Lentiviral Vector Gene Research Articles

MiRNA Lentiviral Vector Integration and Gene Targeting Efficacy in Cardiac Progenitors

Stem and progenitor cardiac cells are challenging for possible cell therapy application. Several research laboratories are exploiting the feasibility of autologous cell therapy approach to get rid of immunosuppressive treatments responsible for undesirable side effects. Recently, we showed that cardiac progenitors isolated from Sgcb null mice, animal model of limb-girdle muscular dystrophy type 2E, undergo an aberrant differentiation in vitro and in vivo due to the dysregulation of miR669. This miRNA family is able to inhibit the skeletal myogenic program directly targeting MyoD 3’ UTR. Using lentiviral technology we provided evidence that it is possible to rescue the dystrophic aberrant phenotype by miRNA669overexpression without gene correction. However, how the viruses carrying the miRNAs were positioned in the genome upon transduction and how their localization site could influence the rescue potential was not analysed. Here we investigate the integration profile of lentiviral vector carrying the pre-miR669 in infected polyclonal and clonal populations derived from Sgcb cardiac progenitors. Our study reveals that the retroviral insertion sites (RIS) are largely restricted to coding genes (65%). Although with the limitation of our analysis, we found no hits for cancer-related genes and several sequenced RIS brought to light genes mainly involved in muscle function. Thus our data show that lentiviral vector insertional profile is cell-specific, however, the chromatin state of target cells positively influences the viral integrations.

Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor β (TGF-β) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein.In this study, we developed an efficient BMP7 delivery system, using a third generation lentiviral vector to produce functional BMP7 protein. The lentiviral transduction of several human cell types, including human embryonic kidney 293 (HEK293) cells, amniotic fluid cells, NTera2 neurons (NT2-N) and primary neuronal cultures resulted in BMP7 expression. The production of BMP7 protein was achieved for at least 4 weeks post-transduction, as determined by enzyme-linked immunosorbent assay (ELISA). SMAD phosphorylation and neuronal differentiation assays verified the bioactivity and functionality of the lentiviral-based BMP7 protein, respectively. In addition, the intracerebroventricular injection of the lentivirus resulted in exogenous BMP7 expression in both neurons and astrocytes in the mouse brain.Taken together, this gene delivery system provides a reliable source of functional BMP7 protein for future in vitro and in vivo studies.

Preconditioning Therapy with Lentiviral Vector-Programmed Dendritic Cells Accelerates the Homeostatic Expansion of Antigen-Reactive Human T Cells in NOD.Rag1−/−.IL-2rγc−/−mice

Dendritic cell (DC)-based immunization is a potent strategy to direct prompt and durable immune responses against viral reactivations after transplantations. Here, we show that overnight lentiviral vector (LV) gene transfer into human monocytes co-expressing granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 induced self-differentiated DCs (SMART-DCs) with stable DC immunophenotype over weeks in culture and secreted several inflammatory cytokines. SMART-DCs injected subcutaneously in immunodeficient NOD.Rag1(-/-).IL2rγ(-/-) (NRG) mice 1 day after LV transduction were stable for a month in vivo. "Conventional" DCs (cDCs) and SMART-DCs were compared with regard to their potency to accelerate the expansion, biodistribution, and antigenic stimulation of autologous human T cells. Peripheral blood cells obtained from human cytomegalovirus (hCMV)-reactive donors and full-length hCMV pp65 antigenic protein or peptides were used. DCs loaded with pp65 were administered subcutaneously into NRG mice as a preconditioning treatment a week prior to intravenous infusion with T cells. Optical imaging analyses demonstrated that in mice preconditioned with SMART-DC-pp65, T cells were directly recruited to the immunization site and subsequently spread to the spleen and other organs. A dramatic expansion of both human CD8(+) and CD4(+) T cells could be observed within a few days after infusion, and this was associated with consistent measurable CD8(+) effector memory T-cell responses against different pp65 epitopes. Thus, this mouse model demonstrates the proof-of-principle for SMART-DCs to accelerate expansion of human lymphocytes, resulting in poly-functional and antigen-specific immune responses against hCMV-pp65.

Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection

AbstractA recent clinical trial for adrenoleukodystrophy (ALD) showed the efficacy and safety of lentiviral vector (LV) gene transfer in hematopoietic stem progenitor cells. However, several common insertion sites (CIS) were found in patients' cells, suggesting that LV integrations conferred a selective advantage. We performed high-throughput LV integration site analysis on human hematopoietic stem progenitor cells engrafted in immunodeficient mice and found the same CISs reported in patients with ALD. Strikingly, most CISs in our experimental model and in patients with ALD cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CISs found in tumor cells from γretroviral vector–based clinical trials and oncogene-tagging screenings in mice always target a single gene and are contained in narrow genomic intervals. These findings imply that LV CISs are produced by an integration bias toward specific genomic regions rather than by oncogenic selection.

