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

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

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

Not reinventing the wheel – applying 3Rs concepts to viral vector gene therapy biodistribution studies

In pharmaceutical drug development, information on where a drug distributes within the body is helpful in understanding the overall chemical and/or biological characteristics of the compound. This is typically assessed preclinically in relevant animal species that are often used for the formal safety studies (toxicology, pharmacokinetics and drug metabolism. One accepted principle of animal studies is the application of the “3 R’s concept” (replacement, refinement and reduction http://www.nc3rs.org.uk/) to reduce animal use as far as makes sense. The focus of this article is to suggest that, for gene therapy biodistribution studies, reductions in animal use could be rationally achieved with no increase in clinical risk

Assessment of Integration-defective HIV-1 and EIAV Vectors In Vitro and In Vivo

The interest in integrase-defective lentiviral vectors (IDLVs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. Here, we directly compared the transducing potential of IDLVs based on the equine infectious anemia virus (EIAV) to the more commonly described HIV-1 IDLVs. IDLVs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. We show that both the single and the triple mutant HIV-1 IDLVs transduce the PC12 cells, but not the C2C12 cells, with similar efficiency to their parental HIV-1 vector. In contrast, the single and triple EIAV IDLVs did not efficiently transduce either differentiated cell line. Moreover, this HIV-1 IDLV-mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. Four weeks following stereotactic administration into adult rat brains, only the single HIV-1 IDLV mutant displayed a comparable transduction profile to the parental HIV-1 vector. In contrast, neither EIAV IDLV mutants showed significant reporter gene expression. This work indicates that the transducing potential of IDLVs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector

Efficient production of germline transgenic chickens using lentiviral vectors

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G(2) generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations