Integrase-deficient Lentiviral Vectors Research Articles

Integrase deficient lentiviral vector: prospects for safe clinical applications.

HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector’s replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. Invitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells invivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.

Persistence of Integrase-Deficient Lentiviral Vectors Correlates with the Induction of STING-Independent CD8+ T Cell Responses

Lentiviruses are among the most promising viral vectors for invivo gene delivery. To overcome the risk of insertional mutagenesis, integrase-deficient lentiviral vectors (IDLVs) have been developed. We show here that strong and persistent specific cytotoxic Tcell (CTL) responses are induced by IDLVs, which persist several months after a single injection. These responses were associated with the induction of mild and transient maturation of dendritic cells (DCs) and with the production of low levels of inflammatory cytokines and chemokines. They were independent of the IFN-I, TLR/MyD88, interferon regulatory factor (IRF), retinoic acid induced gene I (RIG-I), and stimulator of interferon genes (STING) pathways but require NF-κB signaling in CD11c+ DCs. Despite the lack of integration of IDLVs, the transgene persists for 3months in the spleen and liver of IDLV-injected mice. These results demonstrate that the capacity of IDLVs to trigger persistent adaptive responses is mediated by a weak and transient innate response, along with the persistence of the vector in tissues.

Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 components in HEK 293 cells. The Rep 78 and ZFN-based approaches were also compared in HEK 293 cells and in human induced pluripotent stem cells using integrase deficient lentiviral vectors. The targeting efficiencies involving the Rep 78 protein were similar to those involving the AAVS1-specific ZFN, while the targeting specificity for the Rep 78 protein was lower compared to that of the ZFN. It is anticipated that the Rep 78 nickase-based targeting approach may ultimately contribute to the reduction of risks associated with other genome editing approaches involving DNA double-strand breaks.

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing

The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs) are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs) present an attractive means for delivery of CRISPR/Cas9 components because: (1) they are capable of transducing a broad range of cells and tissues, (2) have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors), and (3) they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T) cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer.

Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

Sleeping Beauty Transposition From Nonintegrating Lentivirus

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1-derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).

Targeted Genome Modifications Using Integrase‐deficient Lentiviral Vectors

Gene correction aims at repairing a defective gene directly in the cellular genome, which warrants tissue-specific and sustained expression of the repaired gene through its endogenous promoter. We have developed a novel system based on integrase-deficient lentiviral vectors (IDLVs) that allows us to correct an endogenous mutation using a strategy based on homologous recombination (HR). In a proof-of-concept approach, an IDLV encoding a repair template was co-delivered with an I-SceI nuclease expression vector to rescue a defective enhanced green fluorescent protein (EGFP) gene. Expression of the nuclease created a double-strand break within the target locus, which was crucial for stimulating IDLV-based gene repair. Stable gene correction was realized in up to 12% of the cells, depending on the vector dose, the nuclease expression levels, and the cell type. Genotypic analyses confirmed that gene correction was the result of genuine HR between the target locus and the IDLV repair template. This study presents IDLVs as valuable tools for introducing precise and permanent genetic modifications in human cells.

100. Development of an Integrase Deficient FIV Vector for Transient Gene Expression

Lentiviral vectors mediate long term transgene expression through integration into host genomic DNA. However, integration comes with potential risks of insertional mutagenesis. In cases where transient gene expression is desired, an integrase (IN) deficient retroviral vector might be a useful alternative. Furthermore, IN deficient vectors may provide some advantages over alternative methods of transient gene expression due to the ability to pseudotype these vectors with heterologous envelopes that target specific cell types. Class I IN mutants have point mutations in the catalytic triad amino acids (DX39-58 DX35 E) of IN that disable this protein but leave other viral proteins functional. Transient gene expression from IN deficient vectors is mediated from episomal forms of vector DNA. 1-LTR and 2-LTR circles form through homologous recombination and non-homologous end joining, respectively, of the linear form of the reverse transcribed viral DNA. Although 1-LTR and 2-LTR circles are unable to efficiently integrate in the presence of functional IN protein, they can mediate transgene expression. It was previously reported that IN deficient HIV and FIV vectors could mediate transient gene transfer. In growth arrested cells, transgene expression was maintained. However, in rapidly dividing cells transgene expression was lost, and duration of expression was related to the rate of cell division. Because some gene transfer targets are mitotically quiescent and slowly dividing cells, IN deficient mutants may promote long term, though ultimately transient, gene expression. We hypothesized that IN deficient FIV vectors can be used to maintain transient gene expression in slowly dividing cell types, such as airway epithelia. We developed IN deficient FIV vectors with single (D66V, D118A, E154G), double (D66V/D118A, D66V/E154G, D118A/E154G), and triple (D66V/D118A/E154G) mutations that disrupt the catalytic triad of the IN protein. Preliminary data indicate that the FIV D66V IN mutant has titers similar to wild type IN vectors. Furthermore, we documented the production of 1-LTR and 2-LTR circles in human cell line cultures transduced with FIV D66V IN deficient vector. Ongoing studies are comparing the persistence of transgene expression in IN deficient versus wild type IN FIV in dividing and non-dividing cells. Furthermore, to ensure that transgene expression seen in IN deficient vectors represents episomal and not integrated vector DNA, Southern blotting will be used. IN deficient lentiviral vectors may have applications for transient gene expression, long term expression in slowly dividing cells, and can be used as controls in studies with integration competent vectors.