Lentivirus-mediated Gene Transfer Research Articles

Lentiviral-Mediated Gene Transfer to the Sheep Brain: Implications for Gene Therapy in Batten Disease

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are inherited neurodegenerative lysosomal storage diseases with common clinical features of blindness and seizures culminating in premature death. Gene-therapy strategies for these diseases depend on whether the missing activity is a secreted lysosomal protein taken up by neighboring cells, or an intramembrane protein that requires careful targeting. Therapies are best developed in animal models with large complex human-like brains. Lentiviral-mediated gene delivery to neural cell cultures from normal sheep and sheep affected with an NCL resulted in green fluorescent protein (GFP) expression in neurons and neuroblasts, more efficiently than in astrocytes. Similar transgene expression was obtained from two constitutive promoters, the viral MND promoter and the human EF1α promoter. In vivo studies showed stable and persistent GFP expression throughout the cell bodies, axons, and dendrites from intracortical injections and indicated ependymal and subependymal transduction. The sheep showed no ill effects from the injections. These data support continuing gene-therapy trials in the sheep models of Batten disease.

Lentivirus-mediated Wnt11 Gene Transfer Enhances Cardiomyogenic Differentiation of Skeletal Muscle-derived Stem Cells

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Characterization and biodistribution of human mesenchymal stem cells transduced with lentiviral-mediated BMP2

Recently, the genetic modification of mesenchymal stem cells (MSCs) has led to increased differentiation potential. For the therapeutic application of genetically modified MSCs, it is crucial to evaluate their characteristics and safety. In this study, we investigated the effects of bone morphogenetic protein 2 (BMP2) gene transfer on the characteristics and biodistribution of human MSCs. Lentiviral-mediated BMP2 transduction to MSCs enhanced osteocyte differentiation and decreased adipocyte differentiation. Although there is no significant difference in cell proliferation capacity, MSCs transduced BMP2 proliferate somewhat higher than nontransduced or GFP transduced MSCs. No significant changes were observed in surface antigen expression in genetically modified MSCs. In vivo transplantation of lentiviral-mediated BMP2 gene transferred MSCs to nude mice did not result in tumor formation. To evaluate the biodistribution of genetically modified cells, MSCs carrying BMP2 were injected into the tail vein of femur fractured mice. The introduced MSCs were detected in the spleen, testis and fractured femur 28 days post-implantation. These findings suggest that diverse safety tests for genetically modified MSCs should be considered, particularly when a lentivirus mediated gene transfer method is used.

Anti‐tumor effect of lentivirus‐mediated gene transfer of alphastatin on human glioma

Alphastatin, an endogenous angiogenesis inhibitor, has recently been used as an anticancer agent in several tumor models. This study was to investigate whether local sustained long-term expression of alphastatin could serve to diminish tumor growth of a human xenograft glioma model. We found that the recombinant alphastatin lentiviruses were able to stably infect HUVECs, and infected HUVECs could sustainably secrete alphastatin, which exhibited potent inhibitory effects on HUVECs migration, differentiation but not proliferation induced by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor(bFGF). And the expression of secreted protein alphastatin markedly decreased tumor vascularization and inhibited tumor growth. Additionally, alphastatin inhibited VEGF- or bFGF-induced initial stage of angiogenesis by reducing JNk and ERK phosphorylation in vitro. Taken together, these data demonstrate that secreted protein alphastatin inhibits VEGF- or bFGF-induced angiogenesis by suppressing JNK and ERK kinases activation pathways in HUVECs, and markedly inhibits tumor angiogenesis in vivo. Consequently lentivirus-mediated gene transfer might represent an effective strategy for expression of alphastatin to achieve inhibition of human malignant glioma proliferation and tumor progression.

