Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity

Moreau, Karen; Hong, Saw-See; Ronfort, Corinne

doi:10.1007/s00705-011-1150-5

Cited by 7 publications

(4 citation statements)

References 36 publications

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“…Functional titrations require transduction, which is time-consuming, and efficiencies depend on cell type and transgenes [2,3]. Non-functional approaches thus present an easier alternative for determining the titer of a lentiviral particle batch for optimization and standardization.…”

Section: Introductionmentioning

confidence: 99%

A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR

Mournetas¹,

Pereira

Fernig

et al. 2016

J Biol Methods

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A B S T R A C TGene silencing techniques, including RNA interference methodologies, are widely used in reverse genetics to study the role of specific genes in biological processes. RNA interference has become easier to implement thanks to the RNAi Consortium (TRC), which has developed libraries of short hairpin RNA (shRNA) sequences in pseudotyped lentiviral particles capable of targeting most genes in the human and mouse genomes. However, a problem is the lack of a simple method to titrate the homemade lentiviral particle product, making it difficult to optimize and standardize shRNA experiments. Here we provide a guide describing a quick, non-laborious and reliable method for the titration of TRC pseudotyped lentiviral particles that is based on the detection and measurement of viral RNA using quantitative PCR. Our data demonstrate that purified linearized shRNA plasmids represent more suitable standards than circular or unpurified linearized plasmids. We also show that for precise absolute quantification, it is important to determine suitable plasmid and viral cDNA concentrations in order to find the linear range for quantification, as well as to reduce inhibition and primer dimer amplification. Finally, we show that the lentivirus concentration impacts the level of knockdown in transduced cells. Primers utilized in this non-functional titration can potentially be applied to functional titration of proviral DNA copies or transgene expression, overcoming problems arising from the absence of fluorescent reporter genes in TRC plasmids.

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Section: Introductionmentioning

confidence: 99%

A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR

Mournetas¹,

Pereira

Fernig

et al. 2016

J Biol Methods

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“…1 ). Infectious LV titre was calculated as previously reported [ 72 ]. Briefly, 2 × 10 5 HEK293T cells were seeded and incubated at 37 °C, 5% CO2 overnight to adhere.…”

Section: Methodsmentioning

confidence: 99%

HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival

et al. 2022

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Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation.

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“…We developed LV-OVA and LV-FIX vectors by replacing the GFP gene of the PGK promoter-driven LV-GFP vector ( 23 ) by a SIINFEKL/β2-microglobulin/H-2K b fusion construct ( 24 ) or human FIX cDNA ( 25 ), respectively (Figure 1 ). LV titers (expressed as transducing units, TU/mL) were determined by flow cytometry for LV-OVA and LV-GFP ( 26 ) and by qPCR for LV-FIX ( 10 ). BM cells from female Ly5.1 C57BL/6 (Ly5.1 B6) mice (Charles River Laboratories) were transduced with LV at a multiplicity of infection of 1 ( 17 ).…”

Section: Methodsmentioning

confidence: 99%

Induction of Hematopoietic Microchimerism by Gene-Modified BMT Elicits Antigen-Specific B and T Cell Unresponsiveness toward Gene Therapy Products

et al. 2016

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BackgroundGene therapy is a promising treatment option for hemophilia and other protein deficiencies. However, immune responses against the transgene product represent an obstacle to safe and effective gene therapy, urging for the implementation of tolerization strategies. Induction of a hematopoietic chimerism via bone marrow transplantation (BMT) is a potent means for inducing immunological tolerance in solid organ transplantation.ObjectivesWe reasoned, here, that the same viral vector could be used, first, to transduce BM cells for inducing chimerism-associated transgene-specific immune tolerance and, second, for correcting protein deficiencies by vector-mediated systemic production of the deficient coagulation factor.MethodsEvaluation of strategies to induce B and T cell tolerance was performed using ex vivo gene transfer with lentiviral (LV) vectors encoding coagulation factor IX (FIX) or the SIINFEKL epitope of ovalbumin. Following induction of microchimerism via BMT, animals were challenged with in vivo gene transfer with LV vectors.ResultsThe experimental approach prevented humoral immune response against FIX, resulting in persistence of therapeutic levels of circulating FIX, after LV-mediated gene transfer in vivo. In an ovalbumin model, we also demonstrated that this approach effectively tolerized the CD8+ T cell compartment in an antigen-specific manner.ConclusionThese results provide the proof-of-concept that inducing a microchimerism by gene-modified BMT is a powerful tool to provide transgene-specific B and T cell tolerance in a gene therapy setting.

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Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity

Cited by 7 publications

References 36 publications

A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR

A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR

HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival

Induction of Hematopoietic Microchimerism by Gene-Modified BMT Elicits Antigen-Specific B and T Cell Unresponsiveness toward Gene Therapy Products

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