The 156KELK159 tetrapeptide of HIV-1 integrase is critical for lentiviral gene integration

HIV-1 integrase (HIV-1 IN), a key element of HIV-1-derived lentiviral vectors, is crucial for the stable maintenance of the vector gene by inserting them into host genome. HIV-1 IN has been found to have functions other than integration, such as involving in virion morphology, viral DNA synthesis and viral DNA nuclear import. In our study, the yeast two-hybrid assay identified a tetrapeptide 156KELK159 in HIV-1 IN that was crucial for HIV-1 IN and Daxx interaction. To investigate the functions of the tetrapeptide 156KELK159 of the HIV-1 IN, both the wild type HIV-1 IN and a mutant without 156KELK159 were used to package the EGFP reporter gene contained lentivirus. p24 based titer assay revealed that deleting the tetrapeptide did not affect virus packaging. The result was verified by quantitative real time PCR with viral specific primers. But the 156KELK159 was crucial for lentiviral gene integration. Deleting the tetrapeptide made the percentage of cells expressing the reporter gene significantly decreased and did not affect the level of DNA entered into the cells or nucleus. Real time reverse transcription PCR and FACS were used to detect the lentiviral report gene expression in infection maintaining cells and revealed 156KELK159 did not affect lentiviral vector gene expression. Our results may shed light on the regulatory mechanism of gene integration of lentivirus.

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis.

Lysophosphatidylcholine as an adjuvant for lentiviral vector mediated gene transfer to airway epithelium: effect of acyl chain length

BackgroundPoor gene transfer efficiency has been a major problem in developing an effective gene therapy for cystic fibrosis (CF) airway disease. Lysophosphatidylcholine (LPC), a natural airway surfactant, can enhance viral gene transfer in animal models. We examined the electrophysiological and physical effect of airway pre-treatment with variants of LPC on lentiviral (LV) vector gene transfer efficiency in murine nasal airways in vivo.MethodsGene transfer was assessed after 1 week following nasal instillations of a VSV-G pseudotype LV vector pre-treated with a low and high dose of LPC variants. The electrophysiological effects of a range of LPC variants were assessed by nasal transepithelial potential difference measurements (TPD) to determine tight junction permeability. Any physical changes to the epithelium from administration of the LPC variants were noted by histological methods in airway tissue harvested after 1 hour.ResultsGene transduction was significantly greater compared to control (PBS) for our standard LPC (palmitoyl/stearoyl mixture) treatment and for the majority of the other LPC variants with longer acyl chain lengths. The LPC variant heptadecanoyl also produced significantly greater LV gene transfer compared to our standard LPC mixture. LV gene transfer and the transepithelial depolarization produced by the 0.1% LPC variants at 1 hour were strongly correlated (r2 = 0.94), but at the 1% concentration the correlation was less strong (r2 = 0.59). LPC variants that displayed minor to moderate levels of disruption to the airway epithelium were clearly associated with higher LV gene transfer.ConclusionsThese findings show the LPC variants effect on airway barrier function and their correlation to the effectiveness of gene expression. The enhanced expression produced by a number of LPC variants should provide new options for preclinical development of efficient airway gene transfer techniques.

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.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead

The sequencing of the human genome has greatly facilitated our ability to identify the gene candidates that are critical for developmental regulation and disease progression. However, investigators seeking to use such data to gain a better understanding of the developmental biology and therapeutic potential of pluripotent stem cell types may be frustrated by the limitations of current genetic engineering techniques. Targeted genomic manipulation of mammalian cells is inefficient, and unintended effects of such manipulation on the cells’ developmental and proliferative potential remain uninvestigated. To this end, the recent demonstration of targeted gene correction in human induced pluripotent stem cells (hiPSCs) before and after reprogramming, as reported in this issue of Molecular Therapy, warrants special mention.1

Improvement of lentiviral vector-mediated gene transduction by genetic engineering of the structural protein Pr55Gag

The lentiviral vector is a promising tool for human gene therapy because of its ability to transduce genes into many cell types. However, one of the technical problems associated with the lentiviral vector is that lentiviral titers in current production systems are relatively low compared with the other viral vectors. In this study, we provide genetic evidence that the attachment of heterologous myristoylation (myr) signals on the amino-terminus of human immunodeficiency virus type 1 Pr55(Gag) (Gag) can increase the viral yield up to 10-fold, leading to the enhancement of gene transduction in many cell lines. The myr signal Gag constructs behaved similarly to the wild-type Gag in targeting to detergent-resistant membrane compartments, Vps4-dependence for viral budding, and virion morphology. However, the myr signal Gag constructs showed improved oligomerization efficiency as measured by bioluminescence resonance energy transfer in living cells, contributing to increased viral production and efficient activation of the viral protease responsible for virion maturation. The genetically modified Gag represents the next generation lentiviral vector, and should contribute to the success of many lentiviral vector applications.

