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Lentiviral Vectors (lentiviral + vector)
Selected AbstractsEfficient hepatocyte engraftment and long-term transgene expression after reversible portal embolization in nonhuman primates,HEPATOLOGY, Issue 3 2009Ibrahim Dagher The feasibility of ex vivo gene therapy as an alternative to liver transplantation for the treatment of liver metabolic diseases needs to be analyzed in large animal models. This approach requires appropriate gene transfer vectors and effective hepatocyte engraftment. Lentiviral vectors have the ability to transduce nondividing differentiated cells, such as hepatocytes, and portal vein occlusion increases hepatocyte engraftment. We investigated whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is an effective approach for successful hepatocyte engraftment in nonhuman primates and whether the transgene remains expressed in the long term in transplanted hepatocytes in situ. Simian hepatocytes were isolated after left lobe resection, and the left and right anterior portal branches of animals were embolized with absorbable material. Isolated hepatocytes were labeled with Hoechst dye or transduced in suspension with lentiviruses expressing green fluorescent protein under the control of the human apolipoprotein A-II promoter and transplanted via the inferior mesenteric vein. The whole procedure was well tolerated. The embolized liver was revascularized within 2 weeks. The volume of nonembolized liver increased from 38.7% ± 0.8% before embolization to 55.9% ± 1% after embolization and hepatocytes significantly proliferated (10.5% ± 0.4% on day 3 after embolization). Liver repopulation after transplantation with Hoechst-labeled hepatocytes was 7.4% ± 1.2%. Liver repopulation was 2.1% ± 0.2% with transduced hepatocytes, a proportion similar to that obtained with Hoechst-labeled cells, given that the mean transduction efficacy of simian hepatocyte population was 34%. Transgene expression persisted at 16 weeks after transplantation. Conclusion: We have developed a new approach to improve hepatocyte engraftment and to express a transgene in the long term in nonhuman primates. This strategy could be suitable for clinical applications. (HEPATOLOGY 2009.) [source] Chimeric HIV-1 and HIV-2 lentiviral vectors with added safety insurance,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2007Geetanjali Sachdeva Abstract Lentiviruses are unique in their ability to infect both dividing and non-dividing cells. This makes the vectors derived from them particularly useful for gene transfer into non-dividing cells, including stem cells. Lentiviral vectors are becoming the vectors of choice for si/shRNA delivery. The utility of the lentiviral vectors will be enhanced if additional elements of safety are built into their design. One safety concern is the generation of replication competent virus by recombination. We reasoned that HIV-1 and HIV-2 hybrid or chimeric lentiviral vectors will have added safety insurance in this regard. This is based on the premise that HIV-1 and HIV-2 are dissimilar enough in sequence to curtail recombination, yet similar enough to complement functionally. For hybrid vectors, we found that both HIV-1 and HIV-2 transfer vector RNAs could be packaged to equivalent titer by the HIV-1 packaging machinery. However, HIV-2 packaging machinery was unable to package HIV-1 transfer vector as well as it did HIV-2 transfer vector. This non-reciprocacity suggested that the requirement for HIV-2 vectors was more stringent and that for HIV-1 vectors more promiscuous. When the HIV-1 transfer vector was packaged with the chimeric packaging construct where the leader-gag region of HIV-2 was replaced with that of HIV-1 packaging construct, the titer of the vector went up. This suggests that at least some of the determinants of specificity for vector assembly reside in the leader-gag region. Incorporation of central polypurine tract (cPPT) and woodchuck post-transcriptional enhance element (WPRE) into the HIV-2 vectors had only modest effect on vector titer. Thus, chimeric lentiviral vectors with added safety features can be designed without compromising transduction efficiency. J. Med. Virol. 79:118,126, 2007. © 2006 Wiley-Liss, Inc. [source] Pituitary Transcription Factors: From Congenital Deficiencies to Gene TherapyJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2006M. H. Quentien Despite the existence of interspecies phenotypic variability, animal models have yielded valuable insights into human pituitary diseases. Studies on Snell and Jackson mice known to have growth hormone, prolactin and thyroid-stimulating hormone deficiencies involving the hypoplastic pituitary gland have led to identifying alterations of the pituitary specific POU homeodomain Pit-1 transcription factor gene. The human phenotype associated with rare mutations in this gene was found to be similar to that of these mice mutants. Terminal differentiation of lactotroph cells and direct regulation of the prolactin gene both require interactions between Pit-1 and cell type specific partners, including panpituitary transcriptional regulators such as Pitx1 and Pitx2. Synergistic activation of the prolactin promoter by Pitx factors and Pit-1 is involved not only in basal condition, but also in responsiveness to forskolin, thyrotrophin-releasing-hormone and epidermal growth factor. In corticotroph cells, Pitx1 