Low Levels Of Transgene Expression Research Articles

Development and Validation of an Improved Inducer–Regulator Protein Complex in the pBRES-Regulated Expression System

Widespread adaptation of small molecule-regulated expression systems requires the development of selective inducer molecules that do not have any significant side effects on the endogenous receptors from which the regulated expression system is derived. Here we report the identification and in vitro validation of a novel inducer-receptor pair for the single-plasmid regulated expression system termed pBRES, which contains the ligand-binding domain from the human progesterone receptor (hPR). A small molecule inducer, BLX-913, has been identified as having a 30-fold lower IC(50) for the human progesterone receptor than mifepristone (MFP), the previously best characterized inducer for pBRES. Using modeling-guided protein engineering, compensatory mutations were installed at positions W755 and V729 (hPR numbering) in the ligand-binding pocket of the pBRES regulator protein (pBRES RP) to accommodate the new inducer and allow induction of transgene expression to levels previously seen with MFP. The improved inducer-pBRES RP complex was validated in vitro by monitoring the induction of luciferase, murine secreted alkaline phosphatase, and human interferon beta transgenes in mouse skeletal muscle cells. The engineered pBRES demonstrated low levels of transgene expression in the absence, and high expression levels in the presence, of the new BLX-913 inducer. Findings presented here allow induction of the pBRES-regulated gene expression system by a compound with markedly lower anti-hPR activity than MFP, the previously best characterized inducer.

Quantitative analysis of retroviral and lentiviral gene transfer to murine embryonic stem cells

The effectiveness of retrovirus or lentivirus transduction of embryonic stem (ES) cells is often limited because transgene expression is silenced or variegated. We wondered if other steps of transduction, in addition to gene expression, were restricted in ES cells. We quantitatively compared (1) the amount of virus binding, (2) the number of integrated transgenes, and (3) the resulting level of gene expression. We found that three- to fourfold fewer retroviruses and lentiviruses bound to R1 mES cells than to NIH 3T3 cells, suggesting that both types of viruses bind less efficiently to mES cells. Retroviruses and lentiviruses differed in the efficiency with which they completed post-binding steps of transduction. In R1 mES cells, we detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels than in NIH 3T3 cells, which suggests that the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast, we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels in R1 mES cells than in NIH 3T3 cells, which suggests that lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. The implications of our findings for developing improved viral vectors for transducing mES cells are discussed.

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.

Comparison of the E3 and L3 regions for arming oncolytic adenoviruses to achieve a high level of tumor-specific transgene expression

Arming oncolytic adenoviral vectors with anticancer transgenes that can be expressed in a tumor-selective manner may enable the engineering of vectors with increased potency, while retaining their safety profile. Armed oncolytic adenoviral vectors were constructed in which transgene expression has been linked via modified splice acceptor sequences that did not necessitate the deletion of any part of the adenoviral genome. Several oncolytic adenoviral vectors were compared in which the transgene was inserted in place of either the E3 or the L3 region. While all vectors had similar viral growth and cytotoxicity characteristics, the highest level of transgene expression was observed from a vector in which the transgene had been inserted downstream of the L3 23K protease gene, the Ad-23K-GM vector. Notably, no transgene expression occurred with this vector in the absence of DNA replication either in vitro or in vivo. In contrast, viruses in which the transgene was inserted into E3 locations exhibited a low level of transgene expression even in the absence of DNA replication. In summary, by utilizing the L3 region for arming oncolytic viruses, higher levels of tumor-specific transgene expression can be obtained without the need to delete any parts of the viral genome.

