As for nk92 cells, higher ldlr expression levels enhanced vsvg lentiviral transduction in the presence of ggpp figure 6c, without concerning the viability before or after transduction on primary nk cells figures 6b and 6d. Using a lentiviral based vector system expressing enhanced green fluorescent protein egfp under the control of the murine stem cell virus promoter mscv, we found that multiplicities of infection mois of 1, 10, and 100 transduce 70%, 88%, and 95% of the cells. Their function resembles that of tissue macrophages and, as such, they share many properties with both peripheral macrophages and monocytes. However, these recombinant viral vectors usually have broad specifi city and transduce. Pdf in vivo modulation of gene expression by lentiviral. The invitrogen neon transfection system delivers dna.
Even though more efficient lentiviral techniques are being developed, safety concerns pertaining to human therapeutic use. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Lentiviral expression has many advantages over other viruses, including the ability to infect both proliferating and nonproliferating cells. Lentivirus technologies for modulation of the immune system. In previous studies, we have shown that lentiviral modification of dcs reduces the t helper type 1 activation function. Methods to concentrate viral supernatants to achieve a higher titer are also described. Pdf lentiviral vector gene transfer into human t cells. For viral transduction, lamb cells were replated on laminincoated culture dishes 11. The transfer vector with the transgene and helper plasmids are transiently transfected into a packaging cell line such as hek293 cells, where they get assembled. Cyclosporine h improves the multivector lentiviral.
As main players of the humoral immune response, b lymphocytes. Aug 22, 2019 lentiviral vectors on the other side are less genotoxic and represent a safer option, but the efficiency of lentiviral transduction of primary nk cells has remained variable and may require multiple rounds of transduction andor post transduction cell enrichment to achieve acceptable transgene expression. The role that nk cells play is analogous to that of cytotoxic t cells in that they provide rapid responses to virus. Our results demonstrate that stimulation of nk cells with interleukin il2 and il21 supports efficient transduction using a vsvg pseudotyped lentiviral vector. Adipocyte stem cell isolation, transduction of adiposederived mesenchymal stem cells admscs with recombinant lentiviral particles. Initial cell seeding and cell confluency after 24 hours are determined based on the rate of cell growth of the cells used for transduction. Engineering lentiviral vectors to have the capacity to transduce. Pdf lentiviral gene transduction of mouse and human stem. Restrictions to improve lentiviral transduction and gene editing in human. The number of transductions per kit is based on use of a 24well plate. Transduction with lentiviral vectors altered the expression profile of host micrornas. Moreover, lentiviral vectors have also proven to be effective in transducing brain, liver, muscle, and retina in vivo without toxicity or immune responses. Gfp expression in clones from single cd341cd382 cells confirmed efficient, stable lentiviral transduction in 29% of early and lateproliferating cells. Alteration of t cell immunity by lentiviral transduction of.
Stable transduction of myogenic cells with lentiviral. Hiv1 lentiviral vector immunogenicity is mediated by tolllike. Natural killer cells, or nk cells, are a type of cytotoxic lymphocyte critical to the innate immune system. Cyclosporine h overcomes innate immune restrictions to. Lentiviral delivery of combinatorial carcrispri circuit into human. This was evident by i the participation of the immune system in the affected networks, ii the presentation of immune functions in.
We tested bx795 with t cells collected from multiple donors, and detected the effect of bx795 on t cell transduction, phenotype, cell growth and cell function. In peripheral blood b cells that had been activated with helper t cells murine thymoma el4 b5 and cytokines, multiply attenuated hiv1 derived. Thus, vsvg pseudotyped lentiviral preparations produced by transient transfection were shown to be heavily contaminated with socalled tubulovesicular structures of cellular origin. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then. Cellular innate immunity and restriction of viral infection. Vpxindependent lentiviral transduction and shrnamediated. Key words resting b cells, lentiviral vector, immune regulation, gene therapy, cell therapy, immune tolerance 1 introduction during recent years, different studies have provided evidence for a dual role of b cells in infection and autoimmunity. We provide detailed protocols for producing hightiter lentiviral supernatants by transient transfection and for measuring viral titers. Feb 02, 2021 it is characterized by the absence or lack of function of key immune cells, resulting in a severely compromised immune system and death by 1 year of age if untreated. Production of transgenic rats by lentiviral transduction of male germline stem cells f. Efficient delivery of lentiviral vectors into resting. Surfaceengineered lentiviral vectors for selective. Incubate overnight at 48 c or for 1 h at 37 c if you prepare this transduction plate the next morning. Our viraductin lentivirus transduction kit provides superior lentiviral transduction efficiencies in a variety of cell lines, even when compared to transductions in the presence of polybrene.
