The best known lentivirus is the human immunodeficiency virus, which causes aids. When i infect 293t cells with the viral particles to calculate the viral titer i get a very high efficient gfp signal on facs. Some additives such as polybrene can enhance the transduction efficiency. Enhancing lentiviral and alpharetroviral transduction. The viral rna genome is released and reversetranscribed to produce dna. Lentivirus is prepared and titered as described in rubinson et al. This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. The lentivirus based expression of functional rgr can be used to study rgr in cultured cells and to test in vivo transduction of quiescent rpe. Reconstructing the immune system with lentiviral vectors ncbi. We obtain transduction efficiencies of 15% in the nk92 cell line and 3040% in lnk, yt, and derl7 cell lines. Systemic delivery of lentiviral vector lv in immunocompetent mice leads to. Immune responses to lentiviral vectors bentham science. Thirdgeneration lentivirus vectors efficiently transduce.
Efficient lentiviral transduction of numerous cell lines has been demonstrated previously 11. Protocol for transduction of human embryonic stem cells. Stability of lentiviral vectormediated transgene expression in the brain in the presence of systemic antivector immune responses evelyn abordoadesida,1,2 antonia follenzi,3 carlos barcia,1 sandra sciascia,1 maria g. To obtain the desired infection rate low and high moi. Enhancing lentiviral and alpharetroviral transduction of human. Lentiviral transduction of endothelial cells mellorlab.
This system is ideal for many primary cells as well as immobilized cells. In contrast, lcmv and mulvpseudotyped lentivirus produced transgene expression exclusively in astrocytes. Alteration of t cell immunity by lentiviral transduction. Cyclosporine h overcomes innate immune restrictions. Cold spring harbor laboratory press, cold spring harbor, ny, usa, 2007. Several studies have reported different methods to enhance transduction efficiency in hematopoietic stem cells hsc.
Es cells are cultured according to standard procedures. The lentivirusbased expression of functional rgr can be used to study rgr in cultured cells and to test in vivo transduction of quiescent rpe. Lentiviral transduction of immune cells request pdf. Cyclosporine h overcomes innate immune restrictions to improve lentiviral transduction and gene editing in human hematopoietic stem cells carolina petrillo,1,2 lucy g. Methodology article open access simplified production and concentration of lentiviral vectors to achieve high transduction in primary human t cells adam p cribbs, alan kennedy, bernard gregory and fionula m brennan abstract background.
Pdf reconstructing the immune system with lentiviral vectors. Lentiviral transduction of immune cells springer nature experiments. The scfv displaying lentiviral particles gained relative specificity for transduction of target cells. Innate immune receptors, such as pattern recognition receptors that recognize foreign genomic material, are likely involved in triggering apoptosis of nk cells following viral transduction. Introduction of antigen into dendritic cells dc by using lentiviral vectors leads to antigen. For the past two decades, cells engineered with lentiviral vectors lvs have emerged and are in continuous development for immune therapy of solid and hematologic malignancies 38. We show that these dc are activated by lentiviral transduction in cell cultures and in vivo. Spinoculation of suspension cells this protocol is for transduction of suspension cells jurkat t cells, pbmc, pbl, b cells etc. The day before transduction, trypsinize and count the cells, plating 0.
Spinoculation of suspension cells system biosciences. Hiv1 derived lentiviral vectors lvs transduce dcs at high efficiency but their effects on dc functions have not been carefully studied. Gammaretroviruses and lentiviruses are subtypes of retroviruses, which contain an rna genome that is converted to dna in the transduced cell. Protocol 3 lentivirus transduction into target cell. Lentivirusmediated gene transfer to human epidermis. Antonia follenzi, manuela battaglia, angelo lombardo, andrea annoni, maria grazia roncarolo, luigi naldini. Many such viruses have been the basis of research using viruses in gene therapy, but the lentivirus is. Alteration of t cell immunity by lentiviral transduction of human monocytederived dendritic cells. Cells were infected with gfp lentivirus for 48 hours in the presence of polybrene or the viraductin lentivirus transduction kit. Not all viral particles floating in culture medium can eventually transduce or infect the cells.
