During the course of the transduction, cells are periodically passaged using a fresh working solution of 1 mgml dispase ii diluted in dmemf. Lentiviral transduction of immune cells request pdf. Pdf reconstructing the immune system with lentiviral vectors. Lentivirusmediated gene transfer to human epidermis. Protocol for transduction of human embryonic stem cells hescs using lentiviral vectors. In contrast, primary human t cells are refractory to. If the original lentivirus stock is 109 tuml, then you will need 0. 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. Immune responses to lentiviral vectors bentham science. Enhancing lentiviral and alpharetroviral transduction. Whats the advantages for using lentivirusto generate stable cells. Protocol 3 lentivirus transduction into target cell. Lentiviral transduction of dendritic cells confers protective antiviral.
For example, if you intend to infect 50 thousands cells at moi of 10, you need 10x50,000 5. Gammaretroviruses and lentiviruses are subtypes of retroviruses, which contain an rna genome that is converted to dna in the transduced cell. Es cells are cultured according to standard procedures. Introduction of antigen into dendritic cells dc by using lentiviral vectors leads to antigen. Lentivirus transduction protocols for reference only. Innate immune receptors, such as pattern recognition receptors that recognize foreign. 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. Simplified production and concentration of lentiviral. Most gene therapy lentiviral vector lv production platforms employ. To obtain the desired infection rate low and high moi. Good practice on the assessment of gmorelated aspects in the. In contrast, lcmv and mulvpseudotyped lentivirus produced transgene expression exclusively in astrocytes. 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. Giordano,1,2 francesco piras,1,2 ivan cuccovillo,1 sarah j.
Differences between transfection and transduction blog. 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. Methodology article open access simplified production. 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. Lentiviral transduction of immune cells springer for. Publication processes organization and format errata, author corrections, retractions illustrations and. Influence of promoters and direct comparison of lentiviral vectors with other viral gene delivery systems. Semiautomated closed system manufacturing of lentivirus gene. 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. Cyclosporine h overcomes innate immune restrictions. Publication processes organization and format errata, author corrections, retractions. The lentivirusbased expression of functional rgr can be used to study rgr in cultured cells and to test in vivo transduction of quiescent rpe.
Alteration of t cell immunity by lentiviral transduction of. Thorne,3,5 giulia unali,1,2,5 giulia schiroli,1 anna m. This system is ideal for many primary cells as well as immobilized cells. Enhancing lentiviral and alpharetroviral transduction of human. The reduced efficacy of viral transduction of nk cells compared to t cells may in part be. Frontiers genetic manipulation of nk cells for cancer. Alteration of t cell immunity by lentiviral transduction. Thirdgeneration lentivirus vectors efficiently transduce and phenotypically modify vascular cells. Lentiviral transduction unit tu to be used moi x number of cells to be infected.
However, antitransgene immune response results in clearance of transduced. Clinical use of lentiviral vectors leukemia nature. Lowenstein1,4 abstract lentiviral vectors are promising tools for gene therapy in the cns. Human t lymphocytes transduced by lentiviral vectors in. Randomly integration that such as by transfection demonstrates large variety expression dependent upon the transcription levels at integration sites. 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. 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.
Can use standard mefs, but will need to add exogenous lif. To make stable cell line, taeget has to be intergraded into host cells genome for a stable, constitutive expression. To identify the optimal time of transduction, cells were transduced at different times after stimulation. Many such viruses have been the basis of research using viruses in gene therapy, but the lentivirus is. 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. 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. Production and concentration of lentivirus for transduction. Plate hek 293t cells in a 10 cm dish to be 8090% confluent after 24 h. Anticancer immunotherapy is based on enhancing the immune response to tumor cells. Lentiviral transduction of immune cells springer nature experiments. The day before transduction, trypsinize and count the cells, plating 0.
Moreover, they do not induce a cellular immune response. Dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1. Efficient lentiviral transduction of numerous cell lines has been demonstrated previously 11. We show that these dc are activated by lentiviral transduction in cell cultures and in vivo. It is possible that a selflimited immune response allowed cells expressing low levels of antigen to escape immune clearance. Some additives such as polybrene can enhance the transduction efficiency. 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. C green fluorescence of hek293t cells during lentivirus production. 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. 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. 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. 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. Petit,3 fatima ahsan,3 mahdad noursadeghi,3 simon clare,4.
The viral rna genome is released and reversetranscribed to produce dna. When transducing a lentiviral construct into a cell line for the first time, a range of volume or moi should be tested. Among various technologies available for gene transfer, lentiviral mediated transduction has been successful in introducing genes into nk 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. The transduced arpe19 cells remain able to process all transretinol, and the expressed protein is capable of binding to the alltransretinal chromophore. The scfv displaying lentiviral particles gained relative specificity for transduction of target cells.
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. Duplicate or triplicate wells for each lentiviral construct and control to be used. Simplified production and concentration of lentiviral vectors. Cyclosporine h overcomes innate immune restrictions to improve lentiviral transduction and gene editing in human hematopoietic stem cells carolina petrillo,1,2 lucy g. Various amounts of lentivirus or lvlps ng p24 protein were added to 2. 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. 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. Robust and persisted transduction efficiency of blood cells with lvs, resulted into a broad range of target cells for immune therapeutic.