B cell–specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia

AbstractThe immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Eμ) and Igβ (B29) minimal promoter to drive B lineage–specific human Btk expression in Btk/Tec−/− mice, a strain that reproduces the features of human XLA. After transplantation of EμB29-Btk-LV–transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage–targeted LV gene therapy in human XLA.

Sonoporation-mediated gene transfer into adult rat dorsal root ganglion cells

BackgroundGene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment. However, effective transfection of postmitotic neurons remains a challenge. The aim of this study was to develop a method for gene transfer into rat primary dorsal root ganglion neurons using sonoporation.MethodsDissociated cells from adult rat dorsal root ganglion (DRG) cells were sonicated for 1-8 s at 2.5-10 W to determine the optimal ultrasound duration and power for gene transfection and cell survival. Transfection efficiency was compared between sonoporation, liposome and lentiviral vector gene transfer techniques.ResultsThe optimum ultrasound intensity was 5 W for 2 s and yielded an efficiency of gene transfection of 31% and a survival rate of 35%.ConclusionsSonoporation can be optimized to minimize cell death and yield a high percentage of transfected neurons and that this technique can be easily applied to primary cultures of rat dorsal root ganglion neurons.

From TCR Gene Transfer to TCR Gene Editing of Central Memory T Lymphocytes for Immunotherapy of Leukemia.

Abstract 374▪▪This icon denotes an abstract that is clinically relevant.The transfer of a T cell receptor (TCR) from a high-avidity tumor-specific lymphocyte to polyclonal lymphocytes as been proposed as an attractive approach to overcome the major limitations of adoptive immunotherapy of cancer, such as the difficulty in expanding rare, high-avidity tumor-specific CTLs from cancer patients, in conditions that can preserve their function and prevent exhaustion. However, complete exploitation of this approach is limited by technical and safety issues, like the low and transient transgene expression, unpredictable pairing of the exogenous and endogenous TCR chains and poor survival and expansion potential of gene-modified effector lymphocytes. We developed a strategy aimed at overcoming these limitations. First, to increase TCR expression and facilitate proper TCR pairing we used a codon-optimized high-avidity TCR, specific for an HLA-A2-restricted peptide from the oncogenic Wilms tumor antigen 1 (WT1), modified with point mutations to introduce cysteines into the constant regions of the α and β chains. We cloned genes encoding the TCR in third generation lentiviral vectors under the control of a bi-directional PGK or a bi-directional EF1α promoter. Second, to increase the expected life span and expansion potential of gene modified cells, we transduced lymphocytes upon activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28) and culture with low doses of IL-7/IL-15. Human T lymphocytes were efficiently transduced by both vectors. However, PGK promoter was superior to EF1α in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 70 days, in the absence of further T cell stimulation. The phenotype of transduced cells was consistent with early T cell differentiation (CD62L+, CD28+CD27+, IL7Rα+, IL-2+ γIFN±), a phenotype associated with long-term persistence of adoptively transferred lymphocytes. Most importantly, TCR transduced cells were able to specifically produce γIFN and exhibited cytotoxic activity against WT1+HLA-A2+ primary leukemic blasts from AML patients. Third, to further improve the safety of the strategy and ensure predictable levels of transgene expression, we developed, for the first time, a protocol for site-specific integration of transgenes into primary T lymphocytes by exploiting zinc finger nucleases (ZFNs). Delivery in primary T lymphocytes of ZFNs targeting the CCR5 gene, in association with a GFP expression cassette flanked by CCR5 homology arms, resulted in efficient (6%) site-specific integration of GFP into the CCR5 locus. Extensive molecular analysis on sorted gene-modified cells and clones confirmed site-specific integration and showed a biallelic CCR5 disruption in 87% of GFP+ clones, due to a combined site-specific integration in one CCR5 allele, and non-homologous end joining (NHEJ) repair in the other allele. Finally, we exploited ZFNs to address the major limitation of TCR gene transfer, represented by the co-expression in the same cell of the endogenous and tumor-specific TCR. Such co-expression results in a dilution of tumor-specific TCR, thus reducing the efficacy of gene-modified cells, and might result in autoreactive specificities due to misparing between endogenous and exogenous α and β TCR chains. To this purpose, we used a new set of ZFNs specific for the constant region of the TCR β chain and knock down the expression of endogenous TCR in 20% of Jurkat cell line and 3.5% of primary human lymphocytes. These results show that a complete editing of T cell specificities can be achieved in human lymphocytes. (E.P. and P.G. equally contributed to this work). Disclosures:Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

High Levels of Multilineage Hematopoietic Cell Gene Marking Following Lentiviral Vector Transduction and Transplantation of Steady-State Bone Marrow CD34+ cells in a Non-Human Primate Model.

Abstract 2464Poster Board II-441Non-human primate autologous hematopoietic stem cell (HSC) transplantation models have been invaluable in developing the ex vivo transduction protocols used in human gene transfer clinical trials. Previous studies of HSC gene transfer using gamma-retroviral and lentiviral vectors in these models have almost exclusively utilized cytokine-mobilized peripheral blood (PB) or bone marrow (BM) CD34+ cells as target cells. The administration of the combination of granulocyte colony stimulating factor (G-CSF) plus stem cell factor (SCF) became standard as a priming method prior to HSC collection after it was shown that SCF coupled with G-CSF resulted in significantly improved long-term gene transfer levels, compared to mobilization with G-CSF alone (Hematti et al., Blood 2003 101:2199-2205). However, SCF is not available in the US for human gene transfer trials due to its history of causing severe anaphylactic reactions. For this reason and to define a clinically relevant gene transfer protocol for sickle cell disease, in which G-CSF administration is also contraindicated, we evaluated lentiviral stem cell gene transfer using steady-state BM CD34+ cells in the pigtail macaque transplantation model. Bone marrow was harvested from the long bones and CD34+ cells were enriched using an anti-CD34 antibody and immunomagetic beads (92% purity). Cells were prestimulated overnight in serum-free medium containing 100 ng/ml of FLT-3 ligand, thrombopoietin, and stem cell factor. Cells were then exposed for 14 hours to a VSV-G pseudotyped, HIV-based lentiviral vector encoding GFP at an MOI of 40. Cells were collected, washed, and placed back into culture in serum-free medium containing the above cytokines for an additional 24 hours. Subsequently, cells were collected (15 × 106/kg) and transplanted into an animal which had been conditioned with 950 cGy. Marking of the graft, as assessed by flow cytometry for GFP expression, was 74% and all hematopoietic colonies (CFU) obtained in methylcellulose cultures were GFP positive. Neutrophil recovery (ANC > 500) was observed on day 20. One month following transplant, 36% of peripheral blood granulocytes (GRANS) were GFP positive. At two months post-transplant, marking levels were: GRANS 27%, RBC 43%, and platelets (PLT) 22%. These levels have remained steady out to 6 months post-transplantation: GRANS 21%, lymphocytes 20%, RBC 17%, and PLTS 16%. A BM aspirate was obtained and analysis of BM CFU showed that 186 of 508 (37%) were GFP positive. Southern blot analysis and q-PCR on DNA from peripheral blood leukocytes and BM showed a vector copy number ranging from 0.6 to 1.0 over time, suggesting that the positive cells contained several vector copies. No evidence of clonal dominance has been observed by Southern blot analysis using restriction enzymes which cut only once in the vector. Insertion site analysis using deep sequencing technology is in progress. A second animal has recently been transplanted (11 × 106 cells/kg) with graft marking of greater than 50%. These data are encouraging as they suggest that therapeutic levels of lentiviral vector gene transfer can be obtained using CD34+ cells from steady-state bone marrow as the HSC cell source. This is important since SCF cannot be used in humans and for patients with sickle cell disease, administration of G-CSF is also contraindicated. Disclosures:No relevant conflicts of interest to declare.

Abstract 5436: Generation of Induced Pluripotent Stem Cells From Peripheral Blood of Coronary Artery Disease Patients

Background. Studies have demonstrated that ectopic expression of defined transcription factors can reprogram somatic cells into induced pluripotent stem cells (iPS). However, the sources of patient-derived iPS cells are limited to fibroblasts or mesenchymal cells, which require invasive procedures and long-term manipulation. Accordingly, in this study we aimed to generate human iPS cells from peripheral blood mononuclear cells (PB-MNCs) of coronary artery disease (CAD) patients. Methods and Results. We recruited one healthy volunteer and three male patients aged 58, 62, and 77, respectively, who have CADs confirmed by coronary angiography. PB-MNCs obtained by density gradient centrifugation were cultured in an adherent fashion. The adherent cells were infected with the lentiviral supernatant for the four transcription factors of OCT4, SOX2, KLF4 or c-MYC. We used third generation lentiviral vectors which do not have infectivity. At 10 days post-infection, colonies showing hES cell-like morphology were selected and transferred to feeder layers for expansion. After 3 to 4 passages, we observed tightly packed and flat colonies of cells characterized by large nuclei and scant cytoplasm, highly suggesting successful generation of iPS cells from PB-MNCs of all the patients (HiPS-ECP1, HiPS-ECP2 and HiPS-ECP3) and a healthy volunteer (HiPS-ENB). A series of assays including cytochemistry, real-time RT-PCR and teratoma formation assay revealed identical pluripotency of these iPS cells to human ES cells (H1). Bisulfite sequencing of the OCT4 and NANOG promoters in the iPS cells showed profound demethylation similar to hES cells. We next examined the differentiation capacity of the iPS cells. The iPS cells readily form EBs at 10 days in suspension culture. Upon further cultivation of EBs for another 10 days, these iPS cells spontaneously differentiated into cell types of all 3 embryonic germ layers. In conclusion. This study provides the first evidence that iPS cells can be successfully generated from PB of CAD patients, which have almost identical characteristics to iPS cells derived from healthy volunteers or human ES cells. This advance will facilitate the use of iPS cells for autologous cell therapy for ischemic cardiovascular diseases.

Growth inhibition of colorectal carcinoma by lentiviral TRAIL‐transgenic human mesenchymal stem cells requires their substantial intratumoral presence

Colorectal carcinoma (CRC) constitutes a common malignancy with limited therapeutic options in metastasized stages. Mesenchymal stem cells (MSC) home to tumours and may therefore serve as a novel therapeutic tool for intratumoral delivery of antineoplastic factors. Tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) which promises apoptosis induction preferentially in tumour cells represents such a factor. We generated TRAIL-MSC by transduction of human MSC with a third generation lentiviral vector system and analysed their characteristics and capacity to inhibit CRC growth. (1) TRAIL-MSC showed stable transgene expression with neither changes in the defining MSC characteristics nor signs of malignant transformation. (2) Upon direct in vitro coculture TRAIL-MSC induced apoptosis in TRAIL-sensitive CRC-cell lines (DLD-1 and HCT-15) but also in CRC-cell lines resistant to soluble TRAIL (HCT-8 and SW480). (3) In mixed subcutaneous (s.c.) xenografts TRAIL-MSC inhibited CRC-tumour growth presumably by apoptosis induction but a substantial proportion of TRAIL-MSC within the total tumour cell number was needed to yield such anti-tumour effect. (4) Systemic application of TRAIL-MSC had no effect on the growth of s.c. DLD-1 xenografts which appeared to be due to a pulmonary entrapment and low rate of tumour integration of TRAIL-MSC. Systemic TRAIL-MSC caused no toxicity in this model. (5) Wild-type MSC seemed to exert a tumour growth-supporting effect in mixed s.c. DLD-1 xenografts. These novel results support the idea that lentiviral TRAIL-transgenic human MSC may serve as vehicles for clinical tumour therapy but also highlight the need for further investigations to improve tumour integration of transgenic MSC and to clarify a potential tumour-supporting effect by MSC.

Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

AbstractCorrection of murine models of β-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human γ-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with β-thalassemic HSCs transduced with a γ-globin/MGMT vector initially had subtherapeutic levels of red cells expressing γ-globin. To enrich γ-globin–expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of γ-globin–expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of γ-globin–expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for β-thalassemia.

Late generation lentiviral vectors: Evaluation of inflammatory potential in human airway epithelial cells

Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their potential to induce pro-inflammatory cytokines on delivery into the respiratory tissue remains to be established. Here we tested a third-generation vesicular stomatitis virus (VSV)-G pseudotyped LV vector in the two respiratory epithelial cell lines A549 and CFT1-C2. We observed that the VSV-G LV vector does not induce (a) activation of the nuclear factor (NF)-κB, which intervenes in transcription of pro-inflammatory genes; (b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mild and transient (24 h) increase of IFN-γ mRNA. In contrast, an adenovirus-derived vector strongly activated NF-κB and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-α, IL-6, IL-1β. In conclusion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signals in human airway epithelial cells and appears to be better suited for gene delivery strategies.