Methodology article open access simplified production. Use of lentiviral vectors lvs offers multiple advantages for gene replacement therapy, because they combine efficient delivery, ability to transduce proliferating and resting cells, capacity to integrate into the host chromatin to provide stable longterm expression of the transgene, absence of any viral genes in the vector and absence of. Lentiviral transduction of b cells may202009 hello all, i am transfecting 293t cells with packaging lentiviral plasmids to generate a lentiviral particles carrying gfp and another gene. Jul 15, 2003 human t lymphocytes transduced by lentiviral vectors in the absence of tcr activation maintain an intact immune competence simona cavalieri from the institute for cancer research and treatment, university of torino medical school, candiolo torino. Lentiviral vectors for gene transfection in car tcell. Methodology article open access simplified production and. 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. Feeder free cultures of hescs must be used to avoid loss of lentivirus into the feeder layer. Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols.
Lowenstein1,4 abstract lentiviral vectors are promising tools for gene therapy in the cns. Hiv1 lentiviral vector immunogenicity is mediated by tolllike. Oct 22, 2014 this construct is easily adapted for the delivery of the chimeric antigen receptor consists of an intracellular tcell receptor cd3zeta chain signaling domain that induces tcell activation, a costimulatory 41bb domain that enhances tcell mediated responses and anticd19 antibody fragments that bind to cd19. Transduction of smc with lentivirus was significantly higher than both ad and aav2. Lentiviral particles can efficiently infect a broad range of cell types, including both dividing and nondividing cells. Transduction efficiency varies in different cell lines, and polybrene may be toxic to some cell lines.
To identify the optimal time of transduction, cells were transduced at different times after stimulation. Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and. Gene transfer into mammalian cells has been of crucial importance for studies determining the role of specific genes in the differentiation and cell fate of various hematopoietic lineages. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then. Alteration of t cell immunity by lentiviral transduction of. The reduced efficacy of viral transduction of nk cells compared to t cells may in part be related to the innate properties that characterize nk cells. Human t lymphocytes transduced by lentiviral vectors in. Since, especially the naive t cells will provide a longlasting immune reconstitution to patients these are the cells that need to be transduced for. Simplified production and concentration of lentiviral vectors to. Pdf lentiviral vector gene transfer into human t cells. Can use standard mefs, but will need to add exogenous lif.
Among various technologies available for gene transfer, lentiviral mediated transduction has been successful in introducing genes into nk cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility. Dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1 infection. Giordano,1,2 francesco piras,1,2 ivan cuccovillo,1 sarah j. For example, if you intend to infect 50 thousands cells at moi of 10, you need 10x50,000 5. However, despite these advances, the transduction of primary human t cells is still challenging and methods to achieve efficient gene transfer are often expensive and timeconsuming.
Simplified production and concentration of lentiviral vectors. As important immune modulating cells for immunotherapy and vaccine applications, dcs play critical roles in activating the host immune response. Thorne,3,5 giulia unali,1,2,5 giulia schiroli,1 anna m. Alteration of t cell immunity by lentiviral transduction of human. During the course of the transduction, cells are periodically passaged using a fresh working solution of 1 mgml dispase ii diluted in dmemf. Protocol 3 lentivirus transduction into target cell lines.
But the problem is, when i infect mice spleen cells specially macs separated b cells as my 1st interest nothing really happens anybody ever used lentivirus for transduction of cells. Targeting lentiviral vector expression to hepatocytes limits transgenespecific immune response and establishes longterm expression of human antihemophilic factor ix in mice. Production and concentration of lentivirus for transduction. Since an exact cell cound cannon be determined from the previous days plating due to extreme aggregation of the cells ranging from 50 to 200 cells per clump, a precise moi cannot be calculated. Lentivirusmediated in vitro gene transfer into nile tilapia spermatogonial stem cells s. Petit,3 fatima ahsan,3 mahdad noursadeghi,3 simon clare,4. Moreover, they do not induce a cellular immune response. On the day of transduction, thaw your lentiviral stock and dilute the lentiviral stock into complete. Expression of a recombinant human rgr opsin in lentivirus. Semiautomated closed system manufacturing of lentivirus.
