Loss Of Transgene Expression Research Articles

Parallel Detection of Transduced T Lymphocytes After Immunogene Therapy of Renal Cell Cancer by Flow Cytometry and Real-Time Polymerase Chain Reaction: Implications for Loss of Transgene Expression

Parallel Detection of Transduced T Lymphocytes After Immunogene Therapy of Renal Cell Cancer by Flow Cytometry and Real-Time Polymerase Chain Reaction: Implications for Loss of Transgene Expression

768. Two Distinct Mechanisms, Silencing of RNA Expression and Loss of Vector DNA, Are Responsible for the Loss of Transgene Expression Following Delivery of Foreign Genes to Skeletal Muscle

Plasmid based delivery of foreign transgenes often results in transient expression in vivo. To investigate the cause of this transient expression we used quantitative PCR to measure the copy number of transgene RNA and vector DNA in the skeletal muscle of C57BL6 mice after electroporation of various plasmids. Gene transfer of a plasmid encoding the mouse interferon beta (mIFN-|[beta]|) gene driven by the CMV promoter (pCMV-mIFN) resulted in mIFN RNA levels between 1 and 5 |[times]| 106 copies per microgram (|[mu]|g) of total RNA that were stable for the first 10 days then dropped by only 10-fold by day 44. Plasmid vector DNA levels were also stable for the first 10 days then dropped 7.5-fold between day 10 and day 44. The ratio of mIFN RNA copies per vector DNA copy reached a peak of 1.8 at day 10 and declined slightly to 0.9 at day 44. These results show that following delivery of a non-foreign transgene, a slow decline in RNA levels occurred that was due to a gradual loss of plasmid DNA. Next, we compared plasmids encoding either the mIFN-|[beta]| or human interferon beta (hIFN-|[beta]|) genes driven by identical CMV promoters (pCMV-hIFN and pCMV-mIFN). At 50 days after electroporation of mouse skeletal muscle, the mean copy number of transgene RNA in the muscles that received the mouse or human IFN-|[beta]| plasmids was 1.1 |[times]| 105 and 7.4 |[times]| 103 copies per |[mu]|g of total RNA, respectively. In contrast, the mean plasmid DNA level was slightly higher in the muscles that received pCMV-hIFN. The mean IFN-|[beta]| RNA to vector DNA ratio was 1.3 in the muscles that received pCMV-mIFN, but only 0.04 (31-fold lower, p<0.001) in the muscles that received pCMV-hIFN. This demonstrates that the lower level of transgene RNA expression achieved with the foreign human IFN-|[beta]| gene as compared to the native mouse IFN-|[beta]| gene was due to reduced gene expression, not to reduced persistence of the vector DNA. When a mixture of pCMV-mIFN and a plasmid encoding human secreted alkaline phosphotase driven by the CMV promoter (pCMV-hSEAP) in a 14:1 ratio was delivered to mouse muscle, mIFN RNA levels fell 350-fold between day 1 and day 10 (p<0.01). The level of pCMV-mIFN plasmid DNA in the same muscles fell 100-fold by day 10 (p<0.01) and the ratio of mIFN RNA copies per copy of pCMV-mIFN DNA dropped slightly from 2.8 at day 1 to 1.1 at day 10, suggesting that there was only a small decrease in transcription. This data demonstrates that following co-delivery of pCMV-hSEAP and pCMV-mIFN, rapid clearance of the CMV-mIFN plasmid DNA from the muscle was primarily responsible for the large drop in mIFN RNA expression.

Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms. Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated. The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%). This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes.

Suppression of inflammation by dexamethasone prolongs adenoviral vector-mediated transgene expression in murine nasal mucosa

Conclusion. The results of this study demonstrate that suppression of inflammation by dexamethasone attenuates the host immune response against adenoviral-mediated gene transfection and thereby prolongs transgene expression in murine nasal mucosa. Objectives. Gene transfer using a recombinant adenovirus is a good tool for research and clinical applications, but the immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. In this study we investigated the effects of dexamethasone-induced immunosuppression on adenovirus gene transfer in the nasal mucosa of the mouse. Material and methods. We administered the recombinant adenovirus Ax1CAlacZ, which encodes Escherichia coli β-galactosidase (lacZ gene), to the nasal mucosa of mice treated with or without i.p. dexamethasone and evaluated the expression of the lacZ gene on Days 2, 4, 7, 14 and 28. The nasal mucosa was dissected out, and the mRNA level was measured using a LightCycler. The expression of the exogenous β-galactosidase was detected by means of histochemistry. Results. Dexamethasone treatment significantly increased the mRNA level compared with that in the controls at Days 4, 7 and 14. Histochemistry showed that the expression of β-galactosidase protein persisted in the dexamethasone-treated mice at Days 7 and 14 but had diminished almost to nothing in the control group.