interacts with Tpit. Tpit mutations have turned out to be the main molecular cause of neonatal isolated adrenocorticotrophin deficiency. This finding supports the idea that Tpit plays an essential role in the differentiation of the pro-opiomelanocortin pituitary lineage. The effects of Pit-1 are not restricted to hormone gene regulation because this factor also contributes to cell division and protects the cell from programmed cell death. Lentiviral vectors expressing a Pit-1 dominant negative mutant induced time- and dose-dependent cell death in somatotroph and lactotroph adenomas in vitro. Gene transfer by lentiviral vectors should provide a promising step towards developing an efficient specific therapeutic approach by which a gene therapy programme for treating human pituitary adenomas could be based. [source] Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead ribozymesJOURNAL OF VIRAL HEPATITIS, Issue 4 2005K. L. Nash Summary., Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Current treatments are limited and may be ineffective. Nucleic acid-mediated targeting of viral mRNA is an attractive and specific approach for viral infection and lentiviral vectors provide a means to express antisense sequences or ribozymes stably in target cells permitting continuous production within that cell and its progeny. To demonstrate long-term gene expression by lentiviral vectors in hepatocytes and to introduce lentiviral vectors expressing anti-HBV genes to assess their effect against HBV, lentiviral vectors expressing a reporter gene were assessed for longevity of gene expression in hepatocytes in vitro. Hammerhead ribozymes and antisense sequences targeting the HBV encapsidation signal (,), X or surface antigen on mRNAs were cloned into lentiviral vectors and used to transduce HBV expressing hepatocytes where the effect on HBV mRNA level was assessed using ribonuclease protection. Gene expression in hepatocytes from integrated vectors continued for over 4 months without selection. Antisense RNA targeting HBs mRNA reduced this transcript, whilst antisense RNA to HBX mRNA was ineffective. Sense RNAs corresponding to , and HBX mRNA also reduced HBV mRNA levels. Ribozymes targeting HBs and HBX mRNA effectively reduced HBV mRNA levels compared with inactive constructs indicating their effect to be enzymatic rather than antisense. Lentiviral vectors can produce long-term gene expression in hepatocytes and thus permit prolonged expression of antiviral genes targeting the HBV encapsidation signal, surface and X mRNAs as treatments for chronic HBV infection. [source] Lentivirus-mediated bifunctional cell labeling for in vivo melanoma studyPIGMENT CELL & MELANOMA RESEARCH, Issue 3 2009Chi-Ping Day Summary Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase,green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by fluorescence-activated cell sorter. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. [source] Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cellsTHE JOURNAL OF GENE MEDICINE, Issue 3 2009Min Liang Abstract Background Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed. Methods In the present study, we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors, based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction, we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector, developed in our laboratory, that allows targeted transduction to specific cell receptors via antibody recognition. Results Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. Conclusions Overall, the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. Copyright © 2009 John Wiley & Sons, Ltd. [source] Engineering physiologically controlled pacemaker cells with lentiviral HCN4 gene transferTHE JOURNAL OF GENE MEDICINE, Issue 5 2008Gerard J. J. Boink Abstract Background Research on biological pacemakers for the heart has so far mainly focused on short-term gene and cell therapies. To develop a clinically relevant biological pacemaker, long-term function and incorporation of autonomic modulation are crucial. Lentiviral vectors can mediate long-term gene expression, while isoform 4 of the Hyperpolarization-activated Cyclic Nucleotide-gated channel (encoded by HCN4) contributes to pacemaker function and responds maximally to cAMP, the second messenger in autonomic modulation. Material and Methods Action potential (AP) properties and pacemaker current (If) were studied in single neonatal rat ventricular myocytes that overexpressed HCN4 after lentiviral gene transduction. Autonomic responsiveness and cycle length stability were studied using extracellular electrograms of confluent cultured monolayers. Results Perforated patch-clamp experiments demonstrated that HCN4-transduced single cardiac myocytes exhibited a 10-fold higher If than non-transduced single myocytes, along with slow diastolic depolarization, comparable to pacemaker cells of the sinoatrial node, the dominant native pacemaker. HCN4-transduced monolayers exhibited a 47% increase in beating rate, compared to controls. Upon addition of DBcAMP, HCN4-transduced monolayers had beating rates which were 54% faster than baseline and significantly more regular than controls. Conclusions Lentiviral vectors efficiently transduce cardiac myocytes and mediate functional gene expression. Because HCN4-transduced myocytes demonstrate an increase in spontaneous beating rate and responsiveness to