Integrase Function Research Articles

CRISPR-Cas9 enrichment, a new strategy in microbial metagenomics to investigate complex genomic regions: The case of an environmental integron.

Environmental integrons are ubiquitous in natural microbial communities, but they are mostly uncharacterized and their role remains elusive. Thus far, research has been hindered by methodological limitations. Here, we successfully used an innovative approach combining CRISPR-Cas9 enrichment with long-read nanopore sequencing to target, in a complex microbial community, a putative adaptive environmental integron, InOPS, and to unravel its complete structure and genetic context. A contig of 20 kb was recovered containing the complete integron from the microbial metagenome of oil-contaminated coastal sediments. InOPS exhibited typical integron features. The integrase, closely related to integrases of marine Desulfobacterota, possessed all the elements of a functional integron integrase. The gene cassettes harboured mostly unknown functions hampering inferences about their ecological importance. Moreover, the putative InOPS host, likely a hydrocarbonoclastic marine bacteria, raises questions as to the adaptive potential of InOPS in response to oil contamination. Finally, several mobile genetic elements were intertwined with InOPS highlighting likely genomic plasticity, and providing a source of genetic novelty. This case study showed the power of CRISPR-Cas9 enrichment to elucidate the structure and context of specific DNA regions for which only a short sequence is known. This method is a new tool for environmental microbiologists working with complex microbial communities to target low abundant, large or repetitive genetic structures that are difficult to obtain by classical metagenomics. More precisely, here, it offers new perspectives to comprehensively assess the eco-evolutionary significance of environmental integrons.

RAP44 phage integrase-guided 50K genomic island integration in Riemerella anatipestifer.

Bacteriophages are viruses that infect bacteria. Bacteria and bacteriophages have been fighting for survival. Over time, the evolution of both populations has been affected. Pathogenic Flavobacteriaceae species including Riemerella anatipestifer mainly infects ducklings, geese, and turkeys. However, it does not infect humans, rats, or other mammals, and is a suitable and safe research object in the laboratory. Our previous study showed that there is a 10K genomic island in R. anatipestiferIn this study, we found another integrated 50K genomic islands and focused on the relationship between R. anatipestifer genomic islands and the RAP44 phage genome. The phage RAP44 genome was integrated into R. anatipestifer chromosome, and an evolutionary relationship was evident between them in our comparative analysis. Furthermore, the integrated defective RAP44 phage sequence had the function of integration, excision, and cyclization automatically. Integrases are important integration elements. The integrative function of integrase was verified in R. anatipestifer. The integrase with the attP site can be integrated stably at the attB locus of the R. anatipestifer genome. A recombinant strain can stably inherit and express the exogenous gene. By studying the integration between host bacterium and phage, we have provided evidence for the evolution of the genomes in R. anatipestifer.

Distribution and genome structures of temperate phages in acetic acid bacteria

Acetic acid bacteria (AAB) are industrial microorganisms used for vinegar fermentation. Herein, we investigated the distribution and genome structures of mitomycin C-inducible temperate phages in AAB. Transmission electron microscopy analysis revealed phage-like particles in 15 out of a total 177 acetic acid bacterial strains, all of which showed morphology similar to myoviridae-type phage. The complete genome sequences of the six phages derived from three strains each of Acetobacter and Komagataeibacter strains were determined, harboring a genome size ranging from 34,100 to 53,798 bp. A phage AP1 from A. pasteurianus NBRC 109446 was predicted as an active phage based on the genomic information, and actually had the ability to infect its phiAP1-cured strain. The attachment sites for phiAP1 were located in the 3’-end region of the tRNAser gene. We also developed a chromosome-integrative vector, p2096int, based on the integrase function of phiAP1, and it was successfully integrated into the attachment site of the phiAP1-cured strain, which may be used as a valuable tool for the genetic engineering. Overall, this study showed the distribution of mitomycin C-inducible temperate phages in AAB, and identified the active temperate phage o f A. pasteurianus.

Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.

Identification of Three Clf-Sdr Subfamily Proteins in Staphylococcus warneri, and Comparative Genomics Analysis of a Locus Encoding CWA Proteins in Staphylococcus Species.

