The absence of inflammatory infiltrates in grafts suggests that transduction does not activate the immune system. In terms of vector design, 2nd and 3rd generation lentiviral systems provided by addgene separate transfer, envelope, and packaging components of the virus onto different vectors. In vivo administration of lentiviral vectors triggers a. When cell membranes from the arpe19 cells were incubated with 3halltransretinal, the chro. Pathogenic lentiviruses cause severe immunologic or neurologic diseases in humans and other mammals. Spinoculation of suspension cells system biosciences.
Chimeric antigen receptor redirected t cells cart cells have achieved inspiring outcomes in patients with b cell malignancies, and are now being investigated in other hematologic malignancies and solid tumors. Cold spring harbor laboratory press, cold spring harbor, ny, usa, 2007. The number of cells transduced should be enough that they can grow out in a reasonable amount of time, but not so many that they vastly outnumber the virus particles. Transduction efficiency varies in different cell lines, and polybrene may be toxic to some cell lines. 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. As per the paper, naive t cells are required to be prestimulated with il7 for 612 days for efficient lentiviral transduction, whereas. When transducing a lentiviral construct into a cell line for the first time, a range of volume or moi should be tested. Having established the ability of the system to mediate cd20specific transduction of artificially created cell lines, we next investigated the possibility of specific transduction of primary human blymphoid cells, cells that naturally carry the cd20 antigen. In vivo modulation of gene expression by lentiviral. The use of lentiviral vectors lvs has been explored as possible alternative. Immune system effector cells have the potential to attack and eliminate cancer cells.
Taichman department of oral biology and pathology, stony brook university, stony brook, new york for longterm cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be stably trans. In the first example the human neuroblastoma cell line sknsh will be transduced with mission turbogfp control particles. Protocol 8 lentiviral transduction of hematopoietic stem cells. Replace medium immediately after spin transduction if polybrene is toxic to the cells. On the other hand, transfection or transduction of rna is always transient. Dna is then stably integrated into the host genome at a random position by the viral integrase enzyme.
However, by equipping the same t cells simultaneously with two tcrs, tumor cells could still escape immune recognition by defects in antigen processing or loss of human leukocyte antigen hla expression. Integration of therapeutic genetic material into the haematopoietic stem cell compartment using lvv can mediate longterm correction of haematopoietic lineages, thereby correcting disease phenotypes. Targeting lentiviral vectors to specific cell types in. Lentiviral vectors are efficient gene delivery vehicles suitable for delivering longterm transgene expression in various cell types. On the contrary, immune reactivity against vector components preexisting to lv administration such as following exposure to the parental virus may inactivate the vector, inhibiting transduction, andor attack transduced cells while still. A range of mois will be tested and an increase in gfp positive cells will be observed with an increase in moi. Indeed, using the nk92 cells, we observed almost complete inhibition of cytotoxicity against the k562 chronic myelogenous leukemia cml cell line 48 h after lentiviral transduction in the presence of atorvastatin, fluvastatin, or simvastatin, while cytotoxicity was slightly decreased in the presence of rosuvastatin figure 3a. 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. This protocol was adapted from lentivirus transduction of hematopoietic cells, chapter 7, in gene transfer.
Viral transduction of mammalian cells entered by kevin janes janes lab protocols 5111 2 buffer recipes. The viral rna genome is released and reversetranscribed to produce dna. Effective transduction of the target cells by lentivirus is influenced at each step from virus entry into the cytosol to gene expression within the nucleus. Label plates from 110 and add appropriate puromycincontaining media to cells. The final concentration of puromycin should be from 110. Lentiviral transduction of hematopoietic stem cells protocol for lenti infection of bone marrow sp cells prepare infection media. Nk cell resistance to transduction is a major technical hurdle for developing nk cell immunotherapy. Examine cells each day and change to fresh puromycincontaining media every other day.
As the lentiviral transduction system allows efficient and stable integration of transgenes, nk cell lines can be utilized to develop in vitro models to study and further understand the biology of nk cells. By using baboon envelope pseudotyped lentiviral vectors baevlvs encoding egfp, we obtained a transduction rate of 23. Not all viral particles floating in culture medium can eventually transduce or infect the 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. Use a lentiviral vector to transduce target cells which naturally integrates its dna into the host cell genome in a random manner. Plate target cells in a 12well plate 24 hours prior to viral infection. Transfection is efficient on adherent immortalized cells but primary and stem cells require transduction. Immunization with lentiviral vectortransduced dendritic. Lentiviral transduction of microglial cells balcaitis. Thus, transduction of naive t cells is a crucial prerequisite for any t cell mediated gene therapy trial aimed at providing longlasting immune reconstitution to patients. The infection protocol for suspension cell is recommended for transduction of suspension cells and includes an additional spin step which concentrates the virus at the target cell surface to increase transduction efficiency. The cells should be approximately 50% confluent on.
Here we provide an overview of innovative approaches to target lentiviral vectors to cells of the immune system. Arming t cells with a gp100specific tcr and a cspg4. 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. The first sucessful report utilizing lentiviral vectors in nk cells described the transduction of primary murine nk cells.
Protocol for lentiviral transduction of human cells. Among various technologies available for gene transfer, lentiviral mediated transduction has been successful in introducing genes into nk cells. Protocol for transduction of human embryonic stem cells. Lentiviral vectors have emerged as promising tools for both gene therapy and immunotherapy purposes.
Immune responses to lentiviral vectors bentham science. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Brown, giovanni sitia, andrea annoni, ehud hauben, lucia sergi sergi, anna zingale, maria grazia roncarolo, luca g. Thorne,3,5 giulia unali,1,2,5 giulia schiroli,1 anna m. Il7 based protocol is better for cd4 t cell transduction. After the t cells are expanded and genetically modified, they are. Integrase defective lentiviral vectors idlv represent a promising delivery system for immunization purposes. In vivo administration of lentiviral vectors triggers a type i interferon response that restricts hepatocyte gene transfer and promotes vector clearance. This protocol describes, stepbystep, the preparation and concentration of hightiter lentiviral vectors and the transduction process. Lentivirusmediated gene transfer to human epidermis soosan ghazizadeh,1 anne b. When creating stable cell lines, the optimal antibiotic concentration that is toxic to your cells will need to be determined by performing an antibiotic titration combined with cell density titration.
Incubate the cells for 72 hours at 37c under standard cell culture conditions. Their function resembles that of tissue macrophages and, as such, they share many properties with both peripheral macrophages and monocytes. 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. The organs of the immune system are positioned throughout. The tropism of a lentivirus is determined by the interaction of the glycoproteins on its viral envelop and the cell surface receptors on the target cell. Efficient and highly specific gene transfer using mutated. Duplicate or triplicate wells for each lentiviral construct and control to be used. The transduced arpe19 cells produced 100fold higher amounts of rgr protein than the transduced cos7 cells. Cart cells are generated by the t cells from patients or donors blood. Viral transduction of mammalian cells entered by kevin janes. We are very interested in using the lentivirus system for knocking down genes in primary mouse t cells cd4, cd8, and tregs. Simplified production and concentration of lentiviral vectors. Lentiviral lv vectors offer unique advantages over other gene delivery systems, namely the ability to integrate transgenes into the genome of both dividing and nondividing 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. Fhn to allow efficient transduction of the airways. Phagocytosisshielded lentiviral vectors improve liver. Lentiviral gene therapy combined with lowdose busulfan in. Engineering cart cells biomarker research full text.
Retroviral transduction of tcell receptors in mouse t. Determine the rate of transduced cells per well for each moi and at each time point. Cell biolabs quicktiter lentivirus titer kit lentivirus associated hiv p24 is an enzyme immunoassay developed for detection and quantitation of the lentivirus associated hiv1 p24 core. Wash cells with optimem and then add the peidna mix. 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 time. Lentiviral transduction of immune cells springer for. Lentivirus biosafety information and handling guidelines. Frontiers efficient and robust nkcell transduction with. Cyclosporine h overcomes innate immune restrictions. 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. The immune system is made up of special organs, cells and chemicals that fight infection microbes. In this exercise we will be demonstrating lentiviral transduction on two different cell lines.
Lentiviral gene transduction in human and mouse nk cell lines. Lentivirusmediated gene transfer to human epidermis. Lentiviral vectors lvv are important tools for the treatment of immune system disorders. A number of different modifications in vector design have increased biosafety and transgene expression. Giordano,1,2 francesco piras,1,2 ivan cuccovillo,1 sarah j. Protip transducing too many cells relative to the number of virus particles reduces the transduction efficiency, resulting in massive cell death upon antibiotic selection. To obtain the desired infection rate low and high moi. Retroviral and lentiviral vectors for the induction of. Lentivirus generation and transduction of cd81 and cd41 t cells hla a2 primary t cells were transduced with lentivirus expressing tcrs bearing various affinities for hla a2restricted tumour antigens nyeso1.
Considering overexpression and gene knockdown studies are crucial tools to study the biology of cells, technologies suitable for transfering genes into nk cells are invaluable. Lentiviral transduction of primary mecs lentiviral transductions are performed on freshly isolated primary mammary epithelial cells that have been dissociated into single cells. Nk cells express a fixed set of germ lineencoded activating and inhibitory receptors, upon which they rely on for the recognition of cancer cells. Cells were activated overnight with acd3 cd28 dynabeads invitrogen at a bead to cell ratio of 3.
When i infect 293t cells with the viral particles to calculate the viral titer i get a very high efficient gfp signal on facs. We obtain transduction efficiencies of 15% in the nk92 cell line and 3040% in lnk, yt, and derl7 cell lines. After double transduction at an moi of 3, the vector was detectable by pcr in. The potential for generation of replicationcompetent lentivirus is addressed by the design of the vectors and by safe laboratory practice. Natural killer nk cells are part of the innate immune response against tumors and are emerging as powerful effectors of cancer immunotherapy.
One striking similarity is the difficulty with which these cells can be genetically manipulated via transfection or transduction. Targeted transduction of lentiviral vectors to primary b cells. As a gene delivery system, lentivirusderived vectors can efficienctly deliver transgenes into a host cell genome and infect both dividing and nondividing cells 2, making them powerful molecular biology research tools. Memory t cells expressing an nkg2dcar ef ciently target. Gene therapy using adenoassociated viral vectors aavs has shown safety and efficacy in patients with hemophilia. In order to manipulate the expression of genes of interest, the human hematopoietic progenitor cells can be genetically engineered ex vivo by lentiviral transduction before performing xenograft transplantation. Transduce cells in ultra lowattachment 24well plates.
This approach led to broad immune reconstitution and efficient vector transduction in diverse lineages, including t cells, b cells, nk cells, myeloid cells, and bone marrow progenitors. Petit,3 fatima ahsan,3 mahdad noursadeghi,3 simon clare,4 pietro genovese,1. Dna is then stably integrated into the host genome at. May, 2015 plate hek 293t cells in a 10 cm dish to be 8090% confluent after 24 h. A possibly unique application of lentiviral vectors is the transduction of the elusive longterm repopulating hematopoietic stem cells hsc. The innate immune system in dendritic celltargeted. However, aavs have limitations hindering their efficacy in a subgroup of patients. Transduction efficiency should be checked 72 hrs following.
Efficiency of transduction of highly purified murine. Dendritic cells dcs are the sentinels of the immune system, and thus specialized in transporting foreign antigen to t cells and initiating activation of innate and adaptive immune responses. Optimal lentivirus production and cell culture conditions. The immune system is composed of a variety of different cell types and proteins. Immunization with lentiviral vectortransduced dendritic cells induces strong and longlasting t cell responses and therapeutic immunity yukai he, jiying zhang, zhibao mi, paul robbins, louis d. We have standardized methods of lentiviral infection in human and mouse nk cell lines. Mecs may also be transduced with lentivirus following a hour adenovirus transduction such as ad. Immune responses to lentiviral vectors albert einstein. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. The lowest moi at which all cells or enough cells for the specific approach.
This process was used to produce vector for a clinical trial in patients with wiskottaldrich syndrome. Production and concentration of lentivirus for transduction. Infection protocol for recombinant lentivirus 9601 medical. Recognizing this need, mirus bio has developed a novel transfection formulation, trans. Lentivirusmediated transduction of islet grafts with. Add 1 ml of complete optimal medium with serum and antibiotics and incubate cells overnight. Nov 12, 20 lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and nondividing cells. 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. The immune system and primary immunodeficiency immune. Lentiviral transduction of endothelial cells mellorlab. As written in protocol part 2, in this schematic, the most critical steps of virus production are represented with protocol step numbers and timing. The concentration range for puromycin and blasticidin is usually between 110 gml.
