A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status.
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ABSTRACT:
SUBMITTER: Fusil F
PROVIDER: S-EPMC4817946 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.
| S-EPMC4435596 | biostudies-literature
Project description:BackgroundHIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells.ResultsWe have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency.ConclusionAs with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.
| S-EPMC1797186 | biostudies-literature
Project description:Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation.
| S-EPMC4605225 | biostudies-literature
Project description:In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5?, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5? against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5? overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012.
| S-EPMC3511674 | biostudies-literature
Project description:The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 10(6) transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac239). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished.
| S-EPMC3991964 | biostudies-literature
Project description:Gene transfer is a widely developed technique for studying and treating genetic diseases. However, the development of therapeutic strategies is challenging, due to the cellular and functional complexity of the central nervous system (CNS), its large size and restricted access. We explored two parameters for improving gene transfer efficacy and capacity for the selective targeting of subpopulations of cells with lentiviral vectors (LVs). We first developed a second-generation LV specifically targeting astrocytes for the efficient expression or silencing of genes of interest, and to better study the importance of cell subpopulations in neurological disorders. We then made use of the retrograde transport properties of a chimeric envelope to target brain circuits affected in CNS diseases and achieve a broad distribution. The combination of retrograde transport and specific tropism displayed by this LV provides opportunities for delivering therapeutic genes to specific cell populations and ensuring high levels of transduction in interconnected brain areas following local administration. This new LV and delivery strategy should be of greater therapeutic benefit and opens up new possibilities for the preclinical development of gene therapy for neurodegenerative diseases.
| S-EPMC7902268 | biostudies-literature
Project description:Lentiviral vectors (LVs) are used in cell and gene therapies due to their ability to transduce both dividing and non-dividing cells while carrying a relatively large genetic payload and providing long-term gene expression via gene integration. Current cultivation methods produce titers of 105-107 transduction unit (TU)/mL; thus, it is necessary to concentrate LVs as well as remove process- and product-related impurities. In this work, we used a packaging cell line WinPac-RD-HV for LV production to simplify upstream processing. A direct capture method based on ion-exchange chromatography and cellulose nanofibers for LV concentration and purification was developed. This novel scalable stationary phase provides a high surface area that is accessible to LV and, therefore, has potential for high-capacity operation compared to traditional bead-based supports. We were able to concentrate LVs 100-fold while achieving a two-log removal of host cell protein and maintaining up to a 90% yield of functional vector.
| S-EPMC6804883 | biostudies-literature
Project description:The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system.
| S-EPMC3070093 | biostudies-other
Project description:Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.
| S-EPMC3070105 | biostudies-literature
Project description:The importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.A synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.Transfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.The TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues.
| S-EPMC2765473 | biostudies-literature