Enhanced Cytotoxicity of RIPTK Gene Therapy of Pancreatic Cancer via PDX-1 Co-Delivery

Using in vivo mouse models, we have demonstrated that the insulin promoter-driven suicidal gene therapy (RIPTK) could be used in the treatment of mouse insulinoma and human pancreatic cancer cells. However, limitations of this therapy include tumor cells lack of sufficient PDX-1 protein and low levels of transgene expression mediated by liposome delivery system. The purpose of this study was to determine 1) whether transient transfection of PDX-1 into selected pancreatic cancer cells would lead to increased RIPTK cytotoxicity, and 2) whether an adenoviral delivery system would increase the overall RIPTK gene expression in vitro. RIPlacZ and RSVlacZ plasmid DNA as well as AdCMVlacZ and AdRIPlacZ were used in transfection assays in human pancreatic cancer cell lines PANC-1 and MIA PaCa2 (n = 8). An expression plasmid DNA containing the mouse PDX-1 cDNA was also used. LacZ reporter assays were performed. RIPTK genes constructed either in plasmid or in adenoviral vectors were used in cytotoxic assays. RT-PCR assays were used to determine PDX-1 expression levels. PDX-1 protein was detected in the human pancreatic ductal carcinoma cell line PANC-1, a little in MIA PaCa2 cells. Liposome mediated (L) RSVlacZ and RIPlacZ transfection in PANC-1 cells resulted in 10.1% and 9.3% transgene expression, respectively. Co-delivery of PDX-1 had no significant effect on RSVlacZ expression (9.3%, P = NS) but significantly increased RIPlacZ gene expression (14.9% P < 0.05). Adenoviral mediated (Ad) RIPlacZ transgene was highly expressed in PANC-1 cells (66.1%) and the reporter activity was further enhanced when PDX-1 was co-delivered (70.2%, P < 0.05). Liposomal transfection of MIA PaCa2 cells using RSVlacZ and RIPlacZ reporter genes resulted in 9.3% and 1.0% gene expression, respectively. Co-transfection of PDX-1 in these cells resulted in a significant activation of RIPlacZ gene expression (14.5%, P < 0.05) with no effects on RSVlacZ treated cells (9.8%). AdCMVlacZ and AdRIPlacZ significantly increased reporter activities in MIA PaCa2 cells (63.0% and 9.8%, respectively). Transfection of PDX-1 also significantly enhanced the AdRIPlacZ activities (46.0%, P < 0.05), with no significant effect in AdCMVlacZ treated cells (68.2%). The cytotoxic effect of liposome-RIPTK/ganciclovir (GCV) in PANC-1 cells was 18.6% and increased to 22.8% when PDX-1 was co-transfected into the cells (P = NS). MIA PaCa2 cells treated with RIPTK alone resulted in 4.9% cell death and increased to 18.2% when exogenous PDX-1 was co-delivered (P < 0.05). The AdRIPTK gene delivery with GCV treatment caused significant cytotoxic effect in PANC-1 (29.3%) and MIA PaCa2 (12.4%) compared with untreated cells. The cytotoxic effects were further increased to 43.4% and 29.4% in PANC-1 and MIA PaCa2 cells, respectively, when PDX-1 was co-transfected (P < 0.05 for both). These data demonstrated that adenoviral mediated gene delivery resulted in a significant increase of transgene expression compared with liposomal delivery systems. RIPTK mediated cytotoxicity was also significantly enhanced via co-delivery of exogenous PDX-1 in these cells. Thus, these results also indicated that PDX-1 plays critical roles in insulin promoter activation and demonstrated that PDX-1 production is essential for insulin promoter-directed gene therapy.

Utility of AAV Vectors Derived from Novel Serotypes

AAV vector is derived from nonpathogenic virus and has a number of attractive features as a vector for human gene transfer including safety, broad tissue specificity, and low immunogenicity following gene transfer. Moreover, persistent transgene expression (for years) was demonstrated in multiple animal experiments. For these reasons, applications to a wide spectrum of diseases are expected, and several clinical trials have been conducted. Although it is too early to conclude the outcome, the efficacy of treatment was not sufficiently substantiated in most of the trials despite confirming the safety of the vector. These results are primarily due to low levels of transgene expression. One of the approaches to improve this situation is the use of alternative serotypes of AAV. Traditionally, serotype 2 was considered to be a prototype of AAV, and the majority of studies including human clinical trials have been conducted using this serotype. On the other hand, there are five "classical" serotypes, and several have been additionally discovered from tissues of primates including humans. These serotypes are considered to be valuable resources for vector development to overcome the shortcomings of serotype 2. This review focuses on the difference in expression levels and tissue specificity of various serotype-derived vectors and summarizes current status in the treatment of candidate diseases.

Expression of Recombinant Human Follicle-Stimulating Hormone in the Mammary Gland of Transgenic Mice

In this article, we describe the use of a caprine beta-lactoglobulin (betaLG) expression cassette previously obtained by our group to target the expression of the human follicle-stimulating hormone (hFSH) to the mammary gland of transgenic mice. The hFSH is a pituitary glycoprotein composed of two subunits (alpha and beta). At present, this protein is obtained from mammalian cellular fermentors, and it is extensively used in the treatment of human infertility. Four lines of double (hFSHalpha/beta) transgenic mice that stably transmitted the transgenes were obtained, and hFSHalpha and hFSHbeta mRNA was detected by reverse transcriptase-polymerase chain reaction in the mammary gland of lactating females from all four transgenic lines. The hFSH protein was present in the mammary gland of the lactating females, but could not be detected in the milk by Western blot, probably as a result of low levels of transgene expression.

402. Comparison of Pseudotyped rAAV Vectors for Transduction of Primary Hematopoietic Stem and Progenitor Cells Assayed In Vitro and In Vivo

We evaluated a panel of AAV serotypes for their ability to transduce hematopoietic stem and progenitor cells of human and murine origin. rAAV-2 genomes encoding either dsRed1 or firefly luciferase under the control of the hybrid CBA promoter were packaged into capsids derived from AAV-1, AAV-5, AAV-7, AAV-8 and AAV-9 and purified by successive rounds of cesium chloride gradient centrifugation. Hematopoietic cells were transduced overnight at low cytokine concentrations, following which cells were assayed in vitro for transgene expression or transplanted into sublethally irradiated NOD/SCID mice. For murine marrow cells, AAV-7 and AAV-showed the highest levels of expression as compared with the other serotypes. For analysis of human cells, hematopoietic stem/progenitor cells were purified from cord blood, cytokine-primed peripheral blood and bone marrow samples obtained using IRB approved protocols. CD34 cells were immunomagnetically purified and transduced with rAAV-dsRed. Flow cytometric analyses revealed that AAV-7 consistently showed the highest level of transgene expression in vitro in CD34 cells derived from cord blood and bone marrow with levels ranging from 36-64% 24 hours after transduction. This was followed by AAV-8 and AAV-1, at 10-43% expression at 24 hours post-transduction, while the other serotypes exhibited lower levels of transgene expression. We next compared transduction of the CD34+CD3//Q-population, which is highly enriched for stem cells. CD34+CD3//Q-cells were flow sorted to purity prior to transduction with the different serotypes. Notably, this primitive population yielded higher levels of transduction than the more heterogeneous CD34 population, with approximately 60-70% of CD34+CD3//Q-cells from cord blood and cytokine primed peripheral blood showing dsRed expression 24 hours after transduction with AAV-7. As with CD34 cells, transduction with AAV-8 and AAV-1 were the next most efficient. We have previously demonstrated that although rAAV-2 transduction in vitro results in poor expression at early time points, possibly due to defined limitations in uncoating, nuclear entry and second strand synthesis, long term studies and in vivo assays demonstrate that rAAV-2 efficiently transduces primitive human hematopoietic stem cells capable of serial engraftment in immune deficient mice. Since in vitro assays may not reflect true stem cell activity, we initiated in vivo transplantation studies of stem cells transduced with different AAV serotypes to determine the relative long term engraftment and gene expression potentials of AAV genomes packaged in the different serotype capsids. Initial results following live imaging of luciferase-transduced recipients confirm both the early onset of gene expression with AAV-7 and AAV-8 and the hierarchy of transgene expression seen with the serotypes in vitro. Overall, our results suggest that rAAV genomes pseudotyped in AAV-7 and AAV-8 capsids are very promising for gene transfer into hematopoietic stem cells.