Neurotrophin 3 Improves Delayed Reconstruction of Sensory Pathways After Cervical Dorsal Root Injury

Spinal root avulsion, or section, results in devastating functional sequels. Whereas reconstruction of motor pathways based on neurotization can reduce motor deficit, associated permanent limb anesthesia limits expected benefit. Sensory pathway reconstruction after dorsal root injury is limited by the inability of re-growing central sensory axons to enter the spinal cord through an injured root. To provide evidence for the reconnection of C7 DRG neurons with the central nervous system (CNS) after experimental section of the C7 dorsal root in adult rats. We assessed a new reconstruction strategy in adult rats 9 weeks after transection of C6 and C7 dorsal roots. Re-growing C7 central sensory axons were redirected to the noninjured C5 dorsal root through a nerve graft by end-to-side anastomosis that did not alter the C5 conduction properties. In a subgroup of rats, surgical reconstruction was combined with lentivirus-mediated gene transfer to the nerve graft in order to overexpress neurotrophin 3 (NT-3), a neurotrophic factor that stimulates sensory axon regeneration. Four months after reconstruction, recording of sensory evoked potentials and fluorescent tracer transport showed electrical and physical reconnection of the C7 dorsal root ganglion neurons to the spinal cord through the reconstructed pathway. Sensory perception recovery predominated on proprioception. Axonal regrowth and perception were improved when the nerve graft overexpressed neurotrophin-3 at the time of transplantation. Neurotrophin-3 overexpression did not persist 4 months after transplantation. Efficient and functional reconnection of dorsal root ganglion neurons to the spinal cord can be achieved in rats several weeks after cervical dorsal root injury. Surgical repair of sensory pathways could be considered in combination with motor nerve neurotization to treat persisting severe upper limb disability in humans.

Correction of Murine Bernard Soulier Syndrome by Lentivirus-Mediated Gene Therapy

AbstractAbstract 552Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of developing allo-immunization. Ware and colleagues have developed a murine model of BSS by targeting GPIbα (GPIbαnull), and have shown that the BSS phenotype was rescued by transgenic expression of hGPIbα. We have previously reported successful expression of human GPIbα in human megakaryocytes using a lentiviral vector (LV) encoding hGPIbα under the transcriptional control of integrin aIIb promoter (2bIbα). In this study, we examined the efficacy of this strategy for the gene therapy of BSS using GPIbαnull as a murine model of BSS. GPIbαnull hematopoietic stem cells (HSC) transduced with 2bIbα LV were transplanted into lethally irradiated GPIbαnull littermates. After bone marrow (BM) reconstitution, mice were analyzed. The presence of 2bIbα transgene in recipients was confirmed by PCR amplification of white blood cell derived genomic DNA. Flow cytometry demonstrated that 84.5% ± 9.5% (n = 9) of platelets expressed hGPIbα at 6 weeks after transplantation and stable expression was maintained through the entire observation period of 7 months. Immunofluorescent confocal microscopy demonstrated that transgene protein was expressed on the cell surface of transduced platelets. Tail bleeding times were corrected to normal levels in the GPIbαnull recipients who received LV transduced GPIbαnull HSC (2.3 ± 2.9 minutes, n = 9). On the other hand, recipients who received untransduced GPIbαnull HSC exhibited prolonged bleeding times (8.8 ± 2.4 minutes, n = 4) that were similar to GPIbαnull mice. Macrothrombocytopenia improved with significantly increased platelet counts and decreased platelet sizes in LV transduced GPIbαnull HSC recipients compared to untransduced GPIbαnull HSC recipients (platelet counts; 4.9 ± 1.3×105/μ l, n = 9 vs. 1.8 ± 0.1×105/μ l, n = 4, and mean platelet volume; 6.9 ± 0.7 fL, n = 9 vs. 9.3 ± 0.1 fL, n = 4, respectively). As expected, expression levels of hGPIbα correlated with platelet counts and inversely correlated with the platelet size. Immunoprecipitation followed by Western blot analysis showed that hGPIbα expressed on platelets associated with endogenous murine GPIbβ and GPIX. No antibody to hGPIbα was detected in these recipients. Furthermore, BM mononuclear cells from the primary recipients were transplanted into the secondary GPIbαnull recipients and the results showed sustained expression of hGPIbα leading to the correction of bleeding phenotype as well as macrothrombocytopenia. These results demonstrate that lentivirus mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a promising strategy for gene therapy of BSS in human. Disclosures:No relevant conflicts of interest to declare.