Toxicity and adaptive immune response to intracellular transgenes delivered by helper-dependent vs. first generation adenoviral vectors

The host immune response to intracellular transgenes delivered by helper-dependent (HDV) vs. first generation (FGV) adenoviral vectors has been relatively unstudied. Previous studies showed short-term correction of bovine and murine argininosuccinate synthetase (ASS) deficiency after first generation adenoviral-mediated liver gene therapy. To determine whether the host adaptive immune response against the intracellular transgene human ASS (hASS) contributed to loss of gene expression in this setting, the same vector (FGV-CAG-hASS) was injected into Rag −/− (immunodeficient) mice. As in wild-type C57BL/6 (B6) mice, Rag −/− mice also showed significant loss of hASS expression and vector by week 4 post-injection, with concomitant elevation of liver enzymes and disruption of liver architecture. Therefore, direct toxicity due to vector rather than adaptive immune response against hASS primarily accounted for loss of expression with FGVs. In contrast to hASS, β-galactosidase is strongly immunogenic and activates the host adaptive immune response. Loss of transgene expression was observed in B6 mice with either a FGV or a HDV expressing β-galactosidase. However, the drop in gene expression observed with the HDV was primarily due to the adaptive immune response, since both β-galactosidase expression and vector genome were sustained in immunodeficient mice treated with HDV. As expected, with weakly immunogenic hASS, vector genome and hASS expression were sustained with a HDV in spite of ubiquitous expression of the transgene. Therefore, viral gene expression is a primary determinant of intermediate and chronic toxicities at day 3 and week 4 post-injection. However, even in the absence of viral gene expression, strongly immunogenic intracellular transgenes can stimulate clearance of transduced hepatocytes.

Optimizing regulatable gene expression using adenoviral vectors

Inducible gene expression systems have typically encountered limitations, such as pleitropic effects of the inducer, basal leakiness, toxicity of inducing agents and low levels of expression. However, recently non-toxic, tightly regulated control of transgene expression has been reported for several systems, the most frequently cited being the tetracycline gene control system. We have found that the individual components of the Tet system [the Tet transactivators and tetracycline responsive element (TRE)] function optimally to control gene expression when they are incorporated into separate adenoviral vectors. Furthermore, incorporation of the Woodchuck hepatitis virus post-transcriptional enhancer (WPRE) allows a dual vector Tet-regulatable Ad system to be used at very low titres (2 x 10(4)) that elicit a minimal inflammatory response, with no loss of transgene expression or ability to regulate transgene expression. This and similar regulatable systems will benefit studies investigating neuronal gene function and those seeking to develop effective neuronal gene therapy strategies.

Increased utility in the CNS of a powerful neuron‐specific tetracycline‐regulatable adenoviral system developed using a post‐transcriptional enhancer

In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system. The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP. The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression. We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies.

Cooperativity between Far Upstream Enhancer and Proximal Promoter Elements of the Human α2(I) Collagen (COL1A2) Gene Instructs Tissue Specificity in Transgenic Mice

Interaction between the proximal (-378) promoter and the far upstream (-20 kb) enhancer is essential for tissue-specific expression of the human alpha2(I) collagen gene (COL1A2) in transgenic mice. Previous in vitro studies have shown that three Sp1 binding sites (around -300) are part of a cytokine-responsive element and that two TC-rich boxes (around -160 and -125) and a CBF/NFY consensus sequence (around -80) confer optimal promoter activity by interacting among themselves and with the upstream Sp1 sites. Here we report that mutations of the Sp1 binding sites, TC-rich boxes or CBF/NFY consensus sequence lead to reduced transgene activity, thus underscoring the functional interdependence of the proximal promoter elements. Loss of the Sp1 binding sites was associated with loss of transgene expression in osteoblasts, whereas elimination of the CBF/NFY binding site (alone or in combination with the TC-rich boxes) was correlated with a lack of activity in the ventral fascia and head dermis and musculature. Additionally, transgene expression in skin fascia fibroblasts depended on the integrity of the Sp1 binding sites and TC-rich boxes, and on their physical configuration. Evidence is also presented suggesting cooperativity between cis-acting elements of the far upstream enhancer and proximal promoter in assembling tissue-specific protein complexes. This study thus reiterates the complex and highly combinatorial nature of the regulatory network governing COL1A2 transcription in vivo.

