Hepatitis Virus Post-transcriptional Regulatory Element Research Articles

Development of Chimeric Gene Regulators for Cancer-specific Gene Therapy with both Transcriptional and Translational Targeting

Cancer gene therapy has been of great challenge in achieving maximal high levels of specificity and more rational efficiency in target cancer cell. We herein developed a novel approach for cancer-specific gene therapy using both transcriptional and translational targeting regulation. We integrated the tumor-specific gene promoter of hTERT, the 5'UTR of bFGF-2, the enhancer of woodchuck hepatitis virus post-transcriptional regulatory element (WRE), and/or the 3'UTR of the human EGFR into two major chimeric gene regulators. We found that chimeric gene regulator I (hTERT_5'UTR...WRE_BGHpolyA) enhanced the specificity of expression in hepatocellular carcinoma (HCC) cells up to 300% in total due to increases at both the transcriptional and translational levels but only 120-200% enhancement at the transcriptional level and 120-180% enhancement at the translational level. In addition, chimeric gene regulator II (hTERT_5'UTR...WRE_3'UTR_BGHpolyA) improved the specificity to 550% and also highly strengthened the stability of the mRNA. In vitro cytotoxicity assays demonstrated that HCC cell growth was inhibited by HSV-1 TK expression under the control of both chimeric regulators, with a relative cell viability of approximately 80% for 2 days and approximately 85% for 4 days after transfection, respectively. These observations represent a new approach for highly tumor-specific gene expression and also provide insights into application to cancer gene therapy.

Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.

High-efficiency Transient Transduction of Human Embryonic Stem Cell–derived Neurons With Baculoviral Vectors

Transient genetic manipulation of human neurons without chromosomal integration of the transgene would be valuable but has been challenging due to the quiescent nature of these postmitotic cells. In this study, we developed a set of baculoviral vectors for transient transduction in nondividing neurons derived from human embryonic stem cells (hESCs). Using a baculoviral vector equipped with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), we observed a quick onset of transgene expression as early as day 1 after baculoviral transduction and a high efficiency of up to 80%. Strong transgene expression in the cultured human neurons was observed for more than 1 month and the signal was easily detectable even after 3 months. Using two baculoviral vectors carrying different transgenes, we found that co-transduction at a single neuron level was possible. After transplantation into the brain of nude mice, the baculovirus-transduced human neurons were integrated into the mouse brain and maintained transgene expression for at least 4 weeks, portending the usefulness of this technique in assisting neural transplantation. Therefore, by mediating efficient transient gene expression, baculoviral vectors can provide useful tools for both basic gene function studies in human neurons and therapeutic applications of these cells.

Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters

Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott-Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.

Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3' end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

304 PRODUCTION OF TRANSGENIC CHICKENS EXPRESSING HUMAN ERYTHROPOIETIN IN A TETRACYCLINE-INDUCIBLE MANNER

The use of livestock animals as bioreactors to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to agricultural improvement and medical advancement. We report here the production of transgenic chickens that produce human erythropoietin (hEPO) using replication-defective Moloney murine leukemia virus-based vectors. Because it is well known that constitutive overexpression of some cytokine genes in the transgenic animals may cause serious physiological disturbances, the vectors were designed to express in the presence of tetracycline. In addition, we introduced woodchuck hepatitis virus posttranscriptional regulatory element sequence at 3′ end of hEPO gene to boost the gene expression under the inducible condition. Approximately 5 μL of vector virus solution concentrated as much as 109 cfu mL–1 was injected beneath the blastoderm of non-incubated chicken embryo (stage X). Out of 596 injected eggs, 36 chicks hatched after 21 days of incubation and 23 hatched chicks were found to express vector-encoded hG-GSF gene when fed with doxycycline. Quantitative analysis of the blood samples taken from some Go transgenic chickens resulted in more than 300 IU mL–1 of hEPO in the blood. These transgenic chickens have not exhibited any physiological abnormalities; therefore, it is possible that this controllable gene expression system may be useful in minimizing detrimental side effects when used to produce other transgenic animals. The biological activity of the recombinant hEPO was comparable to its commercially derived Escherichia coli counterpart. The significance of this work stems from the fact that it is the first successful report on the production of transgenic chickens expressing the hEPO gene. This approach can be employed to create a useful transgenic model system for further studies on the chicken embryo development and the efficient production of transgenic chickens as bioreactors. This work was supported by: The BioGreen 21 Program of the Rural Development Administration, Republic of Korea; The SRC/ERC program of MOST/KOSEF (grant no. R11-2002-100-04005-0); The 2006–2011 Technology Development Program for Agriculture and Forestry (TDPAF), Ministry of Agriculture and Forestry, Republic of Korea.

