Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

Tesio, Melania; Gammaitoni, L.; Gunetti, Monica; Leuci, Valeria; Pignochino, Ymera; Jordaney, Noela; Capellero, Sonia; Cammarata, Cristina; Caione, Luisa; Migliaretti, Giuseppe; Tabilio, Antonio; Aglietta, Massimo; Piacibello, Wanda

doi:10.1634/stemcells.2008-0161

Cited by 9 publications

(7 citation statements)

References 24 publications

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“…Other studies have shown that lentiviral vectors transiently arrest transduced cells when used at high concentrations and resulted in a marked decrease in the growth rate of the cells [60], [61]. The lack of observed differences in cell growth and viability in our study may be explained by the relatively low range of MOI (between 2 to 12, due to the limitations of cell number and viral titre) used throughout the study, which is markedly lower than those reported by some other groups [24], [25], [62]. Another important factor to be taken into consideration is the duration in which the cells are in contact with the virus.…”

Section: Discussioncontrasting

confidence: 54%

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

et al. 2009

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BackgroundHematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34+ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.Methodology/Principal FindingsUsing commercially available G-CSF mobilized peripheral blood (PB) CD34+ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.Conclusions/SignificanceThis study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34+ cells.

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Section: Discussioncontrasting

confidence: 54%

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

et al. 2009

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“…BM HSC could be lentivirally modified with long‐term transgene expression without compromising cell differentiation or function as also shown in PB HSC [63]. Recently, lentivirally mediated gene therapy was shown to provide clinical benefits in an inheritable fatal demyelinating disease X‐linked adrenoleukodystrophy [64].…”

Section: Discussionmentioning

confidence: 99%

Production of monocytic cells from bone marrow stem cells: therapeutic usage in Alzheimer’s disease

Magga

Savchenko

Malm

et al. 2012

J Cellular Molecular Medi

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Accumulation of amyloid β (Aβ) is a major hallmark in Alzheimer’s disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aβ burden in mouse models of AD, alleviating the AD pathology. BMM have been shown to be more efficient phagocytes in AD than the endogenous brain microglia. Because BMM have a natural tendency to infiltrate into the injured area, they could be regarded as optimal candidates for cell-based therapy in AD. In this study, we describe a method to obtain monocytic cells from BM-derived haematopoietic stem cells (HSC). Mouse or human HSC were isolated and differentiated in the presence of macrophage colony stimulating factor (MCSF). The cells were characterized by assessing the expression profile of monocyte markers and cytokine response to inflammatory stimulus. The phagocytic capacity was determined with Aβ uptake assay in vitro and Aβ degradation assay of natively formed Aβ deposits ex vivo and in a transgenic APdE9 mouse model of AD in vivo. HSC were lentivirally transduced with enhanced green fluorescent protein (eGFP) to determine the effect of gene modification on the potential of HSC-derived cells for therapeutic purposes. HSC-derived monocytic cells (HSCM) displayed inflammatory responses comparable to microglia and peripheral monocytes. We also show that HSCM contributed to Aβ reduction and could be genetically modified without compromising their function. These monocytic cells could be obtained from human BM or mobilized peripheral blood HSC, indicating a potential therapeutic relevance for AD.

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“…One of the major drawbacks concerning this technique is the relatively low permissiveness of progenitor cells to LV, which results in transduction efficiencies ranging from 19% of the murine MK progeny successfully expressing a therapeutic transgene to 71% of mouse platelets expressing eGFP and from 16% of human CD34 ϩ HSCs containing a GFP sequence to 62% of human platelets expressing eGFP. 88,[90][91][92][93][94][95][96][97][98] Transduction efficiencies can be improved, for example, using higher multiplicities of infection, however, with concomitant increased risk of insertional mutagenesis and lentiviral-mediated cytotoxicity. Therefore, other ways of improving transduction efficiency are currently being explored (see "Perspectives").…”

Section: Transplantation Modelsmentioning

confidence: 99%

Model systems of genetically modified platelets

Thijs

Deckmyn

Broos

2012

Blood

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Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are probably involved in the formation or physiology of platelets under normal and pathologic conditions. This creates an increasing demand for new and improved model systems that allow functional assessment of the corresponding gene products in vivo. Such animal models not only render invaluable insight in the platelet biology, but in addition, provide improved test systems for the validation of newly developed antithrombotics. This review summarizes the most important models to generate transgenic platelets and to study their influence on platelet physiology in vivo. Here we focus on the zebrafish morpholino oligonucleotide technology, the (platelet- specific IntroductionBlood platelets play part in a myriad of processes, such as inflammation, tumor growth and metastasis, immunology and, of course, thrombosis and blood clotting where they provide a first and crucial line of defense against vascular injury, thus maintaining normal hemostasis. 1,2 Primary hemostasis starts when platelets recognize a site of vascular injury where the subendothelial matrix is exposed, bind to collagen, and become activated. 3 The subsequent rise in intracellular calcium triggers conformational changes in integrin receptors, degranulation, exposition of a procoagulant surface, and generation and release of secondary agonists resulting in a thrombus that will cover the site of injury and prevent further blood loss. 4 Platelets are furthermore an important factor in thrombotic events, such as stroke and myocardial infarction. 5 To identify more proteins regulating platelet function that may serve as new targets for the development of anti-thrombotics or in the prevention of bleeding, the platelet research community has seen the completion of several large-scale screening programs and the spectacular rise in the "platelet-omics" field. Several genome-wide association studies and subsequent meta-analysis in patients with coronary artery disease and healthy volunteers identified numerous genetic loci that are possibly involved in regulating platelet formation, count, volume, and function and might confer a risk for coronary artery disease. [6][7][8][9][10][11] On the other hand, gene expression profiling of healthy volunteer platelets, in combination with comparative microarray analysis between in vitro differentiated megakaryocytes (MKs) and closely related cell types, established a comprehensive platelet transcriptome. 6,[12][13][14][15][16][17][18][19] Finally, advanced proteomics studies identified proteins of the platelet sheddome, secretome, interactome, kinome, and phosphoproteome potentially involved in platelet function. 20 The overall result is a large number of newly identified gene products for which we are only beginning to understand their ...

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Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

Abstract: ABSTRACT؉ (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB.

Cited by 9 publications

References 24 publications

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Production of monocytic cells from bone marrow stem cells: therapeutic usage in Alzheimer’s disease

Model systems of genetically modified platelets

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