formyl-Norleu-Leu-Phe-Norleu-Tyr-Lys bound with high and low affinity dissociation constants of 3.7 and 22.6 nM, respectively. The number of receptors was estimated to be 79 000 per cell with 25[percent] being of high affinity. The differentiated FDCP cells, neutrophils had low affinity binding for the peptide fMet-Leu-Phe as compared to human and rabbit neutrophils. Attempts were first made to clone the human and then the murine FPR gene. However, the putative genes were cloned by another group before completion of this work. The muFPR gene, which was transcribed and expressed in murine FDCP cells differentiated to neutrophils, was identified from six putative genes by reverse transcriptase PCR. The time course of transcription was consistent with the appearance of functional FPR during differentiation