Lentiviral Vectors For Gene Therapy Research Articles

Targeting of prostate cancer cells by a cytotoxic lentiviral vector containing a prostate stem cell antigen (PSCA) promoter

The efficacy of prostate cancer gene therapy is limited by the inefficiency of prostate-specific promoters as compared to ubiquitous viral promoters. The purpose of this investigation was to evaluate the specificity and efficacy of a lentiviral vector driven by a PSCA promoter. Prostate cancer (LNCap, C42-B, and LAPC-4) and non-prostate cancer (HeLa, MB231, and MCF-7) cells were transduced with a lentiviral vector expressing either the luciferase or the HSV-TK suicide gene and driven by a short PSCA promoter. Specificity and efficacy were evaluated in vitro and in vivo. Luciferase expression was only detected in prostate cancer cells and was comparable to the universal CMV promoter. Luciferase expression in prostate cancer cells cultured with androgen was higher than that in cells cultured without androgen. In subsequent cytotoxicity experiments in which the luciferase marker gene was replaced with the HSV-TK gene, the lentiviral vector harboring the PSCA promoter induced cytotoxicity in prostate cancer cell lines while demonstrating a minimal effect on non-prostate cells. Cellular toxicity was correlated to increasing concentrations of the prodrug ganciclovir. Androgen had a positive effect on the cytotoxicity of this lentiviral construct. Intratumoral injection of prostate cancer xenografts with the lentiviral construct induced tumor growth inhibition versus saline controls. Our results indicate that a lentiviral gene therapy vector driven by a short PSCA promoter can induce prostate-specific cellular toxicity in vivo and in vitro and may provide a strategy to selectively treat local and advanced metastatic prostate cancer. Prostate 69: 1422-1434, 2009. (c) 2009 Wiley-Liss, Inc.

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

AbstractRetroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

77. CCR5 Positive Cell Specific Targeted Transduction of a CCR5 shRNA Lentiviral Vector for HIV Gene Therapy

77. CCR5 Positive Cell Specific Targeted Transduction of a CCR5 shRNA Lentiviral Vector for HIV Gene Therapy

64. Combination of a Safety-Modified Lentiviral Vector and Artificial Antigen Presenting Cell-Mediated Expansion Generate Potent Antitumor CD8 Lymphocytes for TCR Gene Therapy

64. Combination of a Safety-Modified Lentiviral Vector and Artificial Antigen Presenting Cell-Mediated Expansion Generate Potent Antitumor CD8 Lymphocytes for TCR Gene Therapy

Refinement of lentiviral vector for improved RNA processing and reduced rates of self inactivation repair

BackgroundLentiviral gene therapy vectors are now finding clinical application. In order to fully exploit their potential it is important that vectors are made as efficient and as safe as possible. Accordingly, we have modified a previously reported vector to improve RNA processing, minimise Human Immunodeficiency Virus Type-1 (HIV-1) sequence content and reduce repair of the self inactivating (SIN) deletion.ResultsHIV-1 sequence in the vector was reduced by substituting the polyadenylation signal with a heterologous signal. Mutation of splice donor sites was undertaken to prevent the majority of splicing within the vector genomic RNA. In addition, a number of other sequences within the vector were deleted. The combination of these modifications was able to significantly reduce the rates of both vector mobilisation and repair of the self inactivating deletion after two rounds of marker rescue.ConclusionRNA processing can be improved by mutation of the major and minor HIV-1 splice donor sites in the vector. In addition the rate of vector mobilisation and repair of SIN vectors can be successfully reduced by careful vector design that reduces homology between the 5' and 3' long terminal repeats (LTRs) to a minimum.