461. In Vivo Immunogenicity of Adenoviral Vectors with Improved Dendritic Cell Tropism

Gene therapy using adenoviral vectors possesses advantages of wide tissue-tropism, large capacity for therapeutic genes such as Dystrophin, and relatively easy and efficient vector production to high titer and purity. However, first generation adenoviral vectors still carry and express a variety of viral proteins, which could lead to host cellular immune responses that cause the clearance of vector-transduced cells, resulting in loss of transgene expression, and loss of therapeutic effects.

458. Efficient Transduction of Human and Murine Cells and Circumvention of Preexisting Human Adenoviral Immunity by Porcine Adenoviral Vectors

Adenovirus is a well-known vector for delivering therapeutic genes to tissues. However, it induces an immune response against viral antigens, which results in loss of transgene expression. This has limited the utility of adenovirus in applications in which long-term transgene expression is desired. Fibronectin (FN) is an extracellular matrix glycoprotein that interacts with a variety of cells through integrin and non-integrin receptors. These interactions play a role in cell adhesion and migration during an inflammatory response. In previous studies, we showed that a 33-KD carboxyl-terminal heparin-binding fragment of fibronection (FN-C/H-II) inhibited leukocyte recruitment in mouse arthritis. The present study was designed to determine whether FN-C/H-II could also blunt the inflammatory response to adenovirus. An adenoviral vector (1 × 109 particles) encoding murine IL-10 (AdmIL10) was co-injected with an equal titer of either an adenovirus encoding FN-C/H-II (AdFN) or a control backbone adenovirus (AdBglII) into knee joints of normal C57BL/6 mice. Expression of mIL-10 was significantly higher in the knees co-injected with AdFN over a 2 week observation period. Histological evaluation revealed a marked reduction in infiltration of both neutrophils and lymphocytes in the synovium of mice co-injected with the AdFN vector. These results suggest that adenoviral expression of FN-C/H-II peptide can reduce adenovirus-associated inflammation and enhance the efficacy of adenovirus as a gene transfer vector.

261. Immune Evasion in Gene Therapy

A problem associated with the gene-therapy concept is the T cell-mediated immune response, which is elicited by synthesis and presentation of transgene-derived and vector-derived peptides and which leads to rapid clearance of the transduced cells and loss of transgene expression. One way to avoid the immune response is by blocking the ubiquitin/proteasome pathway. Interference in this pathway is widely applied by viruses, for instance by Epstein-Barr virus (EBV) in its nuclear antigen 1 (EBNA-1). EBNA-1 is required for the maintenance of the viral episomes in the infected cells. Although EBNA-1 specific CTL have been described, they cannot recognize EBV-infected cells. The failure to recognize endogenously expressed EBNA-1 has previously been attributed to a glycine-alanine rich (GAr) stretch in the EBNA-1 sequence that protects the protein from proteasomal degradation. We have shown previously that inclusion of the full length EBNA-1 derived GAr domain could be exploited to abrogate CTL-induced killing of cells expressing the transgene (ref 1). Firstly we demonstrated that inclusion of the EBNA-1 derived GAr domain does not interfere with enzyme function. We tested three different enzymes that all retained their activity. In addition, a recombinant adenovirus expressing GAr-containing β-galactosidase was able to deliver functional enzyme in vivo. However, expression of the GAr-β-gal as well as the control β-gal resulted in the induction of a strong β-galactosidase-specific CTL response. This can be attributed to cross-priming, as vaccination of BALB/c mice (H-2d) with C57Bl/6 (H-2b) fibroblasts expressing GAr-β-galactosidase resulted in the induction of β-gal-specific CTLs. Nonetheless, LacZ-specific CTL did not recognize target cells expressing GAr-LacZ in vitro. These results showed that the GAr can be exploited to create 'stealth' genes by hiding specific foreign antigens from CTL-mediated immune-attack. Currently, we are testing the hypothesis that fusion with the GAr will lead to a prolonged expression of transgenes in vivo. Preliminary evidence indicates that the modification leads to prolonged expression of β-gal in vivo after adenovirus-mediated intra-muscular gene transfer. In addition, we generated stable B16 mouse melanoma cell lines expressing either thymidine kinase (TK) or GAr-TK. The tumor take and survival of these cells in C57Bl/6 mice are being studied either in the presence or in the absence of antigen-specific CTL.