autonomic modulation, this approach may be useful to create a biological pacemaker. Copyright © 2008 John Wiley & Sons, Ltd. [source] Lentiviral vectors that carry anti-HIV shRNAs: problems and solutionsTHE JOURNAL OF GENE MEDICINE, Issue 9 2007Olivier ter Brake Abstract Background HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Methods Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. Results RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Conclusions Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production. Copyright © 2007 John Wiley & Sons, Ltd. [source] Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytesTHE JOURNAL OF GENE MEDICINE, Issue 2 2007Clare Selden Abstract Background Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. Methods We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /, growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. Results Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. Conclusions This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity. Copyright © 2007 John Wiley & Sons, Ltd. [source] Lentiviral vectors for treating and modeling human CNS disordersTHE JOURNAL OF GENE MEDICINE, Issue 9 2004Mimoun Azzouz Abstract Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral-mediated overexpression of mutated human ,-synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non-primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. Copyright © 2004 John Wiley & Sons, Ltd. [source] Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectorsTHE JOURNAL OF GENE MEDICINE, Issue 4 2002Seraphin Kuate Abstract Background Lentiviral vectors allow gene transfer into non-dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing. Methods To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon-optimized gag-pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV-G) under the control of an ponasterone-inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized. Results The RT activity and vector titers of cell clones stably transfected with the inducible gag-pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone-inducible VSV-G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone-induced producer clones vector titers of more than 1×105 transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production. Conclusions The packaging cells described should be suitable for most preclinical applications of SIV-based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd. [source] Development of lentiviral vectors for gene therapy for Usher syndrome type 1BACTA OPHTHALMOLOGICA, Issue 2007T HASHIMOTO 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. [source] Reduction of Dicer impairs Schwann cell differentiation and myelinationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2010Jonathan D. Verrier Abstract The process of Schwann cell myelination requires precisely coordinated gene expression. At the onset of myelination, there is an increase in the expression of differentiation-promoting transcription factors that regulate key Schwann cell genes. Further control of myelin gene expression occurs at the posttranscriptional level and, in part, is mediated by RNA binding proteins and micro-RNAs (miRNAs). miRNAs are small, endogenously derived RNA molecules that repress gene expression by specifically binding to their mRNA targets. In the experiments described here, we tested whether miRNAs were essential in controlling myelination by reducing the levels of Dicer, an essential endoribonuclease in miRNA biogenesis. We decreased the expression of Dicer by about 60% within Schwann cells using a lentiviral vector expressing an shRNA against Dicer. The reduced levels of Dicer led to a decrease in the steady-state expression of selected miRNAs and of the transcription factors Oct6 and Egr2/Krox20, both of which are critical for Schwann cells differentiation and myelination. In contrast, the levels of c-jun and Sox2 were up-regulated by the reduction in Dicer and were associated with an increase in Schwann cell proliferation. In dorsal root ganglion cocultures, Schwann cells transduced with Dicer shRNA synthesized less myelin, which was accompanied by significant reductions in the levels of myelin basic protein and protein zero. These findings support a critical role for Dicer and miRNAs in Schwann cell differentiation and myelination. © 2010 Wiley-Liss, Inc. [source] Silencing of choline acetyltransferase expression by lentivirus-mediated RNA interference in cultured cells and in the adult rodent brainJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2009Julie Santamaria Abstract RNA interference (RNAi) is a potent mechanism for local silencing of gene expression and can be used to study loss-of-function phenotypes in mammalian cells. We used RNAi to knockdown specifically the expression of choline acetyltransferase (ChAT), the enzyme of acetylcholine biosynthesis, both in cultured cells and in the adult brain. We first identified a 19-nucleotide sequence in the coding region of rat and mouse ChAT transcripts that constitutes a target for potent silencing of ChAT expression by RNAi. We generated a lentiviral vector that produces both a small hairpin RNA (shRNA) targeting ChAT mRNAs and the enhanced green fluorescent protein (EGFP) reporter protein to facilitate identification of transduced cells. In the cholinergic cell line NG108-15, there was at least 90% less of the ChAT protein, as