Oxidative stress-inducible lentiviral vectors for gene therapy

Oxidative stress is important in several pathologies, including cardiovascular diseases such as atherosclerosis and cardiac ischemia-reperfusion injury. An important mechanism for adaptation to oxidative stress is induction of genes through the antioxidant response element (ARE), which regulates the expression of antioxidant and cytoprotective genes via the transcription factor Nrf2 (nuclear factor E2-related factor 2). As Nrf2-regulated genes are induced during oxidant stress occurring, for example, in reperfusion after ischemia, we took a novel approach to exploit ARE for the development of oxidative stress-inducible gene therapy vectors. To this end, one, two or three ARE-containing regions from human NAD(P)H:quinone oxidoreductase-1, glutamate-cysteine ligase modifier subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal SV40 promoter. The construct, which was the most responsive to ARE-inducing agents, was chosen for further studies in which a lentiviral vector was produced for an efficient transfer to endothelial cells. Heme oxygenase-1 (HO-1), which has well-characterized anti-inflammatory properties, was used as the therapeutic transgene. In human endothelial cells, ARE-driven HO-1 overexpression inhibited nuclear factor-kappaB activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-alpha. We conclude that the ARE element is a promising alternative for the development of oxidative stress-inducible gene therapy vectors.

Lentiviral-mediated gene transfer – a patent review

Background: Lentiviral vectors are at the forefront of gene delivery systems for research and clinical applications. This special position is mainly due to their capacity to transduce slow dividing and non-dividing cells, to insert large genetic constructs in the host chromatin, and to sustain stable long-term transgene expression. Objective: To review the current literature and patents concerning the lentiviral system, the safety improvements, the production and purification of lentiviral vectors, pseudotyping and preclinical and clinical studies to provide expert opinion about the use of lentiviral vectors for gene therapy. Methods: The National Library of Medicine (PubMed) was searched for studies investigating the lentiviral system and the patents were searched at the World Intellectual Property Organization, European Patent Office and US Patent Office websites/databases. Results/conclusion: Based on the literature, several improvements have been performed regarding the safety, pseudotyping, vector production and purification on the lentivirus system. Clinical trials are underway for five different disorders.

743. New Lentiviral Vectors for Gene Therapy of Thalassemia with the HPFH-2 Enhancer and the -117 HPFH Activating Mutation: Studies on Thalassemic Hematopoietic Stem Cells

743. New Lentiviral Vectors for Gene Therapy of Thalassemia with the HPFH-2 Enhancer and the -117 HPFH Activating Mutation: Studies on Thalassemic Hematopoietic Stem Cells

Optimization of lentiviral vectors for the gene therapy of fanconi anemia

Optimization of lentiviral vectors for the gene therapy of fanconi anemia

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages.Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Development of lentiviral vectors for gene therapy for Usher syndrome type 1B

Abstract Purpose: Usher 1B, one of the major subtypes of a combined blindness and deafness disease, is caused by mutations in the MYO7A gene, which encodes a large unconventional myosin expressed in the retinal pigment epithelium (RPE) and photoreceptor (PR) cells. This study aims at developing viral vectors expressing the wild type human MYO7A at an adequate level in order to rescue cellular phenotypes of MYO7A mutation. Methods: The full‐length (7 kb) human MYO7A cDNA was cloned into the third generation, self‐inactivating lentiviral vector under different promoters and enhancers. Human genomic 4‐kb DNA fragment including exon 1 through 2 was cloned by PCR. Activities of different promoters and enhancers were tested by reporter assays using ARPE‐19 cells. Previously identified Myo7a‐null phenotypes in shaker‐1 mouse were used to test the efficacy of various lentiviruses. Results: Lentiviral vectors could successfully transduce large genes (up to 7.6 kb) in vitro and in vivo for the purpose of gene therapy. Reporter assay indicated that regions with a suppressor activity and an enhancer activity existed within intron 1. The CMV promoter drove excessive MYO7A expression in the RPE, and thus caused cell death. A chimeric promoter that consists of partial CMV promoter with 160‐bp MYO7A enhancer could direct moderate levels of gene expression in RPE and PR in vivo, and rescued a number of phenotypes in the mutant mice. Conclusions: These results illustrate the importance of regulating transgene expression levels in achieving therapeutic outcomes. They demonstrate the efficacy of lentivirus‐mediated expression of the large MYO7A cDNA as a gene therapy strategy for correcting the MYO7A deficiency underlying Usher 1B.

300. Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders

300. Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders

Lentiviral Vectors for T-cell Suicide Gene Therapy: Preservation of T-cell Effector Function After Cytokine-mediated Transduction

Retroviral transfer of the Herpes Simplex thymidine kinase (HSVTK) suicide gene to donor T cells has been used as a safety strategy against graft-versus-host disease following allogeneic stem cell transplantation. The feasibility of this strategy in human studies has been demonstrated, but a number of limitations have become apparent. Preactivation of donor lymphocytes using mitogens or monoclonal antibodies is essential for retroviral transduction, but can compromise subsequent T-cell function in vivo. We report the application of lentiviral vectors for transduction of T cells in cytokine culture, without activation through the T-cell receptor. Using vectors encoding either enhanced green fluorescent protein or a truncated CD34/mutant HSVTK fusion selection/suicide construct, we investigated the properties of T cells after gene modification. We found that following cytokine stimulation, a fraction of T cells undergoes division, and transgene expression occurred predominantly in these cells. Antiviral and alloreactive responses were preserved in these populations, and in contrast to fully activated T cells, there was minimal perturbation of regulatory T-cell numbers. We conclude that the use of interleukin-7 for lentiviral transduction offers the greatest potential for gene transfer to T cells without loss of function, and is favored for the clinical production of suicide gene modified T cells.

Murine Retroviral but not Human Cellular Promoters Induce In Vivo Erythroid-specific Deregulation that can be Partially Prevented by Insulators

We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT) selective gene in a constitutive way. We report that transduction of murine hematopoietic stem cells (HSCs) with a human phosphoglycerate kinase promoter-based vector at low multiplicity of infection (MOI) does not result in a selective in vivo expansion in the presence of alkylating agents. In contrast, by replacing this cellular promoter with the powerful retroviral-derived myeloproliferative sarcoma virus enhancer, negative control region-deleted, dl587rev primer-binding site substituted promoter, the vector allowed efficient chemoprotection of transduced HSCs at low MOI. However, this promoter interacted with the erythroid HS40/ankyrin enhancer/promoter driving green fluorescent protein, leading to an unexpected loss of erythroid specificity. A partial restoration of tissue-specific expression was obtained by interposition of insulator sequences between the expression units. Alternatively, we found that the strong human cellular elongation factor1-alpha promoter allows similar chemoprotection but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data demonstrate that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity.

Tubulovesicular Structures within Vesicular Stomatitis Virus G Protein-Pseudotyped Lentiviral Vector Preparations Carry DNA and Stimulate Antiviral Responses via Toll-Like Receptor 9

Recombinant lentiviral vectors (LVs) are commonly used as research tools and are being tested in the clinic as delivery agents for gene therapy. Here, we show that Vesicular stomatitis virus G protein (VSV-G)-pseudotyped LV preparations produced by transient transfection are heavily contaminated with tubulovesicular structures (TVS) of cellular origin, which carry nucleic acids, including the DNA plasmids originally used for LV generation. The DNA carried by TVS can act as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production by plasmacytoid dendritic cells (pDC). Removal of TVS markedly reduces the ability of VSV-G-pseudotyped LV preparations to activate pDC. Conversely, virus-free TVS are sufficient to stimulate pDC and act as potent adjuvants in vivo, eliciting T- and B-cell responses to coadministered proteins. These results highlight the role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations and suggest possible strategies for diminishing responses to LVs in gene therapy and in research use.

Selection of novel vesicular stomatitis virus glycoprotein variants from a peptide insertion library for enhanced purification of retroviral and lentiviral vectors.

The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering.

