Production Of Lentiviral Particles Research Articles

FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.

High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus.

Mammalian protein expression systems are ideally suited for the high-level production of recombinant eukaryotic secreted and membrane proteins for structural biology applications. Here, we present genetic transduction of HEK293-derived cells using lentivirus as a robust and cost-efficient method for the rapid generation of stable expression cell lines. We describe the features of the lentiviral transfer plasmid pHR-CMV-TetO2, as well as detailed protocols for production of lentiviral particles, determination of functional lentiviraltiter, infection of expression cells, culture and expansion of the resulting stable cell lines, their adaptation to adherent and suspension growth, and constitutive or inducible milligram-scale protein production. The typical lead-time for a full production run is ~3-4weeks, with an anticipated yield of up to tens of milligrams of protein per liter of expression medium.

Development of a laboratory scalable process for enhancing lentivirus production by transient transfection of HEK293 adherent cultures.

Transfection of surface adherent cells remain as a standard methodology for lentiviral production for early phase clinical studies and research purposes. Production today is based on transient co-transfection of three or four plasmids, where the viral elements are encoded separately for safety reasons. Assembly of functional lentiviral particles requires all plasmids to be efficiently transfected into each cell, a notable challenge with many currently available methods for transient transfection. We have previously demonstrated the significant improvement of cationic polymer-based transfection in various cell types using a combination of fusogenic lipids and histone deacetylase 6 inhibitor (Enhancers). In this report, we focused on the transfection step and the feasibility of improving lentiviral production using the Enhancers. After optimization of DNA amount and N/P ratio, transfection using seven commercial gene carriers showed comparable maximal efficiency of production with high cell viability. In the presence of Enhancers, the production of functional lentivirus using LPEI was increased by as much as tenfold and outperformed lentiviral production using Lipofectamine 3000. We demonstrate a scalable and optimized workflow where the use of the Enhancers significantly improved the lentiviral particle production in various HEK293 cell lines.

Real-time transfer of lentiviral particles by producer cells using an engineered coculture system.

Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and quantification process. In this report, we demonstrate the ability for producer HEK293T cells to simultaneously produce lentiviral particles and transduce (i.e., infect) target cells through a membrane-based coculture system in a continuous, real-time mode which negates the need for a separate viral collection and quantification process. The coculture system was evaluated for major design features such as variations in HEK293T seeding density, target cell type densities, as well as membrane porosities to identify key relationships between lentiviral particle production rate and infection kinetics for adherent and suspension cell types. As a proof-of-concept for the creation of an engineered cell immunotherapy, we describe the ability to engineer human T cells isolated from PBMCs under the control of this coculture system in under 6days with a GFP construct. These studies suggest the capability to combine and more closely automate the transfection/transduction process in order to facilitate well-timed and cost-effective transduction of target cell types. These experiments provide novel insight into the forthcoming transition into improved manufacturing systems for viral production and subsequent cell engineering.

Induced Overexpression of THAP11 in Human Fibroblast Cells Enhances Expression of Key Pluripotency Genes

Background: THAP11 is a recently discovered pluripotency factor and described as an important gene that involved in embryonic stem cells self-renewal and embryo development, which works independently with other known pluripotency factors. We aimed to overexpressed the THAP11 gene in primary fibroblast cells to determine the effects of the THAP11 on these cells. Materials and Methods: The THAP11 gene was amplified using PCR followed by ligation into pCDH vector and lentiviral particle production in HEK293T cells by using psPAX2 and pMD2.G helper vectors. The human fibroblast cells were transduced using viral particles and after confirmation overexpression, the key pluripotency factors were estimated using real-time PCR and changes in proliferation rate was measured by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) test. Results: The overexpression of THAP11 in fibroblast cells leads to increase the expression level of Sox2, Oct4, Nanog and Klf4 as key pluripotency genes and a decrease in proliferation rate according to MTT results. Conclusion: Our results confirm that we are faced with a molecule with double features, which could be involved in pluripotency and proliferation suppressor simultaneously. It seems that the roles of THAP11 in pluripotency are so complex and attributed to other regulatory molecules.

Induced Overexpression of THAP11 in Human Fibroblast Cells Enhances Expression of Key Pluripotency Genes

Background: THAP11 is a recently discovered pluripotency factor and described as an important gene that involved in embryonic stem cells self-renewal and embryo development, which works independently with other known pluripotency factors. We aimed to overexpressed the THAP11 gene in primary fibroblast cells to determine the effects of the THAP11 on these cells. Materials and Methods: The THAP11 gene was amplified using PCR followed by ligation into pCDH vector and lentiviral particle production in HEK293T cells by using psPAX2 and pMD2.G helper vectors. The human fibroblast cells were transduced using viral particles and after confirmation overexpression, the key pluripotency factors were estimated using real-time PCR and changes in proliferation rate was measured by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) test. Results: The overexpression of THAP11 in fibroblast cells leads to increase the expression level of Sox2, Oct4, Nanog and Klf4 as key pluripotency genes and a decrease in proliferation rate according to MTT results. Conclusion: Our results confirm that we are faced with a molecule with double features, which could be involved in pluripotency and proliferation suppressor simultaneously. It seems that the roles of THAP11 in pluripotency are so complex and attributed to other regulatory molecules. [GMJ.2019;8:e1308]

Lentiviral Transduction of Primary Human Glioblastoma Cultures.