Lentiviral gene transduction in human and mouse nk cell lines. Lentivirus transduction protocols for reference only many factors can affect transduction efficiency. Clinical use of lentiviral vectors leukemia nature. Infection protocol for recombinant lentivirus 9601 medical. This construct is easily adapted for the delivery of the chimeric antigen receptor consists of an intracellular tcell receptor cd3zeta chain signaling domain that induces tcell activation, a costimulatory 41bb domain that enhances tcell mediated responses and anticd19 antibody fragments that bind to cd19. Now it is clear that use of the survival cytokines, il2 or il7, allows an efficient lentiviral vector gene transfer and could preserve a functional tcell repertoire while maintaining an appropriate proportion of naive and memory t cells. However, antitransgene immune response results in clearance of transduced. Persistence of gfptg apc in secondary lymphoid organs 7.
Lentiviral vectors are efficient gene delivery vehicles suitable for delivering longterm transgene expression in various cell types. This protocol was adapted from lentivirus transduction of hematopoietic cells, chapter 7, in gene transfer. But the problem is, when i infect mice spleen cells specially macs separated bcells as my 1st interest nothing really happens anybody ever used lentivirus for transduction of cells. When these antibiotics are added to the growth medium of the host cells, they kill off any cells that have not incorporated the lentiviral genome and those cells that survive can be expanded to create stable cell lines, which have incorporated the lentiviral genome and harbor the genetic information encoded by that genome. C green fluorescence of hek293t cells during lentivirus production. A distinct subset of highly proliferative and lentiviral vector lv. Differences between transfection and transduction blog. Anticancer immunotherapy is based on enhancing the immune response to tumor cells. Dna is then stably integrated into the host genome at a random position by the viral integrase enzyme.
In contrast, primary human t cells are refractory to. Titration of the ratio of three plasmids on lentivirus yield. Protective antiviral immunity after vaccination with dc transduced with. Now it is clear that use of the survival cytokines, il2 or il7, allows an efficient lentiviral vector gene transfer and could preserve a functional tcell repertoire. On the other hand, transfection or transduction of rna is always transient. Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentivirus lentivirus are a family of viruses that are responsible for notable diseases like hiv, which infect by inserting dna into their host cells genome. Robust and persisted transduction efficiency of blood cells with lvs, resulted into a broad range of target cells for immune therapeutic. Most gene therapy lentiviral vector lv production platforms employ. 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. Lentivirusmediated in vitro gene transfer into nile. Modification of dcs using lvs may lead to important applications in transplantation, treatment of cancer. Protocol for transduction of human embryonic stem cells hescs using lentiviral vectors.
Innate immune receptors, such as pattern recognition receptors that recognize foreign. Protocol 8 lentiviral transduction of hematopoietic stem cells. Influence of promoters and direct comparison of lentiviral vectors with other viral gene delivery systems. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. A simple protocol for producing hightiter lentivirus chen chu.
Publication processes organization and format errata, author corrections, retractions illustrations and. Protocol for lentiviral transduction of human cells. We have standardized methods of lentiviral infection in human and mouse nk cell lines. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Unlike the short term protein expression observed using transient transfection approaches, generating cell lines using lentiviral vectors enables longterm protein expression studies. Lentiviral transduction unit tu to be used moi x number of cells to be infected. Plate hek 293t cells in a 10 cm dish to be 8090% confluent after 24 h. Since, especially the naive t cells will provide a longlasting immune reconstitution to patients these are the cells that need to be transduced for effective gene therapy. Publication processes organization and format errata, author corrections, retractions. Highly efficient transduction of lymphoma cells was achieved, 50% of cells were transduced when moi 1 was used. Thirdgeneration lentivirus vectors efficiently transduce and phenotypically modify vascular cells.
Thirdgeneration lentivirus vectors efficiently transduce and. Lentiviral transduction of dendritic cells confers protective antiviral. To make stable cell line, taeget has to be intergraded into host cells genome for a stable, constitutive expression. Next day cells may be transplanted by injection or cultured further. Semiautomated closed system manufacturing of lentivirus gene. Use a lentiviral vector to transduce target cells which naturally integrates its dna into the host cell genome in a random manner. The cells were incubated with the particlecontaining medium for 1224 h, after which normal medium was replaced.
Frontiers genetic manipulation of nk cells for cancer. Lentivirus lente, latin for slow is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in the human and other mammalian species. Dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1. Addition of puromycin will allow you to select for cells that are stably expressing your shrna of interest. Capable of transduction of nondividing cells can generally transduce hard totransfect cell lines transduction efficiency can reach 100% control of delivery straightforward to deliver a single copy to cells easy to monitor copy number c. Various amounts of lentivirus or lvlps ng p24 protein were added to 2.
Like hiv1, equine infectious anaemia virus eiav is a lentivirus that infects nondividing cells, but it differs from hiv1 in that it is relatively sensitive to. Nov 12, 20 however, despite these advances, the transduction of primary human t cells is still challenging and methods to achieve efficient gene transfer are often expensive and timeconsuming. Transfection is efficient on adherent immortalized cells but primary and stem cells require transduction. The number of transductions per kit is based on use of a 24well plate. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of nonstandard equipment to transduce primary human t cells. Capable of transduction of nondividing cells can generally transduce hard totransfect cell lines transduction efficiency can reach 100% control of delivery straightforward to deliver a single copy to cells easy to. Simplified production and concentration of lentiviral. Randomly integration that such as by transfection demonstrates large variety expression dependent upon the transcription levels at integration sites. Good practice on the assessment of gmorelated aspects in the. The immune response to lentiviraldelivered transgene is modulated. Human t lymphocytes transduced by lentiviral vectors in the absence of tcr activation maintain an intact immune competence. Protocol 8 lentiviral transduction of hematopoietic stem. Duplicate or triplicate wells for each lentiviral construct and control to be used. It is possible that a selflimited immune response allowed cells expressing low levels of antigen to escape immune clearance.
Lentiviral vector gene transfer into human t cells. Whats the advantages for using lentivirusto generate stable cells. Targeting lentiviral vector expression to hepatocytes limits. An external file that holds a picture, illustration, etc. Lentiviral transduction of immune cells springer for. Beads were added at start of the culture according to the user manual. The transduced arpe19 cells remain able to process all transretinol, and the expressed protein is capable of binding to the alltransretinal chromophore. Targeting lentiviral vector expression to hepatocytes. When transducing a lentiviral construct into a cell line for the first time, a range of volume or moi should be tested. Engineering lentiviral vectors to have the capacity to transduce specific cell types is of great interest to advance the translation of lentiviral vectors towards the clinic. 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. Delivering sacas9 mrna by lentiviruslike bionanoparticles. Low viability cells are sensitive to transduction enhancer.
Human t lymphocytes transduced by lentiviral vectors in the. If the original lentivirus stock is 109 tuml, then you will need 0. We first tested whether production of lv by transient transfection induces an innate immune response in hek293t producer cells. Trim5 is an innate immune sensor for the retrovirus capsid. The reduced efficacy of viral transduction of nk cells compared to t cells may in part be. Lentivirus transduction protocols for reference only. The lentiviral particle binds to the cell membrane and enters the host cell. Systematic improvement of lentivirus transduction protocols. Apr 20, 2011 like hiv1, equine infectious anaemia virus eiav is a lentivirus that infects nondividing cells, but it differs from hiv1 in that it is relatively sensitive to human trim5. Lentiviral vector production titer is not limited in.
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