A mutation in the 5' untranslated region of the BRCA1 gene in sporadic breast cancer causes downregulation of translation efficiency

We screened 117 breast tumour samples in Chinese females for mutations in the breast cancer 1 (BRCA1) gene and identified a novel mutation in the 5' untranslated region (5' UTR) in two patients with grade III infiltrating ductal breast carcinoma. We examined whether this 5' UTR mutation affected the translational efficiency of BRCA1 protein. A vector was constructed containing the mutated 5' UTR up-stream of luciferase and we compared its translational efficiency with a wild-type 5' UTR. The expression of BRCA1 protein in breast tumour samples was evaluated using immunohistochemistry. The mutated 5' UTR of BRCA1 resulted in less luciferase activity compared with the wild-type 5' UTR, while there were no significant differences in luciferase mRNA levels. BRCA1 protein was much less expressed in breast tumour tissue from patients with the 5' UTR mutation than in samples from patients without the mutation. Our results show that a mutation in the 5' UTR of the BRCA1 gene downregulates translational efficiency of the protein.     

Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT''s antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.     

Indirect monitoring of endogenous gene expression by positron emission tomography (PET) imaging of reporter gene expression in transgenic mice.

PURPOSE: Repetitive imaging with microPET of endogenous albumin gene expression by using transgenic mice in which the Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene is driven by the albumin promoter (AL-HSV1-tk). METHODS: Transgenic mice were imaged repeatedly on a microPET scanner with approximately 200 microCi of 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) (a substrate for HSV1-TK enzyme). Four transgenic mice were monitored for body weight, serum albumin, and imaged at the end of each of three dietary phases (17%, 0%, and 25% protein diet). Each phase last 14-21 days. The 0% protein diet has been reported previously to reduce albumin gene expression in rats. Twenty non-transgenic mice of the same strain followed a similar feeding schedule and were monitored for serum albumin, body weight, and sacrificed at various time points for determination of their GAPDH normalized albumin mRNA levels. RESULTS: Transgenic mice showed a relatively high FHBG signal from the liver region as expected. Variation of the mean FHBG signal in two mice with a fixed 17% protein diet over a four-month period was <19% s.d. The mean +/- s.e. FHBG liver standardized uptake value (SUV) in four transgenics went from 4.49 +/- 0.32 to 2.17 +/- 0.52 to 6.21 +/- 0.72 as the mice went through the three diets of 17%, 0%, and 25% sequentially. Non-transgenic mice showed GAPDH normalized albumin mRNA that went from 37.68 +/- 6.04 to 26.41 +/- 4.29 to 52.42 +/- 4.09. The FHBG SUV from transgenics was well correlated with GAPDH normalized albumin mRNA from non-transgenics (r(2) = 0.97) supporting that endogenous gene expression of albumin can be indirectly imaged with FHBG. CONCLUSION: Measuring correlated changes in albumin expression in wild type mice and HSV1-TK expression by microPET in transgenic mice in which the reporter gene is driven by the albumin promoter demonstrates that the HSV1-tk gene can be used to monitor, in living animals, modulated expression of transgenes.     

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to approximately 5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.     

Creatine kinase, a magnetic resonance-detectable marker gene for quantification of liver-directed gene transfer