620. A Longer Human Telomerase Reverse Transcriptase Promoter-Based Conditionally Replicative Adenovirus Shows Oncolysis with Less Toxicity for Ovarian Cancer Treatment

Conditionally replicative adenovirus (CRAd) is one of the approaches to allow selective viral replication which lead to infection of neighboring cancer cells with progeny virus. It is reported that the human telomerase reverse transcriptase (hTERT) promoter, which is active in many cancers but mostly inactive in normal organs including the liver, maintains its useful profile in a CRAd construct for ovarian cancers. It is relevant to determine whether low levels of transgene expression driven by the hTERT promoter could occur in normal cells including normally dividing ones, since a leaky promoter that results in low levels of expression of E1 could results in severe toxicity. In many instances, cell-specific promoters have worked well in vitro but have demonstrated leakiness or a lack of specificity in vivo. To improve the specificity of the hTERT promoter to ovarian cancer cells, we therefore constructed a CRAd agents by configuring a longer hTERT promoter (1.5kb, hTERT L) as well as a conventional shorter one (0.4kb, hTERT S) driving an E1 expression cassette in place of the wild type Ad5E1 region and evaluated its oncolytic effect and specificity both in vitro and in vivo. First, transcriptional and translational activity of hTERT in ovarian cancer cell lines were examined by RT-PCR. Then, we employed recombinant Ad vectors containing luciferase reporter genes under the control of the hTERT L or hTERT S promoter (AdhTERT L Luc, AdhTERT S Luc) to evaluate the transductional activity of this promoter in ovarian cancer cell lines in vitro. Ad vectors with these reporter genes under the control of cytomegalovirus (CMV) promoter were used as control vectors (AdCMVLuc). We next examined the biodistribution of these vectors administered intravenously within mice. Finally, we evaluated the oncolytic effect of AdhTERT L E1 and AdhTERT S E1 in ovarian cancer cell lines by an MTS assay and crystal violet assay. RT-PCR analyses showed significant transcriptional and translational activity of the hTERT gene in ovarian cancer cells. hTERT L and hTERT S promoter activity was relatively high in ovarian cancer cells, whereas low activity of them was observed in the hTERT negative cells (normal human keratinocyte, NHEK) with luciferase expression vectors. The specificity (RLU in ovarian cancer cells/RLU in NHEK) of hTERT L was higher than hTERT L. AdhTERT L E1 showed a significant oncolytic effect in all the ovarian cancer cell lines tested as well as AdhTERT S E1, whereas little effect was seen in NHEK cells. Biodistribution of AdhTERT L Luc showed ten times lower luciferase activity in the liver than AdhTERT S Luc, indicating that AdhTERT L vector is more specific to the ovarian cancer cells and less hepatotoxic. These results suggest that hTERT L promoter activity is relatively efficient and more specific in ovarian cancer cells than that of the conventional hTERT S, making it an attractive candidate as a tissue specific promoter for ovarian cancer cells. This specificity-enhanced hTERT L CRAd may be useful for the gene therapy of ovarian cancer.

407. Comparison of Transgene Expression Driven from 4 Different Retroviral Long Terminal Repeats

One of the major limitations of retroviral vectors is the low level of transgene expression in the target cells. To overcome this problem, several strategies have been employed, including the replacement of 3' long terminal repeat (LTR) with other viruses' LTR, the use of different leader sequences, and insertion of RNA export elements. In this study, we tried to evaluate the effect of LTR on transgene expression. For this purpose, we constructed a series of retroviral vectors, MT, MS, MF, and MP that had the same structure except 3' LTR: murine leukemia virus (MLV) LTR for MT, myeloproliferative sarcoma virus (MPSV) LTR for MS, spleen focus-forming virus (SFFV) LTR for MF, and murine stem cell virus (MSCV) LTR for MP. The level of gene expression from these vectors was evaluated using luciferase, stem cell factor (SCF), and enhanced green fluorescence protein (eGFP) as reporters in various cells. We found that luciferase activity driven by MSCV LTR was significantly lower than that driven by other LTRs in K562 and HT1080 cells. When SCF was used as a transgene, the SCF production from the vector containing MPSV LTR was significantly lower than that from other vectors. On the contrary, in experiments using eGFP, MPSV LTR drove the highest level of fluorescence intensity in K562 cells in spite of a low percentage of eGFP expressing cells. Our results strongly indicated that the transgene expression in retroviral vectors is influenced not only by the LTR but also by the nature of the transgenes.