Coagulase-negative Staphylococcus warneri is an opportunistic pathogen that is capable of causing several infections, especially in patients with indwelling medical devices. Here, we determined the complete genome sequence of a clinical S. warneri strain isolated from the blood culture of a 1-year-old nursling patient with acute upper respiratory infection. Genome-wide phylogenetic analysis confirmed the phylogenetic relationships between S. warneri and other Staphylococcus species. Using comparative genomics, we identified three cell wall-anchored (CWA) proteins at the same locus (sdr), named SdrJ, SdrK, and SdrL, on the chromosome sequences of different S. warneri strains. Structural predictions showed that SdrJ/K/L have structural features characteristic of Sdr proteins but exceptionally contained an unusual N-terminal repeat region. However, the C-terminal repetitive (R) region of SdrJ contains a significantly larger proportion of alanine (142/338, 42.01%) than the previously reported SdrI (37.00%). Investigation of the genetic organization revealed that the sdrJ/K/L genes were always followed by one or two glycosyltransferase genes, gtfA and gtfB and were present in an ∼56 kb region bordered by a pair of 8 bp identical direct repeats, named Sw-Sdr. This region was further found to be located on a 160-kb region subtended by a pair of 160-bp direct repeats along with other virulence genes and resistance genes. Sw-Sdr contained a putative integrase that was probably a remnant of a functional integrase. Evidence suggests that Sw-Sdr is improbably an efficient pathogenicity island. A large-scale investigation of Staphylococcus genomes showed that sdr loci were a potential hotspot of insertion sequences (ISs), which could lead to intraspecific diversity at these loci. Our work expanded the repository of Staphylococcus Sdr proteins, and for the first time, we established the connection between sdr loci and phylogenetic relationships and compared the sdr loci in different Staphylococcus species, which provided large insights into the genetic environment of CWA genes in Staphylococcus.

Influence of the amino-terminal sequence on the structure and function of HIV integrase

BackgroundAntiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes.ResultsWe purified HIV-1 IN 1F1–212 and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN1–212. Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strand-transfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers.ConclusionsThe structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.

Reversal of Epigenetic Silencing Allows Robust HIV-1 Replication in the Absence of Integrase Function.

Integration of the proviral DNA intermediate into the host cell genome normally represents an essential step in the retroviral life cycle. While the reason(s) for this requirement remains unclear, it is known that unintegrated proviral DNA is epigenetically silenced. Here, we demonstrate that human immunodeficiency virus 1 (HIV-1) mutants lacking a functional integrase (IN) can mount a robust, spreading infection in cells expressing the Tax transcription factor encoded by human T-cell leukemia virus 1 (HTLV-1). In these cells, HIV-1 forms episomal DNA circles, analogous to hepatitis B virus (HBV) covalently closed circular DNAs (cccDNAs), that are transcriptionally active and fully capable of supporting viral replication. In the presence of Tax, induced NF-κB proteins are recruited to the long terminal repeat (LTR) promoters present on unintegrated HIV-1 DNA, and this recruitment in turn correlates with the loss of inhibitory epigenetic marks and the acquisition of activating marks on histones bound to viral DNA. Therefore, HIV-1 is capable of replication in the absence of integrase function if the epigenetic silencing of unintegrated viral DNA can be prevented or reversed.IMPORTANCE While retroviral DNA is synthesized normally after infection by integrase-deficient viruses, the resultant episomal DNA is then epigenetically silenced. Here, we show that expression of the Tax transcription factor encoded by a second human retrovirus, HTLV-1, prevents or reverses the epigenetic silencing of unintegrated HIV-1 DNA and instead induces the addition of activating epigenetic marks and the recruitment of NF-κB/Rel proteins to the HIV-1 LTR promoter. Moreover, in the presence of Tax, the HIV-1 DNA circles that form in the absence of integrase function are not only efficiently transcribed but also support a spreading, pathogenic integrase-deficient (IN-) HIV-1 infection. Thus, retroviruses have the potential to replicate without integration, as is indeed seen with HBV. Moreover, these data suggest that integrase inhibitors may be less effective in the treatment of HIV-1 infections in individuals who are also coinfected with HTLV-1.