MODELING GENETIC Diseases through Reprogramming of HUMAN Amniotic Liquid Derived CELLS

AbstractAbstract 4789Pluripotency and self-renewal, two key characteristics of induced pluripotent stem cells (IPS), make these cells ideally suited for modeling diseases in vitro and generating biological resources usable for drug screening and cell therapy. However, the reprogramming efficiency of somatic cells greatly varies according to the cell type, to the in vitro proliferation index, the number of passages and the age of the donor. Human amniotic liquid-derived cells (hALDC), collected during amniocentesis for the prenatal diagnosis of genetic diseases, represent an abundant source of primary cells. In preliminary experiments we have shown that hALDC expressed endogenous Oct4 and Sox2 proteins suggesting that could be readily amenable to reprogramming. To this end, we have used two strategies using either hALDC or neonatal fibroblasts: (1) lentivirus mediated gene transfer of OCT4, SOX2, LIN28, NANOG, (2) retroviruses mediated gene transfer of OCT4, SOX2, CMYC, KLF4 and (3) lentiviral transfer of OCT4, SOX2. hALDC transduced by these viruses were placed on MEF and b-FGF (10 ng/ml) with daily medium changes. One to three weeks after infection, typical human ES-like colonies could be picked up for expansion before being characterized. HALDC show an increased reprogramming potential with the [OCT4, SOX2, LIN28, NANOG] and [OCT4, SOX2] cocktails, when compared to reprogramming of neonatal fibroblasts. Twelve hALDC-derived-IPS cells were obtained from 12 different samples of amniotic fluid. All hALDC-IPS cell lines maintained a normal karyotype in culture and displayed the morphology and characteristics of human embryonic stem cells, including the surface expression of Tra-160, SSEA-3, SSEA-4, HESCA-1 and alkaline phosphatase, and formed multi-lineaged teratomas upon injection to NOD-SCID mice. Gene expression profiles of the IPS cell lines reveal a high correlation coefficient between hALDC-iPS cells and human embryonic stem cells, and a low correlation between hALDC-iPS and hALDC. When compared to hES cells H1, H9 and Cl01, these cell lines generated hematopoiesis with a variable efficiency in vitro.Amongst the hALDC-IPS cell lines generated by our laboratory (http://www.hescreg.eu/) four lines carry an inherited trisomy of chromosome 21, and three lines carry the homozygous “S” mutation in the beta-globin gene of sickle-cell anemia. All hALDC-IPS cell are currently banked at the Human Pluripotent Stem Cell Core Facility, France. In conclusion, hALDC can be rapidly and efficiently reprogrammed to pluripotency with a limited number of transgenes. Moreover, hALDC-IPS cell lines derived from patients can be used to modelize in vitro the phenotypic features of monogenic diseases such as sickle cell anemia or more complex, multifactorial disorders such as Down's syndrom. The ability to generate hematopoietic differentiation from these cell lines will facilitate the modelling of these hematopoietic disorders. Disclosures:No relevant conflicts of interest to declare.

