Lentiviral-Mediated RNA Interference

Abbas-Terki, Toufik; Blanco-Bose, William; Déglon, Nicole; Pralong, William; Aebischer, Patrick

doi:10.1089/104303402320987888

Cited by 267 publications

(170 citation statements)

References 12 publications

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“…The black arrow (g) points to an eGFP/ngn3 double-labelled cell. Scale bar 50 μm RNA interference can be efficiently performed using lentiviral vectors [48][49][50].…”

Section: Discussionmentioning

confidence: 99%

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

et al. 2005

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Aims/hypothesis: Gene transfer into pancreatic beta cells, which produce and secrete insulin, is a promising strategy to protect such cells against autoimmune destruction and also to generate beta cells in mass, thereby providing a novel therapeutic approach to treat diabetic patients. Until recently, exogenous DNA has been directly transferred into mature beta cells with various levels of success. We investigated whether exogenous DNA could be stably transferred into pancreatic stem/progenitor cells, which would subsequently differentiate into mature beta cells expressing the transgene. Methods: We designed transplantation and tissue culture procedures to obtain ex vivo models of pancreatic development. We next constructed recombinant lentiviruses expressing enhanced green fluorescent protein (eGFP) under the control of either the rat insulin promoter or a ubiquitous promoter, and performed viral infection of rat embryonic pancreatic tissue. Results: Embryonic pancreas infected with recombinant lentiviruses resulted in endocrine cell differentiation and restricted cell type expression of the transgene according to the specificity of the promoter used in the viral construct. We next demonstrated that the efficiency of infection could be further improved upon infection of embryonic pancreatic epithelia, followed by their in vitro culture, using conditions that favour endocrine cell differentiation. Under these conditions, endocrine stem/progenitor cells expressing neurogenin 3 are efficiently transduced by recombinant lentiviral vectors.Moreover, when eGFP was placed under the control of the insulin promoter, 70.4% of the developed beta cells were eGFP-expressing cells. All of the eGFP-positive cells were insulin-producing cells. Conclusions/interpretation: We have demonstrated that mature rat pancreatic beta cells can be stably modified by infecting pancreatic stem/progenitor cells that undergo endocrine differentiation.

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“…The black arrow (g) points to an eGFP/ngn3 double-labelled cell. Scale bar 50 μm RNA interference can be efficiently performed using lentiviral vectors [48][49][50].…”

Section: Discussionmentioning

confidence: 99%

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

et al. 2005

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“…[79][80][81][82] In general, biosafety is the major concern in the production of retroviral and lentiviral vectors, and currently they are in use in HIV patients only. Adenoviral vectors are widely used both in cells and in vivo; however, their inability to integrate into the genome has proved advantageous in terms of safety but at the cost of inefficient long-term gene silencing.…”