RTE and CTE mRNA export elements synergistically increase expression of unstable, Rev-dependent HIV and SIV mRNAs

Studies of retroviral mRNA export identified two distinct RNA export elements utilizing conserved eukaryotic mRNA export mechanism(s), namely the Constitutive Transport Element (CTE) and the RNA Transport Element (RTE). Although RTE and CTE are potent in nucleocytoplasmic mRNA transport and expression, neither element is as powerful as the Rev-RRE posttranscriptional control. Here, we found that whereas CTE and the up-regulatory mutant RTEm26 alone increase expression from a subgenomic gag and env clones, the combination of these elements led to a several hundred-fold, synergistic increase. The use of the RTEm26-CTE combination is a simple way to increase expression of poorly expressed retroviral genes to levels otherwise only achieved via more cumbersome RNA optimization. The potent RTEm26-CTE element could be useful in lentiviral gene therapy vectors, DNA-based vaccine vectors, and gene transfer studies of other poorly expressed genes.

458. Development of Non-Integrating and Site- Specifically Integrating Lentiviral Vectors

The lentiviral genome non-specifically integrates into the human genome and this can lead to insertional mutagenesis and potentially cell transformation. We are using two strategies to eliminate the risk of insertional mutagenesis thereby improving the safety and efficacy of lentiviral gene therapy vectors. The first is to develop non-integrating lentiviral vectors and the second is to target integration to a non-pathogenic site in the human genome.

466. Comprehensive Analyses of Diverse Lentiviral and Retroviral Vectors for Post-Entry Restriction by TRIM5α and Implications for Gene Therapy

We analyzed HIV-1, FIV, EIAV, N-MLV and NB-(i.e., Moloney)- MLV vectors for post-entry restriction by human (Ref1) and monkey (Lv1) activities and by the respective species-specific TRIM5α proteins that mediate them. RT-normalized preparations were compared. FIV, EIAV and HIV-1 vector infectivity varied widely but concordantly in numerous non-primate mammalian cell lines. In contrast, in diverse human and monkey cells, relative restriction of FIV and EIAV compared to HIV-1 varied from none to substantial, with the greatest nonprimate vector infectivity deficits observed in human T cell lines. Using permissive (restriction factor-lacking) non-primate cells engineered to express human or rhesus TRIM5α, we compared restricted (HIV-1, FIV, EIAV, and N-MLV) and unrestricted (NB-MLV) retroviruses across wide ranges of viral inputs. EIAV, FIV and N-MLV vectors were restricted by rhesus and human TRIM5α, while HIV was only significantly restricted by the rhesus allele and NB-MLV was unrestricted. For FIV, human TRIM5α was less restricting than rhesus TRIM5α, while EIAV was approximately equivalently restricted by both alleles. N-MLV was much more proportionately restricted than FIV or EIAV by human TRIM5α. Rhesus and human TRIM5α proteins restricted in a saturable manner, since endogenous and introduced TRIM5α restrictions could be titrated by co-transduction with FIV, HIV-1 or EIAV virus-like particles. We achieved stable RNAi knockdown of endogenous rhesus TRIM5α in FrhK4 cells, which released the Lv1 restriction, resulting in marked increases in all lentiviral vector infectivities while having no effect on negative control NB-MLV. Interestingly, in canine cells but not feline cells, human TRIM5α supported minimal restriction of FIV compared to significant restriction of EIAV, a phenomenon which is under ongoing mechanistic investigation. We conclude that human and rhesus TRIM5α proteins restrict both FIV and EIAV vectors, with the degree of restriction in different human cell lines varying from none to substantial. Human T cells are more susceptible to transduction by HIV vector than either non-primate vector. Stable knockdown of rhesus TRIM5α releases LV1 restrictions. Differential restrictions in canine cells raise the possibility that these proteins may not function autonomously or that a canine factor may interfere. Further development of lentiviral vector gene therapy will benefit from informed appraisal of the consequences of Ref1/human TRIM5α restriction, which may be advantageous or disadvantageous depending on the particular application.

Oncogenesis Following Delivery of a Nonprimate Lentiviral Gene Therapy Vector to Fetal and Neonatal Mice

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients’ chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.