This chapter provides detailed step-by-step instructions for the production of lentiviral particles and the transduction of primary human glioblastoma cultures. Lentiviruses stably transduce both dividing and non-dividing cells, such as quiescent cancer stem cell populations. The viral envelope is pseudotyped with the vesicular stomatitis virus envelope glycoprotein G (VSV-G), which renders the lentiviral particles pantropic, so that they can infect theoretically all cell types. The third generation packaging system used in this protocol produces lentiviruses with important safety features, including replication incompetence and self-inactivation (SIN). The protocol we describe here leads to transduction of primary human glioblastoma cultures with efficiencies of up to 90%.

Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells.

The present study aimed to construct a lentiviral RNA interference (RNAi) vector targeting the transforming growth factor β1 (TGFβ1) gene of rats, in order to examine its effect on silencing of the TGFβ1 gene and on the expression of collagen type 1 α1 (Col1a1) in HSC‑T6 rat hepatic stellate cells. Three RNAi sites of the TGFβ1 gene were selected according to its CDs sequence. Three pairs of small interfering RNA (siRNA) of these RNAi sites were synthesized and then transfected into HSC‑T6 cells, respectively, to confirm the optimal siRNA sequence via reverse transcription‑polymerase chain reaction analysis. Subsequently, shRNA targeting the sequence of the optimal siRNA was designed, synthesized and annealed to form a double‑stranded structure. The annealed oligonucleotide fragment was cloned into pGreenPuro plasmids to establish the pGreenPuro/TGFβ1 shRNA lentiviral vector, which was then transfected into 293T cells, following identification by restriction enzyme digestion and sequencing for the production of lentiviral particles exhibiting high reactivity. These particles were used to infect HSC‑T6 cells, following which the expression of GFP in the transfected cells was observed under an inverted microscope. The effects on TGFβ1 gene silencing and the expression levels of Colla1 were detected at the mRNA and protein levels. The results provided confirmation of the optimal siRNA sequence. Enzyme digestion and sequencing verified successful construction of the pGreenPuro/TGFβ1 shRNA lentiviral vector. This lentiviral vector effectively silenced the TGFβ1 gene in the HSC‑T6 cells, and inhibited the expression of Col1a1 at the mRNA and protein levels. Taken together, the lentiviral RNAi vector targeting the TGFβ1 gene of rats was successfully constructed, which effectively silenced the TGFβ1 gene of the HSC‑T6 cells and inhibited the expression of Col1a1.

Construction and identification of an RNA interference lentiviral vector targeting the mouse TNF-α gene.

The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-α (TNF-α) gene. Three types of small interfering RNA (siRNA) targeting the mouse TNF-α gene were designed, synthesized and transfected into RAW264.7 cells. Screening was performed to identify the siRNA sequence exhibiting the highest inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and co-transfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNF-α mRNA expression levels of the three siRNA groups were significantly lower than those of the negative control group, with the highest inhibition rate in the siRNA2 group (83.09%). Similarly, the expression levels of TNF-α protein in the three siRNA groups were significantly lower than those of the negative control group, and the highest inhibition rate was found in the siRNA2 group (51.16%). The mRNA expression of interleukin (IL)-1β and IL-6 showed no significant difference among the siRNA groups and the negative control. The recombinant lentiviral shuttle plasmid was constructed, and electrophoresis revealed the polymerase chain reaction product to be 343 bp, while that of the empty vector was 306 bp; DNA sequencing showed partial insertion. The virus titer was calculated to be 2×106 TU/µl. In conclusion, RNAi lentiviral vector particles targeting the mouse TNF-α gene were successfully obtained in the present study. This method may be used to produce lentiviral vector for the in vivo study of RNAi gene therapy targeting TNF-α.

A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

Plasmid-Based shRNA Lentiviral Particle Production for RNAi Applications

Lentiviral vectors have become mainstream gene transfer vehicles for their ability to deliver and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express double-stranded RNA molecules, usually as short hairpin RNA (shRNA), enabling long-term gene silencing, and, when pseudotyped with a broad host glycoprotein envelope, allow a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories, making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focus exclusively on this step and compare the overall impact of the commercial transfection reagent FuGENE 6 with FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard enzyme-linked immunosorbent assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production.