This process can be performed inside a living organism or in cell culture. Innate immune response is elicited early after infection onset, and. The immune response to lentiviraldelivered transgene is modulated. Lentiviral transduction of immune cells springer nature experiments. We used a lentiviral vector encoding functional wasp to genetically correct hspcs from three was patients and reinfused the cells after a reducedintensity conditioning regimen. Direct administration of lentiviral vectors has been shown to result in the transduction of dcs in vivo and therefore has been evaluated for the induction of antigenspecific immune responses in. Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Mar 01, 2003 we studied the transduction of primary human b lymphocytes and myeloma cells with lentiviral vectors.
Protein overexpression following lentiviral infection of. Cts immune cell sr supports lentiviral transduction and expansion of genemodified t cells, and maintains. This protocol may be used with 96well, 48 well, 24well, 12well and 6well plates. One of the more successful variants has proven to be the expression of cars in natural killer nk cells carnk.
Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then be further differentiated into immune lineage cells including t, b, dendritic, or. Plate cells so that cell density will be 2550% confluent at the time of transduction. Lentiviral transduction of microglial cells balcaitis. Systemic delivery of lentiviral vector lv in immunocompetent mice leads to efficient in vivo cell transduction and expression of the encoded protein. Pdf lentiviral transduction of human dendritic cells. This system is ideal for many primary cells as well as immobilized cells. On the day of transduction, thaw your lentiviral stock and dilute the lentiviral stock into complete. We found that bx795 promotes rb3401f lentiviral transduction of human primary t cells, without dramatic change in cell growth and t cell functions. This dc immune cell coculture system encompasses a full repertoire of immune cells. All three patients showed stable engraftment of waspexpressing cells and improvements in platelet counts, immune functions, and clinical scores.
In this study, we aimed to optimize a protocol for transduction of nk cells with vsvg pseudotyped lentiviral vectors that does not rely on stimulation with feeder cells or longterm expansionactivation of the cells in an attempt to define a fast and efficient protocol that can both be practically used for experimental studies of nk cell. This chapter describes the methods we use to transduce mouse and human hematopoietic stem cells hscs and human embryonic stem cells hescs. Inhibition of intracellular antiviral defense mechanisms. In particular, host antiviral factors referred to as restriction factors targeting the retroviral life cycle can hamper lv transduction efficiency. In addition, the effects of cnx on antigen presentation were explored by using lentiviral transduction or peptide loading of dcs. Hiv1 derived lentiviral vectors lvs transduce dcs at high efficiency but their effects on dc functions have not been carefully studied. Lentiviral transduction of apoai into hematopoietic. They have a broad host cell range that also includes cell types such as primary neurons, lymphocytes, and macrophages. Robust and persisted transduction efficiency of blood cells with lvs, resulted into a broad range of target cells for immune therapeutic. Although lentiviral vectors lvs transduce efficiently several types of nondividing cells,14 through the active transport of the viral preintegration complex into the nucleus, 15 quiescent t cells are not permissive to hiv replication 1619 or to hivvector transduction.
Targeted transduction of lentiviral vectors to cell line in vitro. The table below illustrates each of these components. Mustang bio provides updates on its lentiviral gene therapies. Gene transfer into t lymphocytes is currently being tested for the treatment of lymphohematologic disorders. Lentiviral transduction of mammalian cells for fast, scalable and highlevel production of soluble and membrane proteins nat protoc. Modification of dcs using lvs may lead to important applications in transplantation, treatment. We studied the transduction of primary human b lymphocytes and myeloma cells with lentiviral vectors. Mustang bio provides updates on its lentiviral gene. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell. Huang h, zhang c, wang b, wang f, pei b, cheng c, yang w, zhao z. Successfully transfected primary peripherally mobilized. Efficient transduction of myeloid cells by an hiv1derived lentiviral vector that packages the vpx accessory protein. Lentiviral vectors have a broad host cell range that includes cell types such as neurons, lymphocytes, and macrophages.
