Integration-defective Lentiviral Vectors Research Articles

The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes

Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episome levels in IDLV-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells, and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLV LTR circles 6- to 7-fold. The final effect the IS2 element in IDLVs will greatly depend on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1-LTR versus 2-LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2S-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

Optimizing Delivery and Expression of Designer Nucleases for Genome Engineering

Genome engineering can be accomplished by designer nucleases. They are specifically designed to cleave double-stranded DNA at the desired target locus. This double-strand break subsequently engages the DNA repair pathway through nonhomologous end-joining (NHEJ), resulting in either gene disruption or gene repair. Alternatively, the presence of homologous donor DNA allows for targeted integration of this exogenous donor DNA in this target locus through homology-directed DNA repair. The key bottleneck in genome engineering relates to the delivery and expression of the designer nucleases. One of the most attractive vector platforms for genome engineering is based on integration-defective lentiviral vectors (IDLVs). The intrinsic episomal nature of IDLVs is well suited to ensure transient expression of designer nucleases and minimize potential risks associated with their sustained expression. Unfortunately, their expression is compromised because of epigenetic silencing that interferes with the transcriptional competence of IDLVs. In this issue, Pelascini and colleagues now showed that this bottleneck could be overcome by interfering with chromatin remodeling using histone deacetylase (HDAC) inhibitors. HDAC inhibition restored expression of designer nucleases from IDLVs and rescued their ability to achieve efficient targeted gene disruption by NHEJ comparable with that achieved with bona fide integrating lentiviral vectors. This study has implications for the ex vivo use of IDLVs for gene repair and gene targeting.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Histone Deacetylase Inhibition Activates Transgene Expression from Integration-Defective Lentiviral Vectors in Dividing and Non-Dividing Cells

Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone- and cell type-dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles.

Transduction of Human Antigen-Presenting Cells with Integrase-Defective Lentiviral Vector Enables Functional Expansion of Primed Antigen-Specific CD8+ T Cells

Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8(+) T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications.

Toward Integrase Defective Lentiviral Vectors for Genetic Immunization

Genetic immunization with lentiviral vectors is under evaluation as a means for induction of sustained immune response. Lentiviral vectors showed reduced antivector immune responses and efficiently transduce post-mitotic cells in vivo, including antigen presenting cells, such as dendritic cells and macrophages, thus providing a significant benefit over other vector-based antigen delivery platforms. Several evidences indicate that a single immunization with lentiviral vectors induces strong and sustained effector and memory T-cell immune responses, as well as antibody production. New generation of lentiviral vectors with improved biosafety profile are also under development. In particular, integration defective lentiviral vectors have been generated and used as an efficient and safe delivery system for both gene therapy and immunization purposes. Taken together, these evidences support the ongoing development of lentiviral vector-based genetic immunization strategies for safe applications in the clinic.

Protein transduction from retroviral Gag precursors

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.

Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase

BackgroundCircular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription.ResultsWe demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette.ConclusionOur data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile.

793. Integration-Defective Lentiviral Vectors as a Platform for Sleeping Beauty Transposase-Mediated Gene Insertion

793. Integration-Defective Lentiviral Vectors as a Platform for Sleeping Beauty Transposase-Mediated Gene Insertion

391. Effective Gene Therapy with Non-Integrating Lentivirus Vectors

Gene therapy strategies need to achieve efficient transgene expression whilst minimizing toxic effects. Retrovirus vectors are very effective in several systems, but they have been shown to cause malignancies through insertional mutagenesis, one of the most serious side-effects identified to date in pre-clinical studies and clinical trials. The risk of insertional mutagenesis has been considered unavoidable because no gene expression has been demonstrated in vivo in the absence of retroviral integration. However, retrovirus and lentivirus vectors can be rendered integration-defective by mutation in the integrase coding sequence. Following reverse transcription, mutant vectors fail to integrate and instead accumulate in transduced cells as double-stranded DNA circles. These circular forms are non-replicating but very stable and will persist if not diluted by cell division. Contrary to the results from previous in vivo studies, recent reports have shown efficient transgene expression in vitro from integration-deficient lentivirus vectors in post-mitotic and growth-arrested cells. These vectors also led to transient transgene expression or, in combination with episomal systems, to stable expression in proliferating cells in vitro. Given these developments we decided to re-evaluate the proficiency of integration-defective lentiviral vectors for in vivo expression in rodents. We now show that integration-deficient HIV-1 vectors lead to highly efficient, long-term marker gene expression in post-mitotic ocular and brain tissues. We also demonstrate efficient rescue in two rodent models of retinal degeneration, the Rpe65rd12/rd12 mouse and the Mertk-deficient Royal College of Surgeons (RCS) rat. The relevance of these studies for clinical application is highlighted by the fact that we have been able to transduce excised human retinal pigment epithelium with integration-deficient vectors. In conclusion, we demonstrate that integration-deficient HIV-1 vectors transduce post-mitotic tissues in vivo as efficiently as their integrating counterparts. For clinical applications in quiescent tissues these non-integrating vectors should virtually abolish the risk of insertional mutagenesis-induced malignancy.