992. Lentiviral and Retroviral Vectors for Transcriptionally Targeted Expression of Human Beta-Globin

Top of pageAbstract Transgene expression driven by the human cytomegalovirus (hCMV) promoter from adenovectors following intravitreal administration into the eye is characterized by an initial high-level burst of expression that drops quickly and is followed by eventual loss of transgene expression. The decrease in expression could arise from elimination of vector genomes or by loss of promoter activity. In the current studies we investigate the cause for the diminution of activity, evaluate quantitatively vector genomes and expression levels, describe a means in which to follow transgene expression in the eye in the same animal over time, and discover means in which silenced adenovector genomes can be reactivated to express transgenes. In these studies, replication-deficient adenovectors deleted of E1, E3 and E4 that express nothing, GFP, or luciferase from the hCMV promoter were delivered to the eyes of mice by direct intravitreal injection. The amount of vector genome was quantitated by qPCR, the amount of luciferase transgene expression quantitated by luminesence, and the level of GFP transgene expression monitored by fluorescence. From these analyses the loss of transgene expression over time from adenovectors can be attributed to loss of promoter activity. Adenovector genomes from which there was no detectable transgene expression still retained the ability to express their protein product. This was shown by the ability to completely restore expression from a silenced hCMV promoter by multiple means. The administration of non-expressing adenovector particles to the vitreous activated expression from the silenced adenovector genome to levels equal to or greater than that originally observed on day 1. These experiments also showed that quiescent adenovector genomes retain the ability to be reactivated for transgene expression even after being silenced for more than a year. A screen of potential molecules capable of activating the hCMV promoter found retinoic acid (RA) to be a potent activator of expression and systemic administration of RA was found to activate expression from silenced adenovector genomes to day 1 levels. Furthermore, we found that RA could activate expression from silenced adenovector genomes even after 2 months following initial administration into the eye, which further demonstrates that adenovector genomes remain expression competent. These data suggest that adenovectors may provide a means to deliver proteins to the eye requiring repeated expression for the treatment of ocular disease and expands the utility of adenovectors in ocular gene delivery.

10. Cell Cycle-Dependent Transcription from Unintegrated Lentiviral Vector DNA Internal Promoters: Analyses with Class I Integrase Mutants

Top of pageAbstract Background: The circumstances governing the extent to which unintegrated lentiviral DNA can persist and serve as a template for transcription and protein expression are unclear. Methods: We constructed and validated class I mutants for FIV IN (D66V and D66V+D118A) and compared them to analogous class I HIV-1 IN mutants. We documented class I properties, and performed further characterizations with lentiviral vectors and with full length viruses bearing these mutations. Results: The D66V mutation in FIV IN, alone or in combination with D118A, did not affect Gag/Pol precursor expression or proteolytic processing, particle formation, or RT production. The mutations did block integration and transduction of dividing fibroblasts (5 log reduction, RT activity-normalized comparisons). In rats injected subretinally with RT-normalized stocks, retinal pigment epithelium showed extensive transduction, as did some cells in the outer nuclear layer, persisting to at least 3 months for wild-type (WT), compared with only rare positive outer nuclear layer cells for D66V vectors. Results were different in primary post-mitotic rat neurons (retinal ganglion cells), where class I-mutant and WT vectors produced fully equivalent transduction. Transgene expression in growth-arrested fibroblasts was not inhibited at all by class I mutations (97% vs. 99% of cells transduced with WT vector at MOI = 5, comparing RT activity-normalized stocks), and expression level per cell was equal to WT. Northern blotting for reporter gene mRNA fully confirmed the protein expression results and ruled out pseudotransduction, as did use of mock vectors. mRNA levels assayed in Northern blots remained high at 8 days in growth-arrested cells infected with class I mutants. Similar cell cycle-dependent expression was seen with class I mutant HIV vectors and full length FIV. Southern blotting revealed persistence of vector DNA only in arrested cells transduced with the class I mutant vector. Quantitative real-time PCR corroborated this, showing accumulation of 2-LTR circles with the class I mutant vectors. Release from aphidicolin resulted in stable transgene expression in cells transduced with WT FIV and HIV vectors. Cells were monitored for 37 days, and expression was durable to repeated passage. In contrast, release of cells transduced with equal RT units of class I IN mutant FIV or HIV vectors resulted in steady decline of expression, from 97% to 0% of cells by day 10. Loss of transgene expression correlated to a loss of vector DNA evaluated by Southern blotting and a loss of mRNA evaluated by Northern blotting. Conclusions: In non-cycling cells, unintegrated FIV and HIV-1 vector DNAs reside in a compartment that is fully accessible to high-level transcription from internal promoters. Reporter gene expression is equivalent to that of integrated DNA if cells are growth-arrested. mRNA and protein expression levels correlate with abundant, persistent unintegrated DNA. Application of these findings to basic research will be discussed.