Section: Rnai Deliverymentioning

confidence: 99%

siRNA-based approaches in cancer therapy

2006

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The availability of the human genome sequence has revolutionized the strategy of employing nucleic acids with sequences complementary to specific target genes to improve drug discovery and target validation. Development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences offers the possibility of rational design with high specificity, lacking in many current drug treatments for various diseases including cancer, at relatively inexpensive costs. Antisense technology is one such example that has shown promising results and boasts of yielding the only approved drug to date in the genomics field. However, in vivo delivery issues have yet to be completely overcome for widespread clinical applications. In contrast to antisense oligonucleotides, the mechanism of silencing an endogenous gene by the introduction of a homologous double-stranded RNA (dsRNA), transgene or virus is called post-transcriptional gene silencing (PTGS) or RNA interference. PTGS is a natural mechanism whereby metazoan cells suppress expansion of genes when they come across dsRNA molecules with the same sequence. Short interfering RNA is currently the fastest growing sector of this antigene field for target validation and therapeutic applications. Although, in theory, the development of genomics-based agents to inhibit gene expression is simple and straightforward, the fundamental concern relies upon the capacity of the oligonucleotide to gain access to the target RNA. This paper summarizes the advances in the last decade in the field of PTGS using RNA interference approaches and provides relevant comparisons with other oligonucleotide-based approaches with a specific focus on oncology applications. Cancer Gene Therapy (2006) Genomic-based strategiesThe American Cancer Society estimates that, in 2005, a total of 1 372 910 new cancer cases and 570 280 deaths are expected in the United States. 1 These morbid statistics demonstrate the necessity of newer therapeutic modalities for achieving successful cancer treatment and cure. Downregulation of genes that contribute to cancer progression has been the goal of targeted genomics-based strategies, with the expectation that such an approach may lead to selective and specific inhibition of tumor growth with minimal untoward side effects on normal cells. Identification of target genes involved in neoplastic transformation and tumor progression has triggered the idea that nucleotide sequences of cancer-relevant genes could lead to the development of tailored anticancer agents that lack many of the toxic side effects of traditional cytotoxic drugs. This has led to a recent acceleration in the development and optimization of genomic-based strategies for cancer therapy. This idea dates back to the 1960s when RNA sequences were shown to serve as endogenous inhibitors of gene expression in procaryotes. The antigene approaches include the following:(1) Ribozymes: 2,3 These were discovered in the 1970s, and the elucidation of the transactivating hammerh...

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“…Lentiviral RNAi based systems have thus been developed to generate replication incompetent lentiviral vectors that efficiently transduce both dividing and non-dividing mammalian cells and provide stable, long term expression of an miRNA of interest (Abbas-Terki et al, 2002;Stewart et al, 2003). The miRNA expression vectors used here enable bicistronic expression of an EmGFP reporter gene with the miRNA, thus allowing reliable tracking of miRNA expression in mammalian cells.…”

Section: Lentiviral Expression Of Anti-adk Mirna Significantly Reducementioning

confidence: 99%

Lentiviral RNAi-induced downregulation of adenosine kinase in human mesenchymal stem cell grafts: A novel perspective for seizure control

Ren

Lan

et al. 2007

Experimental Neurology

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Cell therapies based on focal delivery of the inhibitory neuromodulator adenosine were previously shown to provide potent seizure suppression in animal models of epilepsy. However, hitherto used therapeutic cells were derived from rodents and thus not suitable for clinical applications. Autologous patient-derived adenosine-releasing cell implants would constitute a major therapeutic advance to avoid both xenotransplantation and immunosuppression. Here we describe a novel approach based on lentiviral RNAi mediated downregulation of adenosine kinase (ADK), the major adenosineremoving enzyme, in human mesenchymal stem cells (hMSCs), which would be compatible with autologous cell grafting in patients. Following lentiviral transduction of hMSCs with anti-ADK miRNA expression cassettes we demonstrate up to 80% downregulation of ADK and a concentration of 8.5 ng adenosine per ml of medium after incubating 10 5 cells for 8 hours. hMSCs with a knockdown of ADK or cells expressing a scrambled control sequence were transplanted into hippocampi of mice 1 week prior to the intraamygdaloid injection of kainic acid (KA). While mice with control implants expressing a scrambled miRNA sequence or sham treated control animals were characterized by KAinduced status epilepticus and subsequent CA3 neuronal cell loss, animals with therapeutic ADK knockdown implants displayed a 35% reduction in seizure duration and 65% reduction in CA3 neuronal cell loss, when analyzed 24h after KA-injection. We conclude that lentiviral expression of anti-ADK miRNA constitutes a versatile tool to generate therapeutically effective adenosine releasing hMSCs, thus representing a model system to generate patient identical autologous adult stem cell grafts.

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Lentiviral-Mediated RNA Interference

Cited by 267 publications

References 12 publications

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

siRNA-based approaches in cancer therapy

Lentiviral RNAi-induced downregulation of adenosine kinase in human mesenchymal stem cell grafts: A novel perspective for seizure control

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