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

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

AbstractTraditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals

Enhanced Efficacy of the CDNF/MANF Family by Combined Intranigral Overexpression in the 6-OHDA Rat Model of Parkinson's Disease

Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.Molecular Therapy (2014); doi:10.1038/mt.2014.206

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described

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 formylated peptide chemotactic receptor

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

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