Comparative analysis of inherited patterns of the transgene in transgenic mud loach Misgurnus mizolepis lines carrying the CAT reporter gene

: Comparative analyses of the inheritance patterns of transgene transmission and expression for up to F5 progenies were carried out in different lines of transgenic mud loach Misgurnus mizolepis containing the chloramphenicol acetyltransferase (CAT) reporter gene. Based on Southern blot hybridization, the analyses revealed different integration sites of transgenes in host chromosomes as well as different transmission modes among transgenic lines, including stable inheritance of integrated transgenic constructs, co-transmission of integrated and extrachromosomally persistant copies, and transgene rearrangement through successive generations. CAT-enzyme-linked immunosorbent assay expression analysis found three different major patterns of inherited expression of the transgene: (i) stable persistence of consistent level of expression; (ii) sudden loss of transgene expression following passage through the germline; and (iii) different levels of reduction in the expression in successive generations.

Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo

The loss of transgene expression has been a major obstacle to the development of nonviral vectors for the treatment of human diseases. We previously demonstrated that bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo. To confirm these studies and develop a means to produce a robust DNA vector that is not silenced in vivo, we developed a phage φC31 integrase-mediated intramolecular recombination technology to prepare minicircle vector DNA devoid of the bacterial backbone and then compared the transgene expression profile of the minicircle with different molecular forms of plasmid DNAs in mice. We demonstrate that minicircular DNAs devoid of bacterial sequences expressed 45- and 560-fold more serum human factor IX and α1-antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver. Our data suggest that minicircles are capable of expressing high and persistent levels of therapeutic products in vivo and have a great potential to serve as episomal vectors for the treatment of a wide variety of diseases.

Alteration of Gene Expression by Chromosome Loss in the Postnatal Mouse Brain

Frequent chromosomal aneuploidy has recently been discovered in normal neurons of the developing and mature murine CNS. Toward a more detailed understanding of aneuploidy and its effects on normal CNS cells, we examined the genomes of cells in the postnatal subventricular zone (SVZ), an area that harbors a large number of neural stem and progenitor cells (NPCs), which give rise to neurons and glia. Here we show that NPCs, neurons, and glia from the SVZ are frequently aneuploid. Karyotyping revealed that approximately 33% of mitotic SVZ cells lost or gained chromosomes in vivo, whereas interphase fluorescence in situ hybridization demonstrated aneuploidy in postnatal-born cells in the olfactory bulb (OB) in vivo, along with neurons, glia, and NPCs in vitro. One possible consequence of aneuploidy is altered gene expression through loss of heterozygosity (LOH). This was examined in a model of LOH: loss of transgene expression in mice hemizygous for a ubiquitously expressed enhanced green fluorescent protein (eGFP) transgene on chromosome 15. Concurrent examination of eGFP expression, transgene abundance, and chromosome 15 copy number demonstrated that a preponderance of living SVZ and OB cells not expressing eGFP lost one copy of chromosome 15; the eGFP transgene was lost in these cells as well. Although gene expression profiling revealed changes in expression levels of several genes relative to GFP-expressing controls, cells with LOH at chromosome 15 were morphologically normal and proliferated or underwent apoptosis at rates similar to those of euploid cells in vitro. These findings support the view that NPCs and postnatal-born neurons and glia can be aneuploid in vivo and functional gene expression can be permanently altered in living neural cells by chromosomal aneuploidy.

1148. Natural Turnover of Fibroblasts in Dermal Constructs Has a Role in the Loss of Transgene Expression by Genetically-Modified Cells In Vivo

1148. Natural Turnover of Fibroblasts in Dermal Constructs Has a Role in the Loss of Transgene Expression by Genetically-Modified Cells In Vivo

1279. Loss of Transgene Expression by Genetically Modified Human Fibroblasts In Vivo Is Not a Function of the Immunodeficient State of the Murine Host: Nude vs. NOD-SCID

1279. Loss of Transgene Expression by Genetically Modified Human Fibroblasts In Vivo Is Not a Function of the Immunodeficient State of the Murine Host: Nude vs. NOD-SCID

Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors.