measured by assaying its enzymatic activity, 3 days postinfection with this vector than in cells infected with a control vector. The vector was used to transduce cholinergic neurons in vivo and reduced ChAT expression strongly and specifically in the cholinergic neurons of the medial septum in adult rats, without affecting the expression of the vesicular acetylcholine transporter. This lentiviral vector is thus a powerful tool for specific inactivation of cholinergic neurotransmission and can therefore be used to study the role of cholinergic nuclei in the brain. This lentiviral-mediated RNAi approach will also allow the development of new animal models of diseases in which cholinergic neurotransmission is specifically altered. © 2008 Wiley-Liss, Inc. [source] Probing platelet factor 4 ,-granule targetingJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2004V. Briquet-Laugier Summary., The storage mechanism of endogenous secretory proteins in megakaryocyte ,-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet ,-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in ,-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify ,-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in ,-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze ,-granule biogenesis and its alterations in the congenital defect gray platelet syndrome. [source] Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead ribozymesJOURNAL OF VIRAL HEPATITIS, Issue 4 2005K. L. Nash Summary., Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Current treatments are limited and may be ineffective. Nucleic acid-mediated targeting of viral mRNA is an attractive and specific approach for viral infection and lentiviral vectors provide a means to express antisense sequences or ribozymes stably in target cells permitting continuous production within that cell and its progeny. To demonstrate long-term gene expression by lentiviral vectors in hepatocytes and to introduce lentiviral vectors expressing anti-HBV genes to assess their effect against HBV, lentiviral vectors expressing a reporter gene were assessed for longevity of gene expression in hepatocytes in vitro. Hammerhead ribozymes and antisense sequences targeting the HBV encapsidation signal (,), X or surface antigen on mRNAs were cloned into lentiviral vectors and used to transduce HBV expressing hepatocytes where the effect on HBV mRNA level was assessed using ribonuclease protection. Gene expression in hepatocytes from integrated vectors continued for over 4 months without selection. Antisense RNA targeting HBs mRNA reduced this transcript, whilst antisense RNA to HBX mRNA was ineffective. Sense RNAs corresponding to , and HBX mRNA also reduced HBV mRNA levels. Ribozymes targeting HBs and HBX mRNA effectively reduced HBV mRNA levels compared with inactive constructs indicating their effect to be enzymatic rather than antisense. Lentiviral vectors can produce long-term gene expression in hepatocytes and thus permit prolonged expression of antiviral genes targeting the HBV encapsidation signal, surface and X mRNAs as treatments for chronic HBV infection. [source] Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2008H.M. Kubisch Abstract A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys. Mol. Reprod. Dev. 75: 1505,1514 © 2008 Wiley-Liss, Inc. [source] Correction of mucopolysaccharidosis type IIIA somatic and central nervous system pathology by lentiviral-mediated gene transferTHE JOURNAL OF GENE MEDICINE, Issue 9 2010Chantelle McIntyre Abstract Background 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. Methods 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. Results 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. Conclusions 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. Copyright © 2010 John Wiley & Sons, Ltd. [source] Single-dose lentiviral gene transfer for lifetime airway gene expressionTHE JOURNAL OF GENE MEDICINE, Issue 10 2009Alice G. Stocker Abstract Background Cystic fibrosis (CF) is caused by a defect in cystic fibrosis transmembrane conductance regulator (CFTR) activity, often resulting in an incurable airway disease. Gene therapy into the conducting airway epithelium is a potential cure for CF; however, most gene vectors do not result in long-lived expression, and require re-dosing. Perversely, intrinsic host immune responses can then block renewed gene transfer. Methods To investigate whether persistent gene expression could be achieved after a single dosing event, thus avoiding the issue of blocking host responses, we used a gene transfer protocol that combined an airway pretreatment using lysophosphatidylcholine with a human immunodeficiency virus type-1 (vesicular stomatitis virus G pseudotype) derived lentiviral vector to test whether an integrating vector could produce gene expression able to last for a substantial part of the lifetime of the laboratory mouse. Results We found that a single dose of LV-LacZ produced immediate as well as lifetime mouse airway expression, confirming our hypothesis that use of an integrating vector extends transgene expression. Importantly, LV-CFTR dosing achieved at least 12 months of CFTR expression, representing partial functional correction of the CFTR defect in CF-null mice. Conclusions These findings validate the potential of this methodology for developing a gene transfer treatment for CF airway disease. Copyright © 2009 John