Embryonic Stem Cell–Based Modeling of Tau Pathology in Human Neurons

Alterations in the microtubule (MT)-associated protein, tau, have emerged as a pivotal phenomenon in several neurodegenerative disorders, including frontotemporal dementia and Alzheimer's disease. Although compelling lines of evidence from various experimental models suggest that hyperphosphorylation and conformational changes of tau can cause its aggregation into filaments, the actual tau species and effective mechanisms that conspire to trigger the degeneration of human neurons remain obscure. Herein, we explored whether human embryonic stem cell-derived neural stem cells can be exploited to study consequences of an overexpression of 2N4R tau (two normal N-terminal and four MT-binding domains; n-tau) versus pseudohyperphosphorylated tau (p-tau) directly in human neurons. Given the involvement of tau in MT integrity and cellular homeostasis, we focused on the effects of both tau variants on subcellular transport and neuronal survival. By using inducible lentiviral overexpression, we show that p-tau, but not n-tau, readily leads to an MC-1-positive protein conformation and impaired mitochondrial transport. Although these alterations do not induce cell death under standard culture conditions, p-tau-expressing neurons cultured under non-redox-protected conditions undergo degeneration with formation of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Our data support a causative link between the phosphorylation and conformational state of tau, microtubuli-based transport, and the vulnerability of human neurons to oxidative stress. They further depict human embryonic stem cell-derived neurons as a useful experimental model for studying tau-associated cellular alterations in an authentic human system.

Histone deacetylase 6 gates the synaptic action of acute stress in prefrontal cortex

The prefrontal cortex (PFC), a region responsible for high-order cognitive functions, such as decision-making, attention and working memory, is highly influenced by stress and corticosteroid stress hormones. Recently it has been shown that acute stress affects PFC functions by potentiating glutamatergic transmission via a mechanism dependent on glucocorticoid receptor (GR) and its downstream target, serum and glucocorticoid-inducible kinase (SGK). To identify the key regulators of stress responses, we examined the role of histone deacetylase 6 (HDAC6), a unique member of the HDAC family that could regulate the GR chaperone protein heat shock protein 90 (HSP90), in the synaptic action of acute stress in PFC. We found that HDAC6 inhibition or knockdown blocked the enhancement of glutamatergic transmission and glutamate receptor trafficking by acute stress in vivo or corticosterone treatment in vitro. In addition, HDAC6 inhibition blocked the up-regulation of SGK in animals exposed to acute stress. HSP90 inhibition or knockdown produced a similar blockade of the acute stress-induced enhancement of glutamatergic signalling. These findings have identified HDAC6 as a key molecule gating the effects of acute stress on synaptic functions in the PFC.

Inhibition of HIV‐1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.

Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

BackgroundA critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells.ResultsWe synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays.ConclusionTat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs.

Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles

Transgenic HIV-1-derived lentiviral particles are at the forefront of current gene therapy and tissue engineering initiatives, which will require optimal protocols for large-scale production of clinical-grade therapeutic lentiviruses. Production of latest-generation self-inactivating lentiviral particles requires cotransfection of mammalian production cell lines with two helper plasmids along with the lentivector, whose transgene-encoding expression cassette is the only genetic information stably transduced into target chromosomes. Capitalizing on a recently designed lentiviral expression vector family, we conducted rigorous analysis of production-relevant parameters including transfection, cell density, media composition, temperature, relative (helper) vector concentrations and genetic configuration. Comparative analysis of lentiviral particle performance (VP) was based on the viral titer (reflecting the number of transduction-competent lentiviral particles) relative to the number of lentiviral particles produced (correlating with p24 production levels) (VP=titer/viral particle number). Optimal lentiviral production parameters, resulting in up to 132-fold greater VP compared to standard protocols, required (i) CaPO 4-based transfection (ii) of helper plasmids and lentivector at a fixed concentration ratio (helper plasmid I:helper plasmid II:lentivector=1:1:2) (iii) into 1×10 5 human embryonic kidney cells/cm 2 (HEK293-T) (iv) cultivated at 37 °C (v) in Advanced D-MEM medium supplemented with (vi) 2% fetal calf serum, (vii) and a culture additive containing 0.01 mM cholesterol, 0.01 mM egg's lecithin and 1× chemically defined lipid concentrate. (viii) Furthermore, constitutive transgene expression units placed in a forward polyadenylation site (pA)-free orientation relative to the lentivector backbone resulted in optimal transgene transduction/expression. Our studies suggest that detailed knowledge of lentivector design and the production of lentiviral particles will advance large-scale manufacturing of clinically relevant lentiviruses for future gene therapy applications.