Simple, 1-Day Manufacturing of Quiescent Chimeric Antigen Receptor T Cells for Adoptive Immunotherapy

The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic "drugs" for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control. Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We showed using a non-optimized process that a mean of 6.5% (range 2%-10%) of freshly isolated quiescent T cells can be transduced using an infrared red fluorescent protein (iRFP)-expressing lentiviral vector with slower kinetics of expression compared with activated T cell transduction (peak at 96 hrs vs. 48 hrs for quiescent and activated T cells, respectively). Although substantially less efficient compared to activated T cells, transduction was detected across all T cells subsets with central memory T cells showing the greatest transduction efficiency with a mean of 4-fold greater transduction compared with naïve T cells. Somewhat unexpectedly, CART19 cells generated from quiescent T cells using a CD19-specific CAR vector showed a 3-5 fold greater transgene expression compared with iRFP vectors transduced at similar MOI. However, we show that CAR expression can occur in quiescent T cells even without reverse transcription or integrase function, so called "pseudotransduction". Importantly, we show that this CAR expression produces T cells with cytolytic activity and effector cytokine production in response to antigen that is similar to activated and transduced CAR T cells. Using the well-characterized Nalm6 model of acute lymphoblastic leukemia, we show that CART19 cells generated by transduction of quiescent T cells for 16 hours followed by washing to remove vector exhibit dose-dependent anti-leukemic activity that is durable with injection of as little as 2x105 total T cells. We estimate the latter to contain ~2x104 T cells with integrated lentiviral vector based upon transduction efficiency determined in studies using non-tumor bearing mice. (Fig 1). In summary, our results support the need for further investigation of CAR T cells that are generated using engineering of quiescent T cells. Taking advantage of the ability of lentiviral vectors to transfer genes to quiescent T cells, the highly abbreviated and simplified manufacturing approach described here has the potential to enhance therapeutic potency while also substantially reducing the materials and labor costs associated with current manufacturing approaches that use activated and expanded T cells. In addition, the rapid nature of this manufacturing has the potential to extend the population of patients that may be treated with these therapies by shortening the interval between aphaeresis collection and re-infusion of CAR T cells, which prevents the treatment of some patients with rapidly progressive disease. Disclosures Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing.

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

Proteae: a reservoir of class 2 integrons?

Our aim was to confirm with a large panel of clinical isolates that class 2 integrons are highly prevalent in Proteae and to analyse their genetic characteristics. Proteae (Proteus spp., Morganella spp. and Providencia spp.) isolates were collected from clinical samples during 2013 at Limoges University Hospital, France. The presence of class 1, 2 and 3 integrons was investigated by quantitative PCR. The presence of a stop codon in the intI2 gene was determined by Sanger sequencing. The gene cassette arrays of class 2 integrons were determined by PCR-RFLP and Sanger sequencing or next-generation sequencing when needed. Of the 327 Proteae collected, 103 (31.5%) harboured a class 2 integron and 45 (13.8%) a class 1 integron. No class 3 integrons were detected. One functional IntI2 integrase was detected in a Morganella morganii isolate. Six different gene cassette arrays were detected. Four had already been described in the literature: dfrA1-sat2-aadA1 (72 isolates), dfrA1-catB2-sat2-aadA1 (17), sat2-aadA1 (6) and lnu(F), dfrA1, aadA1 (1). We identified two new gene cassette arrays: (i) a new variant of the dfrA1 gene cassette (one isolate; the one with the functional IntI2); and (ii) the array dfrA1-gcu115-sat2 harbouring the new gcu115 gene cassette with two ORFs encoding proteins of unknown functions (five isolates). We showed a high frequency of class 2 integrons, as well as a diversity of gene cassette arrays, among Proteae. This work highlights that the Proteae tribe plays an important role as a reservoir of class 2 integrons.

Serine Integrase attP Binding and Specificity

Serine integrases catalyze the site-specific insertion of viral DNA into a host's genome. The minimal requirements and irreversible nature of this integration reaction have led to the use of serine integrases in applications ranging from bacterial memory storage devices to gene therapy. Our understanding of how the integrase proteins recognize the viral (attP) and host (attB) attachment sites is limited, with structural data available for only a Listeria integrase C-terminal domain (CTD) bound to an attP half-site. Here we report quantitative binding and saturation mutagenesis analyses for the Listeria innocua prophage attP site and a new 2.8-Å crystal structure of the CTD•attP half site. We find that Int binds with high affinity to attP (6.9 nM), but the Int CTD binds to attP half-sites with only 7- to 10-fold lower affinity, supporting the idea that free energy is expended to open an Int dimer for attP binding. Despite the 50-bp Int–attP interaction surface, only 20 residues are sensitive to mutagenesis, and of these, only 6 require a specific residue for efficient Int binding and integration activity. One of the integrase DNA-binding domains, the recombinase domain, appears to be primarily non-specific. Several substitutions result in an improved attP site, indicating that higher-efficiency attachment sites can be obtained through site engineering. These findings advance our understanding of serine integrase function and provide important data for efforts towards engineering this family of enzymes for a variety of biotechnology applications.