Inhibitory effects of lentivirus-mediated sFlt-1 gene transfer on retinal neovascularization

目的 研究可溶性fms样酪氨酸激酶-1(sFlt-1)对小鼠视网膜新生血管的抑制作用.方法 将54只C57/6J小鼠随机分为3组,每组18只.将其中2组小鼠于生后第7天开始置于体积分数75%氧环境中,5d后返回正常空气环境中以建立氧诱导的小鼠视网膜新生血管模型,于17d时玻璃体腔内分别注射1μL lenti-GFP和携带sFlt-1基因片段的重组慢病毒,设为模型对照组和lenti.sFlt-1组,18只正常空气环境中生长的小鼠玻璃体腔内注射1μL磷酸盐缓冲液,设为正常对照组.通过小鼠眼球连续切片苏木精-伊红染色和心脏荧光素灌注视网膜铺片法观察小鼠视网膜新生血管的变化情况,采用免疫组织化学染色法检测视网膜血管内皮生长因子(VEGF)和VEGF受体-2(KDR/Flk-1)的表达变化.结果 经RT-PCR扩增的靶基因片段序列与GenBank中的标准序列相吻合.感染后的人RPE细胞能够表达sFlt-1蛋白.正常对照组小鼠视网膜内界膜平整,模型对照组小鼠突破内界膜血管内皮细胞核的数目为(47.26±6.76),而lenti.sFlt-1组为(5.21±1.93)个,差异有统计学意义(P＜0.05).视网膜铺片可见新生血管荧光素渗漏表现.慢病毒携带的sFlt-1基因片段转移至模型小鼠视网膜后,发现突入玻璃体腔的血管内皮细胞核数较模型对照组显著减少,并且视网膜毛细血管扩张、微血管瘤样改变、新生血管等荧光素渗漏表现亦明显减少;同时lenti.sFlt-1组VEGF的表达与模型对照组相比在视网膜神经节细胞层和内核层仍呈现强阳性染色,无明显变化,而KDR/Flk-1的阳性染色显著减少.结论 慢病毒介导sFlt-1基因转移能显著抑制小鼠视网膜新生血管的发生。

Lentiviral Vector-Mediated Gene Transfer and RNA Silencing Technology in Neuronal Dysfunctions

Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition.

Correction of mucopolysaccharidosis type IIIA somatic and central nervous system pathology by lentiviral‐mediated gene transfer

The hallmark of lysosomal storage disorders (LSDs) is microscopically demonstrable lysosomal distension. In mucopolysaccharidosis type IIIA (MPS IIIA), this occurs as a result of an inherited deficiency of the lysosomal hydrolase sulphamidase. Consequently, heparan sulphate, a highly sulphated glycosaminoglycan, accumulates primarily within the cells of the reticulo-endothelial and monocyte-macrophage systems and, most importantly, neurones. Children affected by MPS IIIA experience a severe, progressive neuropathology that ultimately leads to death at around 15 years of age. MPS IIIA pathology was addressed in a mouse model using two separate methods of therapeutic gene delivery. A lentiviral vector expressing murine sulphamidase was delivered to 6-week-old MPS IIIA affected mice either by intravenous injection, or by intraventricular infusion. Therapeutic outcomes were assessed 7 months after gene transfer. After intravenous gene delivery, liver sulphamidase was restored to approximately 30% of wild-type levels. The resultant widespread delivery of enzyme secreted from transduced cells to somatic tissues via the peripheral circulation corrected most somatic pathology. However, unlike an earlier study, central nervous system (CNS) pathology remained unchanged. Conversely, intraventricular gene delivery resulted in widespread sulphamidase gene delivery in (and reduced lysosomal storage throughout) the brain. Improvements in behaviour were observed in these mice, and interestingly, pathological urinary retention was prevented. The CNS remains the last major barrier to effective therapy for children affected by LSDs. The blood-brain barrier (BBB) limits the uptake of lysosomal enzymes from the peripheral circulation into the CNS, making direct gene delivery to the brain a reasonable, albeit more challenging, therapeutic option. Future work will further assess the relative advantages of directly targeting the brain with somatic gene delivery with sulphamidase modified to increase the efficiency of transport across the BBB.

A preclinical approach for gene therapy of β‐thalassemia

Lentiviral-mediated beta-globin gene transfer successfully treated beta-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different beta-globin mutations found in patients. Most mutations can be classified as beta(0) or beta(+), based on the amount of beta-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human beta-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley's anemia patients.

