16,400 research outputs found
High-titre retroviral vector system for efficient gene delivery into human and mouse cells of haematopoietic and lymphocytic lineages
Genetically modified cells of haematopoietic and lymphocytic lineages could provide potentially curative treatments for a wide range of inherited and acquired diseases. However, this application is limited in mouse models by the low efficiency of lentiviral vectors. To facilitate the rapid production of high-titre helper-free retroviral vectors for enhanced gene delivery, multiple modifications to a prototype moloney murine leukemia virus (MoMLV)-derived vector system were made including adaptation of the vector system to simian virus 40 ori/T antigen-mediated episomal replication in packaging cells, replacement of the MoMLV 5′ U3 promoter with a series of stronger composite promoters and addition of an extra polyadenylation signal downstream of the 3′ long terminal repeat. These modifications enhanced vector production by 2–3 logs. High-titre vector stocks were tested for their ability to infect a variety of cells derived from humans and mice, including primary monocyte-derived macrophage cultures. Whilst the lentiviral vector was significantly restricted at the integration level, the MoMLV-based vector showed effective gene transduction of mouse cells. This high-titre retroviral vector system represents a useful tool for efficient gene delivery into human and mouse haematopoietic and lymphocytic cells, with particular application in mice as a small animal model for novel gene therapy tests
Investigating the role of coiled-coil domain containing 124 (CCDC124) in innate antiviral immune response
The innate immune system acts as the first line of defense in a non-specific manner against infectious diseases as well as malignant transformation. Natural Killer (NK) cells are members of innate immune system which are particularly responsible for killing virus-infected cells and tumor cells. Distinct properties of NK cells are remarkable in terms of cancer immunotherapy. Among several approaches, genetic modification of NK cells to enhance their immune function is widely studied with promising results but in vitro gene delivery into NK cells is highly challenging. HIV-1 based lentiviral vector systems for stable gene transfer have been used in most of the studies that aim genetic modification of NK cells. However, viral resistance of NK cells causes low efficiency and reduced stability, but enhancement of gene delivery efficiency is possible to achieve with small-molecule kinase inhibitors, such as BX795. Stress granule assembly is known to be associated with antiviral responses. This study aims to study the effect of CCDC124 gene which may be associated with stress granule formation and antiviral response during lentiviral gene transfer to NK cells. To investigate the mechanism, CRISPR/Cas9 system was used to knock out CCDC124 and other genes that may be involved in the intracellular response against lentiviral vectors in HCT116, NK-92 and YTS cell lines. We compared the responses of different cell lines to lentiviral transduction and observed significant change in transduction efficiencies. Additionally, stress granule formation in CCDC124 knockout NK-92 cells is examined. Our findings present novel insights into the resistance of NK cells to lentiviral gene delivery and provide useful tools to improve genetic modification of NK cell
Accelerated cell line development and improved characterisation of lentiviral vector production through application of MALDI-ToF mass spectrometry and multivariate data analysis.
Ph. D. Thesis.Several cell and gene therapies will be commercially launched within the next
few years using lentiviral vectors as the gene delivery vehicle. Oxford BioMedica’s
Lentivector® platform is an advanced lentiviral-based gene delivery system designed for
improved safety and efficacy. The growing interest in these vectors has created a strong
demand for large scale production of lentiviral vectors as well as for development of
packaging and producer cell lines. This EngD project used a combination of matrix
assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS)
and multivariate data analysis (MVDA) to analyse cell and lentiviral vector samples. A
comparison between mass spectra of samples produced across small and large scale in
adherent and suspension culture was used to identify what aspects of the manufacturing
process had the biggest impact on cell and vector variation. Principal component
analysis was applied to compare different lentiviral vector production methods, assess
data structure of the process parameters and examine whole cell and vector mass
spectrometry data. This approach led to improved characterisation of lentiviral vectors
and HEK293T cells. It demonstrated the capability to differentiate between adherent
and suspension cells as well as cell lines of different levels of performance as defined by
lentiviral vector infectious titre. Partial least squares discriminant analysis (PLS-DA)
was used to calibrate and validate a predictive model of cell line performance based on
mass spectrometry and viral vector titre data obtained from multiple HEK293T cell
lines. PLS-DA model validation resulted in 87.5% accuracy in classification of cell lines
as high or low producers based on a discrimination threshold determined by viral
vector titre. The results of PLS-DA modelling indicated that this method can be used for
accurate cell line performance prediction, accelerating cell line development by several
weeks, improving cell selection and reducing campaign timelines.EPSR
Use of integrase-minus lentiviral vector for transient expression
Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP) gene expression by a fluorescence microscopy. Results: Recombinant and wild lentiviruses titer was about 5�8�10 6 transducing units/ ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting
Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization.
Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single- long terminal repeat (LTR) to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3'SIN-LTR's and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High-titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors
Targeting lentiviral vectors to antigen-specific immunoglobulins
Gene transfer into B cells by lentivectors can provide an alternative approach to managing B lymphocyte malignancies and autoreactive B cell-mediated autoimmune diseases. These pathogenic B cell Populations can be distinguished by their surface expression of monospecific immunoglobulin. Development of a novel vector system to deliver genes to these specific B cells could improve the safety and efficacy of gene therapy. We have developed an efficient rnethod to target lentivectors to monospecific immunoglobulin-expressing cells in vitro and hi vivo. We were able to incorporate a model antigen CD20 and a fusogenic protein derived from the Sindbis virus as two distinct molecules into the lentiviral Surface. This engineered vector could specifically bind to cells expressing Surface immunoglobulin recognizing CD20 (αCD20), resulting in efficient transduction of target cells in a cognate antigen-dependent manner in vitro, and in vivo in a xenografted tumor model. Tumor suppression was observed in vivo, using the engineered lentivector to deliver a suicide gene to a xenografted tumor expressing αCD20. These results show the feasibility of engineering lentivectors to target immunoglobulin-specific cells to deliver a therapeutic effect. Such targeting lentivectors also Could potentially be used to genetically mark antigen-specific B cells in vivo to study their B cell biology
Integrase-deficient lentiviral vectors mediate efficient gene transfer to human vascular smooth muscle cells with minimal genotoxic risk
We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer
Perinatal Gene Transfer to the Liver
The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect.
Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first
weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable.
In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise
the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application
of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic
acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by
assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer
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Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors.
Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. On the basis of their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N and T368A mutations enhanced serum resistance, and additionally K66T, T368A and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy
Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?
The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system
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