Wiley & Sons, Ltd. [source] Diverse genomic integration of a lentiviral vector developed for the treatment of Wiskott,Aldrich syndromeTHE JOURNAL OF GENE MEDICINE, Issue 8 2009Julie Mantovani Abstract Background The genomic integration of a lentiviral vector developed for the treatment of Wiskott,Aldrich syndrome (WAS) was assessed by localizing the vector insertion sites (IS) in a murine model of gene therapy for the disease. Methods Transduced hematopoietic progenitor cells were transplanted into mice or cultured in vitro. The IS were determined in the genomic DNA from blood, the bone marrow of the animals and from cultured cells. Results Sequencing vector,genomic DNA junctions yielded more than 150 IS of which 50,70% were located in transcription units. To obtain additional sequences from the population of cultured cells, we used a vector-tag concatenation technique providing 190 additional IS. Altogether, the profiles confirmed the bias for integration in transcription units. The vector did not congregate as hotspots and did not appear to target specific categories of genes. The diversity of the IS reflected the initial marking of a polyclonal population of cells. However, relatively few vector IS were found in vivo because only 30,40 unique IS were identified in each mouse using this approach. Although four to ten IS were shared by the blood and bone marrow, no common IS was found between mice or between any mouse and the cultured cells. Conclusions Taken as a whole, the pattern of genomic insertion of the WAS lentiviral vector was diverse and similar to that previously described for other HIV-1-derived lentiviral vectors. Testing cells destined for transplantation is unlikely to predict specific IS to be selected in vivo. Copyright © 2009 John Wiley & Sons, Ltd. [source] Partial phenotypic correction and immune tolerance induction to enzyme replacement therapy after hematopoietic stem cell gene transfer of ,-glucosidase in Pompe diseaseTHE JOURNAL OF GENE MEDICINE, Issue 4 2009Gaėlle Douillard-Guilloux Abstract Background Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid ,-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. Even if enzyme replacement therapy (ERT) has already proven some efficacy, its results remain heterogeneous in skeletal muscle, especially in cross reactive immunological material (CRIM)-negative patients. We investigated for the first time the use of hematopoietic stem cell (HSC) gene therapy in a murine model of GSDII. Methods Deficient HSC were transduced with a lentiviral vector expressing human GAA or enhanced green fluorescent protein (GFP) under the control of the retroviral MND promoter and transplanted into lethally irradiated GSDII mice. Animals were then subjected to an ERT protocol for 5 weeks and monitored for metabolic correction and GAA-induced immune reaction. Results GAA was expressed as a correctly processed protein, allowing a complete enzymatic correction in transduced deficient cells without toxicity. Seventeen weeks after transplantation, a partial restoration of the GAA enzymatic activity was observed in bone marrow and peripheral blood cells of GSDII mice, allowing a significant glycogen clearance in skeletal muscle. ERT induced a robust antibody response in GFP-transplanted mice, whereas no immune reaction could be detected in GAA-transplanted mice. Conclusions Lentiviral vector-mediated HSC gene therapy leads to a partial metabolic correction and induces a tolerance to ERT in GSDII mice. This strategy could enhance the efficacy of ERT in CRIM-negative Pompe patients. Copyright © 2009 John Wiley & Sons, Ltd. [source] Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cellsTHE JOURNAL OF GENE MEDICINE, Issue 3 2009Min Liang Abstract Background Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed. Methods In the present study, we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors, based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction, we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector, developed in our laboratory, that allows targeted transduction to specific cell receptors via antibody recognition. Results Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. Conclusions Overall, the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. Copyright © 2009 John Wiley & Sons, Ltd. [source] Synergistic neuroprotective effect via simian lentiviral vector-mediated simultaneous gene transfer of human pigment epithelium-derived factor and human fibroblast growth factor-2 in rodent models of retinitis pigmentosaTHE JOURNAL OF GENE MEDICINE, Issue 12 2008Masanori Miyazaki Abstract Background We previously demonstrated that a new lentiviral vector derived from nonpathogenic simian immunodeficiency virus (SIVagm) was efficient and safe for long-lasting retinal gene transfer, and that it provided the significant therapeutic effect of expressing human pigment epithelium-derived factor (hPEDF) in Royal College of Surgeons (RCS) rats. In the present study, to obtain a more pronounced outcome, we assessed the potential synergistic effect of the simultaneous gene transfer of hPEDF and human fibroblast growth factor-2 (hFGF-2) by improved third-generation SIV on RCS rats and retinal degeneration slow (rds) mice, because the former targets the primary neurons, including photoreceptor cells (PCs), whereas the latter is effective for targeting secondary neural cells, including