Oncoretroviral vectors have been successfully used in gene therapy trials, yet low transduction rates and loss of transgene expression are still major obstacles for their application. To overcome these problems we modified the widely used Moloney murine leukemia virus-derived retroviral vector pMX by replacing the 3'LTR with the spleen focus-forming virus LTR and inserting the woodchuck hepatitis B virus post-translational regulatory element. To compare requirements crucial for efficient transgene expression, we generated the hybrid retroviral vectors pMOWS and pOWS that harbor the complete murine embryonic stem cell virus (MESV)-leader sequence or a shortened MESV-leader not comprising primer binding site (PBS) and splice donor (SD). Applying these retroviral vectors significantly augmented transgene expression in hematopoietic cell lines and progenitor cells. For transduction of murine embryonic stem (ES) cells the retroviral vector pMOWS that harbors the MESV-PBS and -SD was superior resulting in 65% green fluorescent protein (GFP) expressing ES cells. Surprisingly, in murine and human primitive hematopoietic progenitor cells (HPC), the highest efficiency of up to 66% GFP expressing cells was achieved with pOWS, a retroviral vector that retains the negative regulatory element coinciding with the MoMuLV-PBS. In summary our hybrid retroviral vectors facilitate significantly improved transgene expression in multipotent cells and thus possess great potential for reconstituting genes in primary cells of disease models, as well as for gene therapy.

Multipotent Stem Cells from the Brain and Retina of Green Mice

THE RECENT ISOLATION AND AMPLIFICATION of multipotent stem cells in the laboratory (1,2) has enlivened the fields of mammalian development and transplantation. Yet the very plasticity that makes these cells so interesting biologically makes them difficult to track as they integrate into host tissues. Here we describe the isolation of stem cells from the brain and retina of mice expressing the enhanced green fluorescent protein (eGFP) transgene, and the transplantation of these cells to the brain and retina of nontransgenic recipients. The most successful method for labeling stem cells has been transfection with a heritable reporter gene such as beta-galactosidase (b-GAL) (3) or GFP (4). These techniques are performed following isolation and prior to grafting. They are slow, labor-intensive, and require transfection of each cell type isolated, as well as repeated clonal selection for high level expression. Loss of transgene expression following differentiation or transplantation is an additional problem with these techniques. Furthermore, each transfection alters the genome unpredictably, risking unintentional changes in stem cell phenotype. While direct isolation of stem cells from b-Gal1 mice has been described, this label has the disadvantage of requiring an exogenous substrate for visualization. By harvesting from donors transgenic for GFP, it should be possible to obtain stem cell lines exhibiting uniformly high levels of constitutive reporter gene expression. Transgenic animals expressing GFP throughout their body have been generated in a number of species, including mice (5–7). We harvested tissue from newborn GFP transgenic mice and isolated stem cells from the brain (BSCs) and neuroretina (RSCs) using established methods (20 ng/mL EGF and bFGF). In both cases, GFP1 neurospheres appeared within the first 3 days and have now been maintained in culture for more than 1 year. To assess the capacity for self-renewal, spheres were broken up and plated as single cells. Individual cells formed new spheres over a period of 2–5 days, with number and size proportional to time in culture. Spheres labeled positively for nestin, an intermediate filament expressed by neuronal and glial precursors, and for Ki-67, which recognizes actively mitotic cells (Fig. 1a–1c). Spheres cultured in the presence of 10% fetal bovine serum generated cells that maintained their green fluorescence and co-labeled with neural markers. BSCs differentiated into cells labeled with markers of each neural lineage; that is, neurons (MAP-2), astrocytes (GFAP) and oligodendrocytes (GalC) (Fig. 1d–1f). RSCs did not differentiate into oligodendrocytes, a cell type not normally generated in the retina, but did differentiate into cells expressing the photoreceptor-specific marker rhodopsin. Green mouse neurospheres were injected into sites within the brain and eye of mature mice. Transplanted BSCs survived, maintained GFP expression (as evidenced by co-labeling after pulsing with BrdU in vitro), migrated widely and showed morphological evidence of integration with the host central nervous system (Fig. 1g). RSCs grafted to the subretinal space of the mechanically injured mouse retina showed morpho-