We reported previously the in vivo detection of ectopic and transient expression of creatine kinase gene (ck) in the liver by phosphorus-31 magnetic resonance spectroscopy ((31)P MRS). Here we demonstrate the feasibility of using ck as a reporter gene to monitor the transfer of low-density lipoprotein receptor (LDLr) gene to LDLr(/) mice, a preclinical model for familial hypercholesterolemia. A recombinant adenovirus was generated that carries the creatine kinase gene (ck) and human LDL receptor gene (hLDLr) linked by an internal ribosomal entry site sequence. Intravenous injection of the adenovirus into LDLr(/)mice (1 x 10(11) viral particles/mouse) resulted in transduction of more than 90% of hepatocytes in the liver. Simultaneous expression of ck and LDLr was confirmed by Western analysis of the transduced livers. Through precise regulation of transgene expression in hepatocytes in vitro, an excellent correlation (R(2) = 0.96) between LDLr and ck expression was demonstrated over a wide range of viral dose. In vivo 31P MRS was employed to detect the metabolic product (i.e., phosphocreatine) of the creatine kinase protein (CK) reaction. CK activity, which is a true measure of ck gene expression, was quantified in vivo by magnetization transfer. Because ck is expressed abundantly in human muscle and brain but is absent from the liver, ck is useful to monitor any liver directed gene transfer. Use of the ck reporter would facilitate the clinical translation of gene therapy by providing a nondestructive readout of the level and duration of therapeutic gene expression.     

Circadian Regulation of a Viral Gene Promoter in Live Transgenic Mice Expressing Firefly Luciferase

PURPOSE: This study was conducted to test for possible circadian control of viral infection in live animals using bioluminescence imaging of a firefly luciferase transgene. METHODS: Transgenic mice expressing the firefly luciferase gene under the control of the promoter and enhancer of the human cytomegalovirus major immediate-early gene (CMV::luc) were examined through whole-animal imaging. Mice were crossed with HRS/J hairless albino mice to improve imaging of deep structures. RESULTS: Transgene expression in the extremities and head was elevated around dusk in mice maintained in cycles of light and dark. Signal was also elevated during the animal's night in mice maintained in extended darkness. The viral promoter was induced during the active phase of the circadian locomotor rhythm in several tissues. Both the acinar cells and islets expressed the transgene in dissociated pancreas cultures. CONCLUSIONS: These results suggest that viruses may exploit the circadian system for optimal timing of infection at particular phases in several tissue types.     

Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phosphatase vectors as helper viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo.

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.     

Large liver cell dysplasia in hepatitis B virus x transgenic mouse liver and human chronic hepatitis B virus-infected liver

OBJECTIVES: Large liver cell dysplasia (LCD) is frequently associated with hepatitis B virus (HBV), but it remains uncertain whether it is reactive, senescent or preneoplastic. METHODS: The HBX transgenic mice and normal control mice were sacrificed at 1, 3, 5, 7, 9, 11, 13 and 15 months after birth. Twenty-three cases of human B viral chronic hepatitis/cirrhosis with prominent LCD were selected. The immunohistochemical stain of proliferating cell nuclear antigen (PCNA), transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and senescence-associated beta-galactosidase (SA-beta-Gal) were evaluated. RESULTS: In HBX transgenic mice, LCD was developed since 3 months and formed small nodules of hepatocellular adenoma, which progressed to hepatocellular carcinoma. The hepatocytes with LCD in HBX transgenic mice showed significantly higher PCNA-labeling index (LI) and lower TUNEL-LI than normal hepatocytes of control mice (p < 0.05). In the majority of human B viral chronic hepatitis/cirrhosis, the hepatocytes with LCD revealed higher PCNA-LI and lower TUNEL-LI than those without, when compared in each case using the same tissue block. SA-beta-Gal staining showed no difference between hepatocytes with and without LCD. CONCLUSION: It is suggested that LCD, related to HBV, might not be just an innocent bystander, but closely related to hepatocarcinogenesis.     

Role of hepatocyte direct hyperplasia in lentivirus-mediated liver transduction in vivo