Muller cells. Methods Vector solution (SIV-hPEDF, SIV-hFGF-2, a 1 : 1 mixture of SIV-hPEDF and SIV-hFGF-2, or SIV-enhanced green fluorescent protein) was injected into the peripheral subretinal space of 3-week-old RCS rats or rds mice. Histopathological and electroretinographic assessments were made at several points after gene transfer. Results Administration of SIV-hPEDF or SIV-hFGF-2 significantly delayed the histological PC degeneration and electrical deficit in RCS rats, and these delays were synergistically and significantly pronounced by SIV-hPEDF + SIV-hFGF-2 (1 : 1 mixture). In rds mice, functional therapeutic effects were observed even by SIV-PEDF, or SIV-FGF-2 alone and, moreover, both SIV-PEDF and SIV-FGF-2 showed higher therapeutic effects. Conclusions These synergistic rescues of retinitis pigmentosa (RP) model animals are the ,proof concept' that the ,dual' expression of hPEDF and hFGF-2 dramatically improved therapeutic efficacy by keeping lower titers. This strategy may contribute to safer and more effective gene therapy for RP. Copyright © 2008 John Wiley & Sons, Ltd. [source] Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit modelTHE JOURNAL OF GENE MEDICINE, Issue 9 2008Rafael Moreno Abstract Background Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors. Methods Rabbit fetuses received 8.5 × 106 HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post- in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein. Results Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP. Conclusions At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptorsTHE JOURNAL OF GENE MEDICINE, Issue 12 2007Marjorie Nicoud Abstract Background We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. Methods Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. Results All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. Conclusions It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target. Copyright © 2007 John Wiley & Sons, Ltd. [source] Lentiviral vectors that carry anti-HIV shRNAs: problems and solutionsTHE JOURNAL OF GENE MEDICINE, Issue 9 2007Olivier ter Brake Abstract Background HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Methods Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. Results RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Conclusions Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production. Copyright © 2007 John Wiley & Sons, Ltd. [source] Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skippingTHE JOURNAL OF GENE MEDICINE, Issue 5 2007Maria B. Asparuhova Abstract The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS. Copyright © 2007 John Wiley & Sons, Ltd. [source] Brain engraftment and therapeutic potential of stem/progenitor cells derived from mouse skinTHE JOURNAL OF GENE MEDICINE, Issue 4 2006Patrizia Tunici Abstract Skin stem/progenitor cells (SKPs) derive from the dermis and in culture can generate mesodermal and neural progenies. To investigate their potential for the treatment of brain diseases, we first injected SKPs into the brain of syngeneic mice. Brain histology indicated that most SKPs remained undifferentiated and clustered at the injection site, while, in vitro, 17% of SKPs expressed neural markers, as assessed by flow cytometry. After labeling with magnetodendrimers, murine and human SKPs were detected by magnetic resonance imaging even 5 months after brain injection. To evaluate their therapeutic potential on malignant gliomas, IL-4 SKPs (i.e. SKPs transduced by a lentiviral vector carrying the cDNA of the anti-glioma cytokine interleukin-4) were injected into GL261 experimental gliomas. IL-4-SKPs prolonged significantly the survival of tumor-bearing mice: furthermore, GL261 gliomas attracted SKPs originally injected into the contralateral hemisphere. Thus, prolonged survival, capacity for transgene expression, and lack of uncontrolled proliferation suggest that SKPs warrant further consideration as therapeutic tools for brain tumors and, possibly, other neurological disorders. Copyright © 2006 John Wiley & Sons, Ltd. [source] Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID miceTHE JOURNAL OF GENE MEDICINE, Issue 10 2004Jiro Kikuchi Abstract Background An Erratum has been published for this article in Journal of Gene Medicine 7(6), 2005, 836. Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. Results We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 ± 0.1%) at MOI of 5 × 103 vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 ± 20.1 ng) from 106 CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5,10 × 105) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 ± 0.9 ng/mL, maximum 3.6 ± 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright © 2004 John Wiley & Sons, Ltd. [source] Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epitheliumTHE JOURNAL OF GENE MEDICINE, Issue 12 2003Nils Loewen Abstract Background In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2,5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol -encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. Methods We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (,-galactosidase) expression by an observer blinded to vector assignments. Results Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed ,-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. Conclusions These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd. [source] | |||||||||||||