They all have antigen binding sites which combine specifically with the foreign antigen. Human t lymphocytes transduced by lentiviral vectors in. Lentiviral vectors have emerged as efficient tools for investigating t cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. We have developed a lentiviral vector which is specifically pseudotyped with the sendai virus envelope proteins f and hn rsiv. Adjust the number of cells plated to accommodate a confluency of 70% upon transduction. The efficiency of lentivirus infection of target cells, however, tends to be lower. Select transduced cells 50% confluence is recommende d with medium containing appropriate antibiotics or by flow cytometry to sort out fluorescencepositive cells if necessary. In contrast, during trained immunity innate immune cells display. Quicktiter lentivirus titer kit lentivirusassociated hiv. Cyclosporine h overcomes innate immune restrictions to improve lentiviral transduction and gene editing in human hematopoietic stem cells carolina petrillo,1,2 lucy g. Lentiviral transduction of immune cells request pdf. 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.
Viral and nonviral engineering of natural killer cells as. Since this greatly hinders the therapeutical benefits of gene therapy by lvs. Rosuvastatin enhances vsvg lentiviral transduction of nk. They have been used for some time for gene therapy and the development of gene vaccines. The adaptive immune system, also referred as the acquired immune system, is a subsystem of. Stability of lentiviral vectormediated transgene expression. Protocol 3 lentivirus transduction into target cell lines. Indeed, lv lack viral genes, thus viral proteins are not actively produced by lv transduced cells. Recombinant factor viii expression in hematopoietic cells. Some additives such as polybrene can enhance the transduction efficiency. Lentivirus transduction protocols for reference only many factors can affect transduction efficiency. Schematic of lentivirus production for organoid transduction.
Recently retroviral and lentiviral vectors have been used to generate tolerogenic dendritic cells, key professional antigen presenting cells. To achieve selective transduction, gene vectors must avoid binding to offtarget cells while quickly binding to target cells with high specificity and must efficiently deliver dna to the nucleus following cell entry. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility. Selective transduction of only target cells and tissues represents a major goal of therapeutic gene delivery. Moreover, differentiated cells such as macrophages can be trained as well e. Spinoculation of suspension cells this protocol is for transduction of suspension cells jurkat t cells, pbmc, pbl, b cells etc. Microglial cells are the resident immune cells of the central nervous system. Modulation of immune responses in lentiviral vectormediated. The lentiviral particle binds to the cell membrane and enters the host cell. However, pravastatin did not alter the cytotoxic capacity of nk92 cells on k562 cells. Retroviral transduction of tcrs into in vitro activated mouse t cells is a quick and relatively easy method to obtain t cells of desired peptidemhc specificity. Simian immunodeficiency virusvpx for improving integrase. Central to the immune systems ability to mobilize a response to an invading. Differences between transfection and transduction blog.
When someone might have bacterial infection, doctors can order a blood test to see if it caused the body to have lots of neutrophils. Using this mouse model, the human immune system can be assessed for both fundamental and preclinical purposes. Cells were infected with gfp lentivirus for 48 hours in the presence of polybrene or the viraductin lentivirus transduction kit. In this work, we first explore how dcs sense viral pathogens and stimulate antigenspecific t cell responses. Acquire pictures of the cells by fluorescence microscopy after 24, 48 and 72 hours. A protocol for lentiviral transduction and downstream. Lowenstein1,4 abstract lentiviral vectors are promising tools for gene therapy in the cns. The cells producing the antibody remember the infection and provide. Lentiviral expression has many advantages over other viruses, including the ability to infect both proliferating and nonproliferating cells.
Since this greatly hinders the therapeutical benefits of gene therapy. Primary t cell lentiviral transduction researchgate. Lentivirus technologies for modulation of the immune system. 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. However, one of the major barriers to stable gene transfer by lvs and other viral vectors is the development of innate and adaptive immune responses to the delivery vector and the transferred therapeutic transgene. Dilute puromycin in the preferred culture media for your target cells. Expression of a recombinant human rgr opsin in lentivirus. Retroviral and lentiviral vectors have proven to be particularly efficient systems to deliver genes of interest into target cells, either in vivo or in cell cultures. Antigenspecific t cells can be generated in one week and used in any downstream applications. Such lentiviral vectors have the potential to infect a wide variety of cell types. They exhibit several advantages over other viral systems in that they are less immunogenic and.