Lentiviral vectors have been used for gene transfer into the liver, but the ability of these vectors to efficiently transduce quiescent hepatocytes remains controversial. Regardless, lentivirus-mediated gene transfer is greatly enhanced when delivered during hepatocellular cycling. For this reason, the present study was designed to determine the role of hepatocyte proliferation in the enhancement of lentiviral transduction by using three different modes of liver regeneration: (1) compensatory regeneration stimulated by two-thirds partial hepatectomy, (2) direct hyperplasia after intragastric administration of the primary mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), and (3) a combination of modes 1 and 2. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vector expressing beta-galactosidase was administered to mice via the peripheral circulation after a regeneration stimulus. Gene transfer as measured by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) staining showed 30-fold higher levels of liver transduction in groups 1 and 2 as compared with the non-liver-manipulated control group (p < 0.005). The combination of TCPOBOP and partial hepatectomy (group 3) resulted in an ~80-fold increase in transduction efficiency compared with the control animals. The enhanced transduction was consistent with higher levels of hepatocellular proliferation observed in animals that received both treatments compared with either single treatment alone. Importantly, the hepatocytes were the predominant cell type transduced, although transgene expression was observed in a low number of nonparenchymal cells regardless of which liver stimulus was received. Biodistribution studies confirmed that most of the gene transfer was limited to the liver and spleen. Taken together, this study suggests that disease-induced cellular proliferation in the liver will enhance the utility of this vector in treating diseases such as viral hepatitis, liver cirrhosis, and cancer.     

High overexpression of the human alpha-galactosidase A gene driven by its promoter in transgenic mice: implications for the treatment of Fabry disease.

BACKGROUND: Human alpha-galactosidase A (alpha-Gal A) is the lysosomal enzyme that cleaves alpha-galactosyl residues from glycoconjugates and is the deficient enzyme in Fabry disease. To date, there have been no studies on the regulation of this "housekeeping" gene. METHODS: Transgenic mice were established with either 1) a 13.3-kilobase (kb) human genomic fragment that contained 246 bp of 5'- and approximately 2.8 kb of 3'- untranslated sequences, or 2) an "intronless" construct derived from the genomic sequence with the 5' and 3' flanking regions intact. Tissues that expressed high levels of alpha-Gal A activity were examined by light and electron microscopy. RESULTS: Transgenic mice were generated with 2 and 12 copies of the genomic sequence (Lines 1 and 2) or about 60 copies of the intronless construct (Lines 3 and 4). In mice hemizygous for the genomic transgene (Lines 1 and 2), tissue alpha-Gal A activities were 12 to 155 times higher than those in the respective wild-type tissue, depending on tissue and transgene copy number. Of note, the high overexpression did not alter the cellular or subcellular cytoarchitecture. In contrast, alpha-Gal A activities expressed by mice that carried the intronless construct were only two- to sixfold more than in wild-type tissues in which the genomic transgene was highly expressed. CONCLUSIONS: The remarkably high levels of alpha-Gal A expression in transgenic mice carrying the intact genomic sequence versus the intronless construct suggested that the genomic sequence contained a strong intronic enhancer element. Identification of this regulatory element or elements may be useful in efforts to overexpress human alpha-Gal A for gene therapy endeavors. In addition, overexpression of human alpha-Gal A did not affect cellular morphology, which indicates that its overexpression in gene therapy endeavors should be safe.     

TWEAK induces liver progenitor cell proliferation

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.     

Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice.

Circulating alpha 1-antitrypsin is synthesized primarily in the liver and secreted into the bloodstream, where it serves as the major protease inhibitor. The PiZ variant of alpha 1-antitrypsin is associated with decreased levels of the protein in sera as a result of its retention within hepatocytes. Homozygosity for the variant allele predisposes individuals to the development of pulmonary emphysema and an increased risk for liver disease. We and others have previously demonstrated that the normal PiM human alpha 1-antitrypsin gene can be properly expressed in the livers of transgenic mice. The PiZ variant of the human alpha 1-antitrypsin gene was introduced into the germline of mice to determine whether the mutant protein would accumulate in mouse hepatocytes and if such accumulation would result in the development of liver damage in an animal model. As expected, the mutant human protein was abundantly synthesized in the livers of the transgenic animals and accumulated within the rough endoplasmic reticulum of hepatocytes as it does in human patients. PiZ mice developed significantly more liver necrosis and inflammation than PiM transgenic mice or control littermates. The degree of liver damage was correlated with the amount of PiZ alpha 1-antitrypsin accumulated in the liver of the different pedigrees of mice. Although 40% of PiZ mice tested were seropositive for mouse hepatitis virus (MHV), the degree of liver damage was not influenced by the MHV seropositivity; rather, it was related only to the presence of accumulated PiZ protein.     