Evidence that the mouse 3' kappa light chain enhancer confers position-independent transgene expression in T- and B-lineage cells.

One of the major obstacles for successful application of murine leukemia virus (MLV) vectors to genetic therapy of lymphocyte disorders is low levels of transgene expression or the eventual loss of expression. To overcome this problem, an improved retroviral vector was constructed utilizing the myeloproliferative sarcoma virus (MPSV) long terminal repeat (LTR), which provided a significantly higher level of transgene expression in human lymphoid cells than did MLV vectors. Nevertheless, transgene expression remained low in a large percentage of transduced cells. To address whether lymphocyte enhancer elements might improve transgene expression mediated by retroviral vectors in lymphocytes, we cloned the mouse immunoglobulin 3' kappa light chain enhancer gene (mE3') into the MPSV vector. We found that the mE3' conferred a higher, more uniform and sustained level of expression in transduced T- and B-cell lines, and in primary T cells, than did the control vector lacking this element. Integration sites were diverse and a single copy of the proviral genome was present in all examined transduced cells. The mE3' failed to enhance transgene expression in most nonlymphoid cells, indicating it is relatively lineage-specific. Taken together, these results provide strong evidence that the mE3' functions as a locus control region (LCR) in conferring enhanced integration-site-independent expression of a retroviral transgene.

Targeted expression of the dominant-negative prolactin receptor in the mammary gland of transgenic mice results in impaired lactation.

The F3-short form of the rat PRL receptor (F3-SPRLR) form acts as a dominant negative inhibitor in vitro. We have developed a transgenic mouse model in which the rat F3-SPRLR was expressed in mammary epithelium under the control of the mouse mammary tumor virus promoter. Two lines of mice were characterized and shown to express the transgene in the mammary gland. No developmental abnormalities or differences from wild-type littermates were observed on the basis of size, activity, or fertility. Mice with a low level of transgene expression had a mammary phenotype similar to the wild type. However, mice overexpressing the transgene (levels much higher than those of the endogenous long PRLR transcript) had impaired mammary gland differentiation and lactation. In these mice, whole-mount and histological analyses demonstrated normal ductal development, but severely reduced lobuloalveolar outgrowth. signal transducer and activator of transcription-5 phosphorylation and expression of beta-casein and whey acidic protein gene were decreased. In vivo bromodeoxyuridine incorporation at midpregnancy showed that the reduction in mammary development was not due to an inhibition of ductal growth and side-branching. This model demonstrates for the first time in vivo a function of the SPRLR and a local and targeted effect of PRL on the mammary gland that are essential for its function, but not for its development.

Integration and expression of Sorghum C 4 phosphoenolpyruvate carboxylase and chloroplastic NADP +-malate dehydrogenase separately or together in C 3 potato plants

We have integrated two cDNAs expressing Sorghum photosynthetic phosphoenolpyruvate carboxylase (C 4-PEPC) and NADP-malate dehydrogenase (cpMDH), two key enzymes involved in the primary carbon fixation pathway of NADP-malic enzyme-type C 4 plants, separately or together into a C 3 plant (potato). Analysis of the transgenic plants showed a 1.5-fold increase in PEPC and cpMDH activities compared to untransformed plants. Immunolocalization confirmed an increase at the protein level of these two enzymes in the transgenic plants and indicated that the Sorghum cpMDH was specifically addressed to the chloroplasts of potato mesophyll cells. However, integration of either or both of the cDNAs into the potato genome did not appear to significantly modify either tuber starch grain content or the rate of photosynthetic O 2 production compared to control untransformed plants. The low level of transgene expression probably explains the lack of influence on carbon metabolism and photosynthetic rates. This general observation suggests that some complex mechanism may regulate the level of production of foreign C 4 metabolism enzymes in C 3 plants.

Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.

Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.

A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo.

Transient transfection studies have shown that the probasin (PB) promoter confers androgen selectivity over other steroid hormones, and transgenic animal studies have demonstrated that the PB promoter will target androgen, but not glucocorticoid, regulation in a prostate-specific manner. Previous PB promoters either targeted low levels of transgene expression or became too large to be conveniently used. The goal was to design a PB promoter that would be small, yet target high levels of prostate-specific transgene expression. Thus, a composite probasin promoter (ARR2PB) coupled to the bacterial chloramphenicol acetyltransferase reporter (ARR2PBCAT) was generated and tested in prostatic and nonprostatic cell lines and in a transgenic mouse model. In PC-3, LNCaP, and DU145 prostate cancer cell lines, the ARR2PB promoter gave basal expression and was induced in response to androgen and glucocorticoid treatment after cotransfection with the respective steroid receptor. Basal expression of ARR2PBCAT in the nonprostatic COS-1, MCF-7, ZR-75-1, and PANC-1 cell lines was very low; however, CAT activity could be induced in response to androgens and glucocorticoids when cells were cotransfected with either the AR or GR. In contrast to the transfection studies, ARR2PBCAT transgene expression remained highly specific for prostatic epithelium in transgenic mice. CAT activity decreased after castration, and could be induced by androgens and, in addition, glucocorticoids. This demonstrates that the necessary sequences required to target prostate-specific epithelial expression are contained within the composite ARR2PB minimal promoter, and that high transgene expression can now be regulated by both androgens and glucocorticoids. The ARR2PB promoter represents a novel glucocorticoid inducible promoter that can be used for the generation of transgenic mouse models and in viral gene therapy vectors for the treatment of prostate cancer in humans.

Transplant immunosuppression increases and prolongs transgene expression following adenoviral-mediated transfection of rat lungs

Background: Gene therapy provides the potential to modify donor organs to better withstand transplantation, but this has yet to be realized. In vivo gene transfer using adenoviral vectors has had limited success because of host immune response that induces inflammation and limits the amount and duration of transgene expression. We hypothesize that transplantation immunosuppression can attenuate the post-transfection host-immune response to allow for improved gene transfer following adenoviral-mediated transfection. Methods We intratracheally transfected with adenovirus containing the β-galactosidase gene and randomized the rats to either the immunosuppression group, receiving daily cyclosporine, azathioprine, and methylprednisolone, or the control group, receiving no immunosuppression. We evaluated transgene expression and post-transfection inflammation at time points ranging from 1 day to 5 weeks. Results Following transfection, control rats showed relatively low levels of transgene expression, which rapidly decreased to non-detectable levels. In contrast, immunosuppressed rats demonstrated significantly higher levels of transgene expression overall ( p < 0.00005), peaking at almost 3 times that of the control group ( p < 0.02), and showing prolonged and elevated transgene expression at 5 weeks ( p < 0.02). On histologic sections of the lungs, immunosuppressed rats exhibited overall lesser grades of post-transfection inflammation. Conclusions Transplant immunosuppression provides the means to attenuate the severe immune response to adenoviral-mediated gene transfection and thereby increase and prolong transgene expression.