Structural Implications of Genotypic Variations in HIV-1 Integrase From Diverse Subtypes.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) integrates viral DNA into the host genome using its 3′-end processing and strand-transfer activities. Due to the importance of HIV-1 IN, it is targeted by the newest class of approved drugs known as integrase strand transfer inhibitors (INSTIs). INSTIs are efficient in maintaining low viral load; however, as with other approved antivirals, resistance mutations emerge in patients receiving INSTI-containing therapy. As INSTIs are becoming increasingly accessible worldwide, it is important to understand the mechanism(s) of INSTI susceptibility. There is strong evidence suggesting differences in the patterns and mechanisms of drug resistance between HIV-1 subtype B, which dominates in United States, Western Europe and Australia, and non-B infections that are most prevalent in countries of Africa and Asia. IN polymorphisms and other genetic differences among diverse subtypes are likely responsible for these different patterns, but lack of a full-length high-resolution structure of HIV-1 IN has been a roadblock in understanding the molecular mechanisms of INSTI resistance and the impact of polymorphisms on therapy outcome. A recently reported full-length medium-resolution cryoEM structure of HIV-1 IN provides insights into understanding the mechanism of integrase function and the impact of genetic variation on the effectiveness of INSTIs. Here we use molecular modeling to explore the structural impact of IN polymorphisms on the IN reaction mechanism and INSTI susceptibility.

A heterogeneous human immunodeficiency virus-like particle (VLP) formulation produced by a novel vector system

First identified as the etiological agent behind Acquired Immunodeficiency Syndrome (AIDS) in the early 1980s, HIV-1 has continued to spread into a global pandemic and major public health concern. Despite the success of antiretroviral therapy at reducing HIV-1 viremia and preventing the dramatic CD4+ T-cell collapse, infected individuals remain HIV positive for life. Unfortunately, it is increasingly clear that natural immunity is not, and may never be, protective against this pathogen. Therefore, efficacious vaccine interventions, which can either prevent infection or eradicate the latent viral reservoir and effect cure, are a major medical priority. Here we describe the development of a safe vaccine platform, currently being utilized in on-going prophylactic and therapeutic preclinical studies and consisting of highly heterogeneous virus-like particle formulations that represent the virus diversity within infected individuals. These VLPs contain no 5′LTR, no functional integrase, and have a severely mutated stem loop 1—thereby preventing any potential reverse transcription, integration, and RNA packaging. Furthermore, we demonstrate that these VLPs are morphologically identical to wild-type virus with polyvalent Env in a functional form. Finally, we show that the VLPs are antigenic and capable of generating strong immune recall responses.

A C-terminal “Tail” Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

Gene tagging in Physcomitrella patens: an addition to the genetic toolbox.

This article is a commentary on Vives et al., 212: 759–769.

The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae

Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins.

Getting IN on Viral RNA Condensation and Virion Maturation

The retroviral enzyme integrase plays an essential role in the virus replication cycle by catalyzing thecovalent insertion of newly synthesized viral DNA into the host cell chromosome early after infection. Now, Kessl etal. report a second function of integrase: binding to the viral RNA genome in virion particles late in the virus replication cycle to promote particle maturation.

Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

Retroviral infection requires integration of the viral genome into the host genome. Recombinant integrase proteins may be purified following bacterial expression. A bulk biochemical assay of integrase function relies on the conversion of supercoiled plasmids to linear or relaxed circles. Single molecule molecular tweezer assays of integrase also evaluate the conversion of supercoiled DNA to nicked and broken species. A bacterial nuclease that co-purifies with retroviral integrase may affect the quantitation of integration activity in bulk or single molecule assays. During purification of retroviral integrase from bacteria, fractions may be screened for contaminating nuclease activity. In order to efficiently separate the nuclease from integrase, the binding affinities of each protein must differ. We find that a co-purifying nuclease may be efficiently separated from integrase based on heparin affinity, but not ionic affinity.

Will LEDGIN molecules be able to play a role in a cure for HIV infection?

Will LEDGIN molecules be able to play a role in a cure for HIV infection?

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal genetransfer.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19+ target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo. We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity.