Polyadenylation of pre-mRNAs requires the conserved hexanucleotide AAUAAA, as well as sequences located downstream from the poly(A) addition site. The role of these sequences in the production of functional mRNAs was studied by analyzing a series of mutants containing deletions or substitutions in the SV40 early region poly(A) site. As expected, both a previously defined GU-rich downstream element and an AAUAAA sequence were required for efficient usage of the wild-type poly(A) addition site. However, when either of these elements was deleted, greatly increased levels of SV40-specific RNA were detected in the nuclei of transfected cells' Evidence is presented that this accumulation of RNA resulted from a failure of transcription termination, leading to multiple rounds of transcription of the circular templates. We conclude that the sequences required for efficient cleavage/polyadenylation of the SV40 early pre-mRNA also constitute an important element of an RNA polymerase II termination signal. A model proposing a mechanism by which the act of pre-mRNA 3' end formation is signaled to the elongating RNA polymerase, resulting in termination, is presented.

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIIIdeficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A. IntroductionHemophilia A is a severe, X-linked bleeding disorder caused by a deficiency of blood coagulation factor VIII (FVIII). Hemophilia A has an incidence approaching 1 in 4000 males in all populations, 1 and in its severe form, is a life-threatening, crippling disease. Infusion of plasma-derived or recombinant FVIII protein in response to bleeding crises is currently the most widely accepted therapy 1 and has dramatically increased the life expectancy and quality of life for many patients with hemophilia. However, the high cost and short supply of FVIII replacement products has resulted in their availability being limited to less than 10% of the world's hemophilic population.Gene therapy for hemophilia A would provide prophylactic expression of FVIII and correction of the coagulation defect. Considerable progress has been made recently in the development of adenoviral vector-mediated gene therapy for hemophilia A. 2,3 Potent adenoviral vectors encoding a human FVIII complementary DNA (cDNA) have been developed that mediated expression of physiologic levels of FVIII in mice, 4-7 monkeys, 8 and dogs, 9 and sustained human FVIII expression in normal 5 and hemophilic mice. 7 Treatment of hemophilic mice and dogs resulted in human FVIII expression and complete phenotypic correction, verifying the feasibility of adenoviral vector administration for the treatment of hemophilia A. 7,[9][10][11] Expression in the hemophilic mice was sustained for at least 1 year, 7,11 whereas the duration of expression in the hemophilic dogs was short-term, limited by a rapid antibody response to the human FVIII protein. 9 Canine hemophilia A was first described 50 years ago, 12,13 and FVIII-deficient dogs have been used to support the development of FVIII pharmaceutical products. [14][15][16][17][18][19] However, human FVIII is highly immunogenic in dogs when the protein is delivered intravenously 20 or v...

Endostatin, a proteolytic fragment of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis that also inhibits choroidal neovascularization. In this study, we assessed the effects of increased intraocular expression of endostatin on vascular endothelial growth factor (VEGF)-induced changes in the retina. After subretinal injection of a pair of gutless adenoviral vectors (AGV) designed to provide tamoxifen-inducible expression of endostatin, diffuse endostatin immunoreactivity was induced thoroughout the retina by administration of tamoxifen. Induction of endostatin in double transgenic mice with doxycycline-induced expression of VEGF in the retina resulted in significant suppression of leakage of intravascular [3H]mannitol into the retina. The ability of endostatin to reduce VEGF-induced retinal vascular permeability was confirmed by using [3H]mannitol leakage and two other parameters, fluorescein leakage and retinal thickness, after subretinal injection of a bovine immunodeficiency lentiviral vector coding for endostatin (BIV-vectored endostatin, or BIVendostatin). Subretinal injection of BIVendostatin resulted in more discrete, less intense staining for endostatin in the retina than that seen with the inducible AGV system, which suggested lower levels and allowed visualization of sites where endostatin was concentrated. Endostatin staining outlined retinal blood vessels, which suggested endostatin binding to a component of vessel walls. More prolonged or higher level expression of VEGF in the retina resulted in neovascularization and retinal detachment, both of which were also significantly reduced by BIVendostatin. These data suggest that endostatin may be an endogenous inhibitor of vasopermeability as well as neovascularization. In patients with diabetic retinopathy, endostatin gene transfer may provide a way to decrease the risk of three causes of visual loss: macular edema, neovascularization, and retinal detachment.