Inhibition of intrahepatic hepatitis B virus replication by antiviral drugs in a novel transgenic mouse model.

Transgenic mice which carry the integrated human hepatitis B virus (HBV) genome and produce HBV particles in the liver were treated with antiviral compounds, and the amount of viral DNA in the liver and serum was monitored by Southern analyses. Mouse alpha interferon (200,000 U per injection, twice daily for 3 days) and a novel nucleoside, oxetanocin G (15 mg/kg of body weight per injection, twice daily for 7 days), inhibited viral DNA synthesis in the liver almost completely. This animal model should prove useful in evaluating anti-HBV agents.     

Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of murine hepatocytes. Viral second-strand DNA synthesis continues to be a rate-limiting step for efficient transduction by the single-stranded AAV (ssAAV) vectors. This is due, in part, to the presence of a cellular chaperone (FK506-binding) protein, FKBP52, phosphorylated forms of which interact with the D-sequence in the inverted terminal repeats of AAV2 genome and inhibit the viral second-strand DNA synthesis. Our previous studies have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at serine/threonine residues by protein phosphatase 5 (PP5) enhances viral second-strand DNA synthesis and consequently, the transgene expression. We have also reported that coinfection with a self-complementary AAV (scAAV)-TC-PTP vector results in up to sixfold increase in the transduction efficiency of conventional ssAAV2 vectors in primary murine hepatocytes in vivo. We reasoned that coinfection with scAAV-TC-PTP and scAAV-PP5 vectors may lead to a further increase in the transduction efficiency of ssAAV2 vectors. We demonstrate here that this strategy does indeed lead to approximately 16-fold increase in the transduction efficiency of conventional ssAAV vectors in primary murine hepatocytes in vivo following tail-vein injections. Neither scAAV2-TC-PTP nor scAAV2-PP5 vectors alone or together had any adverse effect on the hepatocytes. Thus, this coinfection strategy may be useful for achieving expression from recombinant ssAAV2 vectors containing larger genes, such as coagulation factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.     

Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors.

Safe and efficient genetic modification of liver cells could enable new therapies for a variety of hepatic and systemic diseases. Lentiviral vectors are promising tools for in vivo gene delivery. Previous data suggested that recruitment into the cell cycle was required for transduction of hepatocytes in vivo. We developed an improved vector design that enhanced nuclear translocation in target cells and significantly improved gene transfer performance. Using the new vector and a panel of internal promoters, we showed that rat hepatocytes were transduced ex vivo to high frequency without requirement for proliferation. On intravenous administration of vector into adult severe combined immunodeficient (SCID) mice, we found high levels (up to 30%) of transduction of parenchymal and nonparenchymal cells of the liver, integration of the vector genome in liver DNA and stable expression of the marker green fluorescent protein (GFP)-encoding gene without signs of toxicity. Coadministration of vectors and 5'-bromo-2'-deoxyuridine in vivo proved that cell cycling was not required for efficient transduction of hepatocytes. In addition to the liver, the spleen and the bone marrow were transduced effectively by systemic delivery of vector. GFP expression was observed in all these organs when driven by the cytomegalovirus promoter and by the phosphoglycerate kinase gene promoter. Using the promoter of the albumin gene, we could restrict expression to hepatocytes. By a single vector injection into the bloodstream of SCID mice, we achieved therapeutic-range levels of the human clotting factor IX, stable in the plasma for up to 1 year (the longest time tested), indicating the potential efficacy of improved lentiviral vectors for the gene therapy of hemophilias and other diseases.