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 degrees C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3' untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31 degrees C as compared to expression at 37 degrees C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).

Reconstituting retroviral (ReCon) vectors facilitating delivery of cytotoxic genes in cancer gene therapy approaches

We have previously described the generation of reconstituting retroviral (ReCon) vectors designed for cancer gene therapy using cytotoxic gene products. The unique vector structure with a promoter physically separated from the transgene allows generation of stable virus producer cells irrespective of the toxic gene. The mechanism of synthesis of DNA from retroviral RNA dictates that infection leads to the reconstitution of functional expression cassettes in the target cell. To improve vector titres, a cytomegalovirus enhancer was inserted upstream of the 5'-long-terminal repeat (LTR); the Woodchuck hepatitis virus post-transcriptional regulatory element and an elongated attachment site upstream of the 3'-LTR were included. In addition, a bacterial origin of replication was deleted and a functional internal polyadenylation signal mutated. Transcriptional targeting was attempted by introducing mammary tissue-specific promoters such as the U3 region of mouse mammary tumour virus or the promoter of the whey acidic protein encoding gene. All modifications were analysed in detail with respect to virus production and infectivity. Finally, the vector was armed with the lambda-holin encoding gene and transduced cells were analysed for cytotoxic effects. Distinct modifications of the vector resulted in a titre improvement of more than 560-fold. Compatibility of the optimized vector with targeted cellular promoters was demonstrated. When equipped with the cytotoxic gene, stable producer cells could be successfully established and high titre virus infection resulted in rigorous target cell killing. The ReCon vector in its optimized form is an attractive tool for cancer gene therapy approaches.

Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell-type-specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression. Addressing this problem, we have integrated the human cytomegalovirus (CMV) immediate-early enhancer into an 'advanced' generation lentiviral vector. Expression cassettes with the reporter gene green fluorescent protein (GFP), combined with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under control of a ubiquitous phosphoglycerate kinase (mouse PGK), cardiomyocyte- (human atrial natriuretic factor (ANF), human ventricular myosin light chain (MLC2v)), or type II alveolar epithelial cell (AT-2)-specific human surfactant protein C (SP-C) promoter, were introduced. As insertion of an enhancing element can interfere with the promoter's specificity, expression levels conferred by our enhancer/promoter constructs were evaluated in target and non-target cells. Transduction of target cells with human CMV enhancer containing lentiviral vectors resulted in a multiple-log increase in GFP expression compared to corresponding vectors lacking the human CMV enhancer. In the case of the ANF, the MLC2v, and the SP-C promoters, tissue-specific reporter gene expression in cardiomyocytes and in lung AT-2 cells was maintained, as expression in non-target cells increased only up to 7-fold. The results of this study indicate that lentiviral vectors with the human CMV enhancer conferring efficient cell-type-specific gene expression may be useful tools for gene therapy purposes or cell tracing, e.g. to analyze stem cell differentiation in transplantation and co-culture settings.

The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors

The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression from a variety of viral vectors, although the precise mechanism is not known. WPRE is most effective when placed downstream of the transgene, proximal to the polyadenylation signal. We hypothesized that WPRE likely reduces viral mRNA readthrough transcription by improving transcript termination, which in turn would increase viral titers and expression. Using a Cre-lox-mediated plasmid-based assay, we found significant readthrough transcription from gamma-retroviral vector (RV) long terminal repeat (wt RV-LTR) and RV LTR with a self-inactivating deletion (SIN RV-LTR). WPRE, when placed upstream of the RV LTRs, significantly reduced readthrough transcription. Readthrough, present at much lower levels with the SIN HIV-1 LV-LTR, was also reduced with WPRE. When placed in RV vectors, WPRE increased total RV genomic mRNA; and increased viral titers from transiently transfected 293T cells and stable PG13 producer cells by 7- to 15-fold. The mechanism of increased titers and expression was not due to increased nuclear mRNA export, increased rate of viral transcription or a significant increase in viral mRNA half-life. Our results showed that WPRE improved vector genomic transcript termination to increase titers and expression from RVs.

Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells

Baculoviruses can express transgenes in a wide range of vertebrate cells. However, in some cells transgene expression is weak. To enhance transgene expression, we studied the effect of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on baculovirus (BV)-mediated gene expression of several transgenes. A significant increase in BV-mediated gene expression was detected in several cell lines. A 10-fold increase in transgene expression was observed with the WPRE as determined by the percentage of positive cells and mean fluorescence intensity (MFI). Furthermore, a combination of optimized cell culture medium and WPRE virus led to more than a 60-fold increase in gene expression. In accordance, elevated mRNA and protein levels were detected in WPRE-virus transduced cells. In HepG2 and RaaSMC, WPRE-mediated enhancement was comparable to the previously shown positive effect of sodium butyrate on BV-mediated gene expression. Thus, inclusion of the WPRE into a baculovirus vector provides a simple means to improve BV-mediated gene expression in vertebrate cells.

275. Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery

275. Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery

Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector

An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTet bidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level (∼ 4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) ∼ 2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis.

176 DOXYCYCLINE-INDUCIBLE EXPRESSION OF THE hEPO GENE IN A RETROVIRUS VECTOR

The use of 'bioreactors' to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to medical advancement. However, it is well known that constitutive overexpression of some cytokine genes may cause serious physiological disturbances in vivo and in vitro. The main objective of this study was to test the feasibility of controllable gene expression of hEPO, a glycoprotein hormone that stimulates red blood cell production. We constructed and tested retrovirus vectors designed to express the hEPO (human erythropoietin) gene under the control of tetracycline-inducible promoters. Advantages of this system over other counterparts, whose gene expressions are controlled by steroid hormones or heavy metals, are little or no cytotoxicity of the inducer and efficient turn-on/off transition. However, the most critical problem of the tetracycline-controllable gene expression system is that overall gene expression level is low. As a solution for this problem, we introduced a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence into the retrovirus vector at a downstream region of either the hEPO gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator) of Tet-On system. In cooperation with tetracycline, the rtTA activates the tetracycline response promoter which controls the expression of the hEPO gene. Transformed cells derived from pig and chicken were cultured in medium supplemented with or without 1 µg mL–1 of doxycycline (a tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the hEPO gene expression level using RT-PCR, Western blotting, and ELISA. Western blotting analysis showed that, among several vectors tested, the vector carrying the WPRE sequence at the 32 side of the hEPO gene was the best in both chicken and porcine cells. Quantitative analysis using ELISA resulted in a more than 10-fold increase in overall hEPO gene expression without sacrificing the efficiency of turn-on/off transition. These results should be very helpful in establishing transgenic livestock as bioreactors, making it possible to produce commercially valuable and biologically active proteins in large quantities.

Recombinant Bovine Herpesvirus 4 (BoHV-4) Expressing Glycoprotein D of BoHV-1 Is Immunogenic and Elicits Serum-Neutralizing Antibodies against BoHV-1 in a Rabbit Model

Several biological characteristics of bovine herpesvirus 4 (BoHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types coming from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Starting from BoHV-4 cloned as a bacterial artificial chromosome (BAC), we used MuA transposase-mediated in vitro transposition to generate recombinant BoHV-4 expressing the immunodominant glycoprotein D (gD) of BoHV-1, one of the most important pathogens of cattle. Although a cis-acting element from woodchuck hepatitis virus (the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) in the 3' end of the gD expression cassette was required for maximal gD expression from plasmids in transient transfection assays, this element was not necessary for efficient expression of gD from recombinant BoHV-4 genomes. BoHV-4 recombinants containing gD expression cassettes with or without the WPRE expressed gD at similarly high levels. Several cell lines originating from different animal species expressed gD when infected with BoHV-4 recombinants. When rabbits were immunized with one of the recombinants, high levels of serum neutralizing antibodies against BoHV-1 were generated. This work is one of the first demonstrations of the use BoHV-4 as a vector for vaccine purposes and may provide the basis for BoHV-1 vaccination of cattle with recombinant BoHV-4.

773. Chemoprotection of Mouse Marrow Transplant Recipients by Ex Vivo Lentiviral Transduction of Murine tyr22 Dihydrofolate Reductase Conferring Resistance to Methotrexate

Top of pageAbstract Methotrexate (MTX) impedes tumor development by inhibiting dihydrofolate reductase (DHFR), an enzyme that catalyzes the conversion of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate (THF). MTX depletes cells of THF, a required precursor for de novo purine and thymidine nucleotide synthesis, and has toxic effects on normal cells of the hematopoietic and gastrointestinal systems. To decrease MTX sensitivity, we introduced a MTX-resistant murine DHFR variant into mouse bone marrow by lentiviral transduction, so as to efficiently transduce non-dividing hematopoietic stem cells. We constructed HIV-1-based lentiviral vectors containing an enhanced green fluorescent protein (eGFP) gene with or without a murine tyr22 DHFR cDNA under transcriptional control of the human EF1-|[alpha]| promoter. These VSV-G-protein pseudotyped lentiviral vectors contained a self-inactivating 3|[prime]| LTR, rev response element (RRE) and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to enhance transduction efficiency. Viral vector was produced by co-transfection in 293T cells and concentrated 100-fold by centrifugation to a titer of 1 x 108 IU/ml, as determined by MTX-resistant colony formation and by flow cytometry for GFP expression. We transduced unfractionated bone marrow from normal Bl/6 Ly5.1 mice with either DHFR-GFP or GFP virus for 12 hours at a multiplicity of infection of 10 in the presence of cytokines (mSCF, IL-6, IL-3). Congenic Ly5.2 recipients received sub-lethal irradiation (700 cGy) prior to transplantation of 3 x 106 transduced marrow cells. MTX or PBS were administered daily starting twenty- four hours after transplantation in three recipient cohorts. Animals that received DHFR-virus transduced marrow recovered to normal hematocrit levels by week 3 in spite of MTX administration (44.7% +/- 2.5). In contrast, animals that received GFP-transduced marrow with subsequent MTX administration exhibited extremely reduced hematocrits (16.5% +/- 0.5) at week 3 and did not survive MTX dose-escalation to 2 mg/kg/day. Animals on MTX therapy that received DHFR-GFP transduced marrow exhibited increased donor cell (Ly5.1) engraftment and significant GFP marking in lymphoid (CD3, B220) and myeloid (GR-1) populations, while recipients that received GFP-transduced marrow exhibited lower levels of peripheral blood mononuclear cells and GFP+ leukocytes. Studies are currently underway to determine transgene copy number in repopulated marrow cells and assess MTX toxicity for the gastrointestinal tract. We conclude that transplantation of lentivirally transduced MTXr-DHFR transgenic marrow supports bone marrow repopulation during MTX chemotherapy in irradiated recipients, providing significant chemoprotection that permits MTX dose-escalation. These pre-clinical results support the development of a clinical gene therapy protocol to provide chemoprotection by lentiviral gene transfer.

216. Long Term Portal Vein Administration of AAV-WPRE Vector Results in Increased Incidence of Neoplastic Disease and Hepatic Pathology