However, it takes weeks to generate sizable stable cell colonies for counting and calculating the titer results. The day before transduction, trypsinize and count the cells, plating 0. Although largescale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentivirusinduced dendritic cells idc for immuneregenerative. These data demonstrate that dispersion of islets prior to lentiviral transf. This was evident by i the participation of the immune system in the affected networks, ii the presentation of immune functions in the described canonical pathways, iii the increase of the participation of the immune. Microglial cells are the resident immune cells of the central nervous system. Lentiviral mediating genetic engineered mesenchymal stem. Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. We next examined whether cd20bearing virus can transfer genes into cells expressing cd20 in a cell specific manner. Oct 01, 2015 stable transduction of quiescent t cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins blood, 112 2008, pp.
Immune modulatory cell therapy for hemophilia b based on. A distinct subset of highly proliferative and lentiviral. Transduction efficiency can be optimized using the plko. Frontiers engineering nk cells for car therapyrecent. Lentiviral transduction of mammalian cells for fast, scalable.
Production of transgenic rats by lentiviral transduction of. Lentiviral vectors containing the human immunodeficiency. Thorne,3,5 giulia unali,1,2,5 giulia schiroli,1 anna m. Lentiviral delivery of combinatorial carcrispri circuit into. On the day of transduction, thaw your lentiviral stock and dilute the lentiviral.
The development of chimeric antigen receptor car t cell therapy has introduced a new and effective strategy to guide and promote the immune response against tumors in the clinic. Jul 15, 2003 although lentiviral vectors lvs transduce efficiently several types of nondividing cells,14 through the active transport of the viral preintegration complex into the nucleus, 15 quiescent t cells are not permissive to hiv replication 1619 or to hivvector transduction. Combined lentiviral and rnamediated crisprcas9 delivery. Overcoming the challenge of transduction of human tcells. One striking similarity is the difficulty with which these cells can be genetically manipulated via transfection or transduction. Reconstructing the immune system with lentiviral vectors. Lentiviral vectors for immune cells targeting ncbi nih. Lentiviral transduction of immune cells springerlink. Modulation of immune responses in lentiviral vectormediated gene. Lentiviral transduction of cell lines for the transduction of cell lines, 2105 cells per well were seeded into 24well plates. Aug 22, 2012 integrase defective lentiviral vectors idlv represent a promising delivery system for immunization purposes. Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment.
Therefore, transduced t cells are unlikely to carryover viral ags and induce antilv immune response by exposing lvderived epitopes in classi major. Cd38 human hematopoietic cells by hiv1based lentiviral vectors. Rv transduction often results in an inversion of the physiologic cd4cd8 ratio, enrichment in memory cells associated. Lentiviral delivery of combinatorial carcrispri circuit. Lentiviral transduction of the murine lung provides. Here, we discuss optimization of technologies for human immunodeficiency virus hivbased gene transfer into murine and human progenitor and immune cell. K562based aapc can be used off the shelf and represent an inexhaustible, costefficient, single reagent for t cell expansion. In this report, we studied lvmodified dcs supraphysiologically expressing cnx employing an autologous immune cell stimulation system. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. Use one well per lentiviral construct to be used, for a maximum of 5.
Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. Above all, among 5 kinds of statins, rosuvastatin was the most potent compound to both upregulate the ldlr on nk. Pdf cellular innate immunity and restriction of viral infection. Healthy and subconfluent cells are required for successful transduction with lentiviral activation particles. Giordano,1,2 francesco piras,1,2 ivan cuccovillo,1 sarah j.
This new formulation neutralizes electrostatic repulsions between membrane and viral particles. Pdf utilization of a lentiviral system for the generation. Day 2 prepare a mixture of complete medium with polybrene sc4220 at a. In comparison, t lymphocyte manipulation by lentiviral transduction. Lentiviral transduction of immune cells request pdf. Gfp expression was used to measure the transduction efficiency. Although their b cells are normal in number, they are not functional. Cyclosporine h overcomes innate immune restrictions to improve.
Patients with xscid have no t cells or natural killer nk cells. Production of transgenic rats by lentiviral transduction. Based on encouraging results from macrophages, we hypothesized that lentiviral vectors might provide a valuable tool in the transduction of microglial cells. The human immunodeficiency virus type 1 hiv1based lentiviral vectors are capable of transducing both dividing and nondividing cells and have become the tool of choice because of longterm stable gene transfer both in vitro and in vivo. Sep 23, 2020 background adoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary t cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long dna sequences. Lentivector expression systems guide to packaging and. We evaluated the effect of lentiviral vectors on myeloid dendritic cells.