Rescue of Fatal Neonatal Hemorrhage in Factor V Deficient Mice by Low Level Transgene Expression

Factor V (FV) is a critical component of the coagulation cascade. FV-deficient patients suffer moderate to severe bleeding, though residual FV activity is detectable in nearly all cases. In contrast, FV-deficient mice die either during mid-embryogenesis, or of massive perinatal hemorrhage. In order to examine the requirements for FV in murine embryogenesis and hemostasis, we generated transgenic mouse lines expressing a Fv minigene under control of either the tissue-specific albumin (Malb) or rat platelet factor 4 (Rpf4) promoter. A total of 12 Malb and 3 Rpf4 lines were analyzed. Though expression in the target tissue was detectable in most lines by RT-PCR, only low levels of transgene expression were achieved (<3% of endogenous Fv in all lines). Despite a low level of Fv transgene expression, rescue of the lethal Fv-/- phenotype was observed with one of the Malb transgenic (Tg+) lines. However, rescue appeared to be incomplete with continued loss of >1/2 of expected Tg+,Fv-/- mice in early embryogenesis. Rescued Tg+,Fv-/- mice have undetectable FV (<0.1%) in both plasma and platelet compartments, but survive the perinatal period and mature to adulthood without spontaneous hemorrhage. We conclude that FV present at <0.1% is sufficient to support postnatal survival. Failure of the Malb transgene to rescue the midembryonic block suggests that FV expression is required during mammalian development at higher levels or with a different tissue-specific or temporal pattern. Taken together, these data may explain the observation of residual FV activity in most human FV-deficient patients due to early embryonic lethality in those absolutely deficient, and suggest that minimal levels of FV expression, below the level of detection, also may be sufficient to support survival in humans.

Transgenic expression of an immunologically privileged retinal antigen extraocularly enhances self tolerance and abrogates susceptibility to autoimmune uveitis.

Interphotoreceptor retinoid-binding protein (IRBP) is an immunologically privileged retinal antigen that can elicit experimental autoimmune uveitis (EAU). The nature and extent of tolerance to immunologically privileged self antigens is poorly understood. To investigate whether transgenic expression of IRBP extraocularly enhances tolerance and protects from EAU we prepared mice that express half of the mouse IRBP gene, containing a potent uveitogenic epitope (residues 161 - 180), under control of MHC class II promoter. Transgene mRNA was detectable in many tissues. Transgenic protein was undetectable by conventional assays, but was detected in thymic tissue by lymphocyte proliferation assay after induction of the promoter. Transgenic mice challenged with p161 - 180 did not develop EAU and had reduced immunological responses, but remained susceptible to EAU induced by whole IRBP, that contains additional uveitogenic epitopes. Disease was also induced by wild type T cells specific to p161 - 180. Thus, extraocular expression of a privileged retinal antigen enhances self tolerance, supporting the notion that sequestration contributes to immune privilege. Exceedingly low levels of transgene expression result in tolerance that is both profound and epitope specific, implying anergy or deletion of the endogenous uveitogenic repertoire. The same level of expression is, however, insufficient to tolerize wild-type effector T cells in the periphery.

Protein tyrosine phosphatase epsilon increases the risk of mammary hyperplasia and mammary tumors in transgenic mice.

Accurate phosphorylation of tyrosine residues in proteins plays a central role in regulation of cellular function. Although connections between aberrant tyrosine kinase activity and malignancy are well-established, significantly less is known about the roles of protein tyrosine phosphatases (PTPases) in tumorigenesis. We have previously shown that the transmembranal form of PTPase Epsilon (PTPepsilon) is upregulated in mouse mammary tumors initiated specifically by ras or neu, suggesting that PTPepsilon may play a role in transformation by these two oncogenes. In order to test this notion in vivo, we created transgenic mice that express elevated levels of PTPepsilon in their mammary epithelium by use of the MMTV promoter/enhancer. Following several cycles of pregnancy female MMTV-PTPepsilon mice uniformly developed pronounced and persistent mammary hyperplasia which was accompanied by residual milk production. Solitary mammary tumors were often detected secondary to mammary hyperplasia. The sporadic nature of the tumors, the long latency period prior to their development, and low levels of transgene expression in the tumors indicate that PTPepsilon provides a necessary, but insufficient, signal for oncogenesis. The results provide genetic evidence that PTPepsilon plays an accessory role in production of mammary tumors in a manner consistent with its upregulation in mammary tumors induced by ras or neu.

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.