A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors

Vector injection into the perivitelline space has emerged as the standard delivery method to transduce lentivirus to mammalian oocytes or one-cell embryos, but its application is limited by the need for high titers of lentivirus. Herein we developed a new method by using a Piezo impact micro-manipulator for injecting low titer of lentivirus into the subzonal space of two-cell embryos or the perivitelline space of one-cell embryos that were shrunk with a highly concentrated sucrose solution. The survival rate of embryos was greater than 98% using this micromanipulation strategy, which was increased compared to the normal one-cell embryo injection method. More than 90% of injected embryos were GFP positive after subzonal injection of a lentivirus vector carrying the GFP gene with titers of 2 × 10⁸ I.U./ml. Even when a low titer of lentivirus (2 × 10⁶ I.U./ml) was used, 53.26% and 40.85% transgenic embryos were obtained after two-cell embryonic injection and one-cell sucrose treated embryonic injection, respectively. The GFP-positive rates were also greater than in the conventional method of injecting one-cell embryos (25.39%). In addition, blastocysts from the two-cell embryo injection group displayed stronger GFP fluorescence than the one-cell embryo injection groups treated with or without the sucrose solution. Increased expression of GFP suggests that the embryos obtained from this injection method have higher exogenous gene expression levels compared to previous methods. Therefore, in contrast with the traditional injection method, we have demonstrated a simplified and efficient lentivirus-mediated gene transfer method based on a low-titer virus preparation.

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.

Lentivirus-mediated Gene Transfer of Small Interfering RNA against the Chemokine Receptor CXCR3 Suppresses Cytokine Indicators of Acute Graft Rejection in a Rat Model

Despite improvements in immunosuppressive therapy, acute rejection remains an important cause of morbidity and late graft loss in patients undergoing liver transplantation. Increasing evidence supports an important role for chemokines and their receptors in transplant immunology. An acute liver graft rejection model in rats was used to study the role of the chemokine receptor CXCR3 in acute transplant rejection after liver transplantation by lentivirus-mediated gene transfer of small interfering RNA (siRNA) against CXCR3. Using reverse transcription-polymerase chain reaction it was first shown that three lentivirus-CXCR3 siRNA vectors inhibited the in vitro expression of CXCR3 in activated T-cells bearing CXCR3. Then, it was shown that treatment of the animals with lentivirus-CXCR3 siRNA before liver transplantation reduced CXCR3 mRNA and protein, and protein levels of interleukin (IL)-2, IL-4, IL-6 and IL-10 and interferon-gamma measured as indices of acute graft rejection. Based on the results from this animal model, targeting chemokines by the use of siRNA may become a feasible option for therapy after transplantation.

Inhibition of experimental lung metastasis by systemic lentiviral delivery of kallistatin

BackgroundAngiogenesis plays an important role in the development and progression of tumors. Kallistatin exerts anti-angiogenic and anti-inflammatory activities that may be effective in inhibiting tumor metastasis. We investigated the antitumor effect of lentivirus-mediated kallistatin gene transfer in a syngeneic murine tumor model.MethodsLentiviral vector encoding kallistatin (LV-Kallistatin) was constructed. The expression of kallistatin was verified by enzyme-linked immunosorbent assay (ELISA), and the bioactivity of kallistatin was determined by using cell proliferation, migration, and invasion assays. In addition, antitumor effects of LV-Kallistatin were evaluated by the intravenous injection of virus into tumor-bearing mice.ResultsThe conditioned medium from LV-Kallistatin-treated cells inhibited the migration and proliferation of endothelial cells. Meanwhile, it also reduced the migration and invasion of tumor cells. In the experimental lung metastatic model, tumor-bearing mice receiving LV-Kallistatin had lower tumor nodules and longer survival than those receiving control virus or saline. Moreover, the microvessel densities, the levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, and nuclear factor κB (NF-κB) transcriptional activity were reduced in the LV-Kallistatin-treated mice.ConclusionResults of this study showed that systemic administration of lentiviral vectors encoding kallistatin inhibited the growth of metastatic tumor and prolonged the survival of tumor-bearing mice. These results suggest that gene therapy using lentiviruses carrying the kallistatin gene, which exerts anti-angiogenic and anti-inflammatory activities, represents a promising strategy for the treatment of lung cancer.