Dec 29, 2004 microglial cells are the resident immune cells of the central nervous system. The choice of sbis lentiviral system for experimental studies is driven by functional considerations, including increased productivity and transduction efficiency. Efficient delivery of lentiviral vectors into resting human. Lentiviral transduction of mammalian cells for fast. In peripheral blood b cells that had been activated with helper t cells murine thymoma el4 b5 and cytokines, multiply attenuated hiv1derived vectors pseudotyped with vesicular stomatitis virus vsv genvelope protein achieved the expression of green fluorescence protein gfp in 27%.
Human t lymphocytes transduced by lentiviral vectors in. However, the t cell receptor tcr activation and sustained proliferation required for retroviral vector transduction may impair the halflife and immune. Nov 01, 2004 background dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1 infection. Simplified illustration of the lentiviral transduction mechanism. Lentiviral hematopoietic stem read the full ar ticle. Lentiviral transduction of human dendritic cells springerlink. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce cd341 and cd341cd382 cells. Engineered lentivector targeting of dendritic cells for in vivo. D elivery solutions for immune cells thermo fisher scientific. Targeting lentiviral vectors to specific cell types in vivo. Lentiviral calnexinmodified dendritic cells promote.
Oct 28, 2011 a further disadvantage of vsvg expressing lentiviruses in the transduction of immune cells is the potential activation of these cells by lentiviral particles. Frontiers efficient and robust nkcell transduction with. Article information, pdf download for cell loss during pseudoislet formation. Cell loss during pseudoislet formation hampers profound. Cts immune cell sr for serum free culture and expansion of. Alteration of t cell immunity by lentiviral transduction. Delivery of genes or other genetic materia by a vector is termed transduction and the infected. Adipocyte cells were isolated from liposuction tissue with mechanical and enzyme digestion. Dendritic cells dcs are potent antigenpresenting cells apcs that play a pivotal role in stimulating antigenspecific t cells in vivo 1, 2. Biosafety level s2 is needed for lentiviral transduction. Prospects in innate immune responses as potential control. Stable transduction of quiescent t cells without induction.
Frontiers recent advances in lentiviral vaccines for hiv. The reasons behind poor permissiveness of primary human hematopoietic cells to gene transfer partly reside in the retroviral origin of lentiviral vectors lvs. Lentiviruses in gene therapy clinical research nature. Delivery solutions for immune cells thermo fisher scientific. Since, especially the naive t cells will provide a longlasting immune reconstitution to patients these are the cells that need to be transduced for. More recently, in an attempt to enhance its utility, this method has been expanded to novel cell types. Human t lymphocytes transduced by lentiviral vectors in the. Efficient transduction of primary human b lymphocytes and. Viral titer can be determined by transduction of ht1080 or hela cells, and followed by antibiotic selection of stable clones. The efficiency of lentivirus infection of target cells, however, tends to be lower. Immune cells neon electroporation system lipofectamine 3000 reagent for lentiviral production crisprcas9 speci. However, efficient transduction of therapeutic gene into human t cells and further cell expansion are challenging.
Protocol is the same as with retroviral transduction except the packaging plasmids differ. Lentiviral transduction of microglial cells wiley online library. Pdf lentiviral calnexinmodified dendritic cells promote. Implications for lentiviral gene therapy in human hematopoietic cells. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graftversushost disease. Using a lentiviral based vector system expressing enhanced green fluorescent protein egfp under the control of the murine stem cell virus promoter mscv, we found that multiplicities of infection mois of 1, 10, and 100 transduce 70%, 88%, and 95% of the cells, respectively. Cyclosporine h overcomes innate immune restrictions to improve lentiviral transduction and gene editing in human hematopoietic stem cells carolina petrillo,1,2 lucy g. Cts immune cell sr supports expansion of dynabeads cd3cd28 ctsactivated polyclonal t cells and rem expanded t cells when supplemented into commonly used t cell culture media. Simian immunodeficiency virusvpx for improving integrase. Petit,3 fatima ahsan,3 mahdad noursadeghi,3 simon clare,4.
Pdf efficient transduction of primary human b lymphocytes. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then be further differentiated into immune lineage cells including t, b, dendritic, or natural killer cells. Rna, and protein into cells while avoiding the challenges faced by reagent and viral methods. Rosuvastatin enhances vsvg lentiviral transduction of. Multipotency of the cells was confirmed by their differentiation into adipocyte and osteocyte cells. For b10r cells, treatment with 6lm bx795 was started 30min prior to transduction and continued during the transduction process since we observed in preliminary experiments that this increases ef.
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