The cells were incubated with the particlecontaining medium for 1224 h, after which normal medium was replaced. 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. The best known lentivirus is the human immunodeficiency virus, which causes aids. Protocol for transduction of human embryonic stem cells. Systematic improvement of lentivirus transduction protocols. Protective antiviral immunity after vaccination with dc transduced with. Dna is then stably integrated into the host genome at a random position by the viral integrase enzyme. Lentivirusmediated in vitro gene transfer into nile tilapia spermatogonial stem cells s.
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. Cells were infected with gfp lentivirus for 48 hours in the presence of polybrene or the viraductin lentivirus transduction kit. Lentiviral gene transduction in human and mouse nk cell lines. Hiv1 derived lentiviral vectors lvs transduce dcs at high efficiency but their effects on dc functions have not been carefully studied.
When i infect 293t cells with the viral particles to calculate the viral titer i get a very high efficient gfp signal on facs. Simplified production and concentration of lentiviral vectors to. Lentiviral vectors are efficient gene delivery vehicles suitable for delivering longterm transgene expression in various cell types. Hiv1 lentiviral vector immunogenicity is mediated by tolllike. Beads were added at start of the culture according to the user manual. Transfection is efficient on adherent immortalized cells but primary and stem cells require transduction. Thirdgeneration lentivirus vectors efficiently transduce. Lentiviral vectors for gene transfection in car tcell. Persistence of gfptg apc in secondary lymphoid organs 7.
Transduction of smc with lentivirus was significantly higher than both ad and aav2. Highly efficient transduction of lymphoma cells was achieved, 50% of cells were transduced when moi 1 was used. Spinoculation of suspension cells system biosciences. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility. Protocol 8 lentiviral transduction of hematopoietic stem. Low viability cells are sensitive to transduction enhancer. Targeting lentiviral vector expression to hepatocytes limits. Systemic delivery of lentiviral vector lv in immunocompetent mice leads to. Addition of puromycin will allow you to select for cells that are stably expressing your shrna of interest. Lentivirus transduction protocols for reference only many factors can affect transduction efficiency. Delivering sacas9 mrna by lentiviruslike bionanoparticles. 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.
Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and. Semiautomated closed system manufacturing of lentivirus. Lentiviral vector production titer is not limited in. As important immune modulating cells for immunotherapy and vaccine applications, dcs play critical roles in activating the host immune response.
Infection protocol for recombinant lentivirus 9601 medical. Pdf lentiviral vector gene transfer into human t cells. Dendritic cells dcs are professional antigenpresenting cells that play important roles during human immunodeficiency virus type 1 hiv1 infection. Targeting lentiviral vector expression to hepatocytes limits transgenespecific immune response and establishes longterm expression of human antihemophilic factor ix in mice. Targeting lentiviral vector expression to hepatocytes. 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. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells 80% that can then.
Lentiviral transduction of endothelial cells mellorlab. Lentiviral vector gene transfer into human t cells. We have standardized methods of lentiviral infection in human and mouse nk cell lines. 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. Trim5 is an innate immune sensor for the retrovirus capsid. The immune response to lentiviraldelivered transgene is modulated.
Protocol for lentiviral transduction of human cells. Next day cells may be transplanted by injection or cultured further. A simple protocol for producing hightiter lentivirus chen chu. 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. Human t lymphocytes transduced by lentiviral vectors in the absence of tcr activation maintain an intact immune competence. Expression of a recombinant human rgr opsin in lentivirus. 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.
Protocol 3 lentivirus transduction into target cell lines. Since, especially the naive t cells will provide a longlasting immune reconstitution to patients these are the cells that need to be transduced for. Unlike the short term protein expression observed using transient transfection approaches, generating cell lines using lentiviral vectors enables longterm protein expression studies. The lentiviral particle binds to the cell membrane and enters the host cell. We first tested whether production of lv by transient transfection induces an innate immune response in hek293t producer cells. Lentivirusmediated in vitro gene transfer into nile. 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. On the day of transduction, thaw your lentiviral stock and dilute the lentiviral stock into complete. Antonia follenzi, manuela battaglia, angelo lombardo, andrea annoni, maria grazia roncarolo, luigi naldini. Thirdgeneration lentivirus vectors efficiently transduce and. 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. 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. An external file that holds a picture, illustration, etc.
Feeder free cultures of hescs must be used to avoid loss of lentivirus into the feeder layer. Protocol 8 lentiviral transduction of hematopoietic stem cells. Modification of dcs using lvs may lead to important applications in transplantation, treatment of cancer. A distinct subset of highly proliferative and lentiviral vector lv. On the other hand, transfection or transduction of rna is always transient. Not all viral particles floating in culture medium can eventually transduce or infect the cells. 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. This protocol was adapted from lentivirus transduction of hematopoietic cells, chapter 7, in gene transfer. The number of transductions per kit is based on use of a 24well plate. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. 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. 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.
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. Alteration of t cell immunity by lentiviral transduction of human monocytederived dendritic cells. This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. 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. Alteration of t cell immunity by lentiviral transduction of human. Human t lymphocytes transduced by lentiviral vectors in the. 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. Titration of the ratio of three plasmids on lentivirus yield. Use a lentiviral vector to transduce target cells which naturally integrates its dna into the host cell genome in a random manner. Methodology article open access simplified production and. Lentiviral particles can efficiently infect a broad range of cell types, including both dividing and nondividing cells. Lentivirus is prepared and titered as described in rubinson et al.
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