Abstract 3239: Synergy of PIM1 kinase with c-MYC in prostate cancer

Abstract The oncogenes PIM1 and c-MYC are frequently up-regulated in human prostate tumors. We found that PIM1 and c-MYC were co-expressed in human prostate cancer and were associated with higher Gleason scores. We next used a model of prostate regeneration coupled with lentiviral-mediated gene transfer to directly show that Pim1 dramatically enhanced c-MYC tumorigenicity in naïve adult mouse prostatic epithelium in a kinase-dependent manner. Microarray analysis revealed that many signaling pathways including MAPK, focal adhesion calcium signaling pathway were activated in MYC/Pim1 tumor. Knockdown of Pim1 in MYC/Pim1 tumor derived cell line reduced ERK and Akt phosphorylation. We further showed that targeting Pim1 by shRNA inhibited tumorigenicity in vitro and in vivo. Pim1 kinase inhibitor quercetagetin also inhibited cell growth in vitro. These results directly demonstrate that MYC and PIM1 cooperate in promoting tumor development. PIM1 can be considered as a drug target for human prostate cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3239.

Fibrin-mediated lentivirus gene transfer: Implications for lentivirus microarrays

We employed fibrin hydrogel as a bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 and 7.5 mg/ml. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially-localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine.

Pim1 kinase synergizes with c-MYC to induce advanced prostate carcinoma

The oncogenic PIM1 kinase has been implicated as a cofactor for c-MYC in prostate carcinogenesis. Here we show that in human prostate tumors, coexpression of c-MYC and PIM1 is associated with higher Gleason grades. Using a tissue recombination model coupled with lentiviral-mediated gene transfer we find that Pim1 is weakly oncogenic in naïve adult mouse prostatic epithelium. However, it cooperates dramatically with c-MYC to induce prostate cancer within 6-weeks. Importantly, c-MYC/Pim1 synergy is critically dependent on Pim1 kinase activity. c-MYC/Pim1 tumors showed increased levels of the active serine-62 (S62) phosphorylated form of c-MYC. Grafts expressing a phosphomimetic c-MYCS62D mutant had higher rates of proliferation than grafts expressing wild type c-MYC but did not form tumors like c-MYC/Pim1 grafts, indicating that Pim1 cooperativity with c-MYC in vivo involves additional mechanisms other than enhancement of c-MYC activity by S62 phosphorylation. c-MYC/Pim1-induced prostate carcinomas demonstrate evidence of neuroendocrine (NE) differentiation. Additional studies, including the identification of tumor cells coexpressing androgen receptor and NE cell markers synaptophysin and Ascl1 suggested that NE tumors arose from adenocarcinoma cells through transdifferentiation. These results directly demonstrate functional cooperativity between c-MYC and PIM1 in prostate tumorigenesis in vivo and support efforts for targeting PIM1 in prostate cancer.

349: Lentivirus-Mediated Long-Term Gene Transduction in the Lung Attenuates Allograft Airway Obliteration after Mouse Intra-Pulmonary Trachea Transplantation

IL-10 is a potent anti-inflammatory and immune-modulating cytokine. Lentiviral vectors (LVs) facilitate stable, long-term gene transduction to many tissues and organs. Prolonged low-level IL-10 production in the lung graft would be a very attractive strategy to achieve a form of “local” systemic sparing immunosuppression in the graft. We hypothesized that lentivirus-mediated IL-10 gene transfer could modulate the alloimmune responses in the transplant setting.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.