Utilizing the Pahenu2 mouse model for phenylketonuria, we developed several expression vectors containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element added in a rAAV-mPAH construct (rAAV-mPAH-WPRE). While these modifications resulted in increased efficacy in reducing hyperphenylalanemia, we observed severe hepatic pathology and increased incidence of hepatic and systemic neoplastic disease at the termination of these studies. Group 1 received AAV- CBmPAHdimer-WPRE with doses ranging from 1.5x109 to 4e10 IU and was sacrificed at 11 months. 44% (4/9) developed neoplastic disease that included 2 hepatocellular carcinomas, 1 hepatoma, and 1 bronchiogenic carcinoma. 6/9 (66%) had severe hepatic morphology which included myriad cytoplasmic inclusion bodies (1/9) and hepatocellular metaphase arrest. Group 2 was sacrificed at 8 months and had received a similar dose range and vector as group 1 but was ovariectomized and received either a testosterone or placebo pellet. 46% (6/13) developed the most unusual and poorly differentiated forms of neoplasia which included a peritoneal carcinosarcoma, a mesothelioma-like neoplasm of the pleura and epicardium, bilateral renal carcinoma, pulmonary and splenic lymphosarcoma, and two blastoma-like tumors with hepatic sinusoidal involvement. 7/13 (54%) also had pathologic changes in the liver as in group 1. Experimental group 3 was sacrificed at 6 months and received 4e9 AAV-CB-mPAHhd-WPRE as well as 4x109 to 2x1010 AAV-CB-RzI209 vector. None developed neoplastic disease but 60% (3/5) did develop altered hepatocellular morphology as above. Mice from group 4 were sacrificed at 6 and 12 months and received between 4e9 and 3e10 IU of AAV-CB-mPAHhrd+RzI209 (no WPRE). None of these mice (0/8) developed neoplastic disease, but 37.5% (3/8) developed mild to moderate changes in hepatocellular morphology that were consistent with increased metabolic activity. The woodchuck hepatitis virus (WHV) contains a highly conserved 154 amino acid HBV X protein (HBx) that is implicated as a key element in the development of hepatocellular carcinoma. HBx is a trans- activating protein that is often expressed in hepatocellular carcinoma (HCC) and surrounding nontumor liver tissue. The WPRE element we used included an amino terminal 60 amino acid fragment of the HBx protein (1503-1680). To determine if vector was present in neoplastic tissue, and more specifically to look for the presence of HBx, PCR (TAQMan|[copy]|) was employed using HBx probes. HBx DNA was not detected in tumoral tissue although it was present in transduced liver. Immunohistochemical detection of Hbx antigen was observed on the periphery of 1 liver tumor and scattered randomly throughout an intestinal tumor. These results imply that AAV-vector is not integrated into the tumor, but rather it is possible the Hbx element is acting in a |[ldquo]|hit and run|[rdquo]| fashion when administered systemically and may activate cellular oncogenes at random.

929. Functional Potential of Human T Cells Following Lentiviral Suicide Gene Transduction

Top of pageAbstract Clinical trials of retroviral T cell suicide gene therapy following allogeneic haematopoietic stem cell transplantation (HSCT) have shown that T cells causing graft versus host disease (GVHD) can be eliminated following the administration of the prodrug Ganciclovir (GCV). This strategy offers the possibility of harnessing powerful donor lymphocyte immunity in patients undergoing alternative donor HSCT (in both the HLA matched or mismatched settings) who would otherwise be at high risk of intractable GVHD. A number of limitations have been identified, including the detection of GCV resistant alternate splice variants, evidence of impaired anti-viral immunity, and immune response against gene-modified cells. We have generated lentiviral vector systems capable of mediating gene transfer to T cells in the absence of T cell receptor or CD3 activation. The HIV-1 based, self-inactivating constructs encode a central polypurine tract (cPPT) element and an X-protein start site mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). A dual suicide gene/selection marker encoding an enhanced HSVTK mutant (with corrected splice site) and truncated CD34 (tCD34) has been incorporated. Virus was pseudotyped with the Vesicular Stomatitis Virus (VSV-G) or a chimaeric RD114 envelope encoding the Molony leukaemia virus cytoplasmic tail . We compared transduction protocols using either full activation of T cells (anti-CD3/CD28 conjugated beads plus Interleukin-2) or pre-treatment of T cells with IL-2 or IL-7 cytokines alone or in combination. We show that the culture of T cells in media containing cytokines for 96h prior to exposure to viral supernatant renders cells permissive to lentiviral transduction. Transuction efficiency in IL-2 or IL-7 averaged 25% comapred to over 70% for fully activated cells.We observed reduced cell division on the basis of CFSE dye dilution in the cytokine stimulated cells, and flow cytometry confirms greater retention of CD45RA+ naive populations, with less skewing towards the CD45RO+CD27+ subgroups. T cells transduced in the presence of IL-7 retained the highest levels of viability. Preservation of virus specific responses was confirmed by tracking CMV specific T cells through the transduction procedure using tetramer staining. After enrichment on the basis of CD34 expression, transduced cells mediated intact responses against autologous dendritic cells pulsed with CMV. Similarly, proliferative responses against allogeneic dendritic cells were best preserved following IL-7 stimulation. Primary T cells, and virus specific T cells, specific for Adenovirus or CMV, were readily eliminated by exposure to GCV or the less toxic agent Aciclovir and there was no evidence splice variants resistant to prodrug action. The vectors offer the potential of high efficiency transduction and selection, efficient prodrug mediated elimination with preservation of T cell effector responses using cytokine pre-stimulation.