Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy.

Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide who depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential reinducible vector DNAs in the trabecular meshwork (0.4 v.g. per cell) and negligible copies in six major internal organs (0.00002-0.005 v.g. per cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid-induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted self-complementary adeno-associated vectors (scAAV) vectors in a large animal model.

Angiogenesis is confined to the transient period of VEGF expression that follows adenoviral gene delivery to ischemic muscle.

Therapeutic angiogenesis involves the introduction of exogenous growth factor proteins and genes into ischemic tissues to augment endogenous factors and promote new vessel growth. Positive results from studies in animal models of peripheral arterial disease (PAD) and coronary artery disease over the past decade have supported the implementation of clinical trials testing vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and genes. Although several clinical trials reported positive results, others have been disappointing and results of a recent Phase II trial of VEGF delivered by adenovirus (the RAVE trial) were negative. It has been suggested that the duration of gene expression following delivery by adenovirus may be insufficient to produce stable vessels. Here we present direct evidence in support of this using the rabbit ischemic hindlimb model injected with adenovirus encoding VEGF165. Immunohistology indicated an activation of endothelial cell cycling and proliferation 2-3 days after VEGF delivery that coincided closely with transient VEGF expression. Ki-67-positive endothelial nuclei were evident at high levels in capillaries and large vessels in muscles from treated animals. Angiography indicated increased density of both large and small vessels in Ad-VEGF-treated muscle at 1 week, but no significant differences thereafter. The early burst of endothelial proliferation was accompanied by increased nuclear fragmentation and condensation in VEGF-treated muscles, suggesting coincident apoptosis. No further endothelial cell proliferation took place after 1 week although there was still evidence of apoptosis. The results suggest that angiogenesis is confined to the short period of VEGF expression produced by adenovirus and early gains in collateralization rapidly regress to control levels when VEGF production ceases.

Inhibition of beta-globin gene expression by 3'-azido-3'-deoxythymidine in human erythroid progenitor cells.

3'-Azido-3'-deoxythymidine (AZT) treatment in HIV-infected patients is limited by bone marrow suppression including neutropenia and anemia. Previous studies had shown a direct effect of high concentrations of this drug on globin gene expression in K-562 erythroleukemia cells. To better define the mechanism(s) of AZT-induced bone marrow toxicity, the present study evaluates these effects in more relevant human erythroid progenitor liquid cultures, because AZT is 100 times more toxic to human bone marrow cells than K-562 cells. At a clinically relevant concentration of 1 microM, AZT inhibited specifically erythroid cell growth by approximately 58% as compared with untreated cells. The percentage of cells synthesizing hemoglobin was decreased also by 47% in AZT-treated cells with beta-globin mRNA levels accounting for 0.27 pmol in treated cells as compared with 1.44 under control conditions while beta-actin levels remained unchanged. Under the same conditions, AZT inhibited the beta-globin chain synthesis by approximately 60% as compared with the control. Consistent with the data described above was the finding that a concentration as low as 0.1 microM of AZT decreased by almost 40% the binding level of the erythroid-specific transcription factor GATA-1. These findings demonstrate that AZT, at clinical relevant concentrations, specifically inhibits beta-globin gene expression in human erythroid progenitor liquid cell culture.

5H-pyrrolo[1,2-b] [1,2,5]benzothiadiazepines (PBTDs): a novel class of non-nucleoside reverse transcriptase inhibitors.

With the aim of developing novel inhibitors of human immunodeficiency virus, various derivatives (10-17) related to 5H-pyrrolo[1,2-b] [1,2,5]benzothiadiazepine (PBTD) were prepared and tested in vitro. The title tricyclic derivatives were obtained by intramolecular cyclization of the open-chain intermediate arylpyrrylsulfones, followed by N-alkylation at position 10. Among test derivatives some 10-alkyl-5H-pyrrolo[1,2-b] [1,2,5]benzothiadiazepin-11(10H)-one-5,5-dioxides were found to exert potent and specific activity against HIV-1. In particular, 7-chloro derivatives 11i and j showed a potency comparable to that of nevirapine. However, when the chloro atom was shifted to the 8 position, the related products were scarcely active or totally inactive. Replacement of the pyrrole with pyrrolidine led to inactive products and the reduction of SO2 to S strongly diminished the antiviral potency. PBTD derivatives active in cell cultures were also inhibitory to the recombinant HIV-1 RT in enzyme assays, thus allowing the conclusion that PBTDs are a new class of non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Synthesis and anti-HIV activity of 10,11-dihydropyrrolo [1,2-b][1,2,5]benzothiadiazepine-11-acetic acid 5,5-dioxide derivatives and related compounds.

The synthesis and the in vitro anti-HIV-1 activity of novel pyrrolo annulated benzothiadiazepine acetic acids and some related derivatives are reported. The new compounds share chemical features with pyrrolo[1,2-d][1,4]benzodiazepin-6-one 1 and Ro 5-3335 pyrrylbenzodiazepinone 4, two inhibitors of HIV-replication at the level of reverse transcriptase (RT) and transcriptional transactivation by Tat, respectively. Two derivatives, namely methyl 10,11-dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine-11-acetic-5,5 -dioxide (5a) and 1,12b-dihydro-2H-azeto[2,1-d]pyrrolo[1,2-b][1,2,5]benzoth iadiazepin-2-one 8,8-dioxide (7a), were found to exhibit a significant, although not very potent, activity against human immunodeficiency virus Type 1 (HIV-1).

3'-Azido-3'-deoxythymidine inhibits erythroid-specific transcription factors in human erythroid K562 leukemia cells.

The present study examines genetic mechanism(s) possibly involved in the observed 3'-azido-3'-deoxythymidine (AZT)-induced inhibition of globin gene transcription by evaluating the direct phenotypic erythroid effects of AZT on erythroid-specific transcription factors which regulate globin gene promoters. In vitro binding of GATA-1 or NFE-2 to its consensus sequence was decreased in the presence of AZT reaching a maximum inhibition as early as 24 h after AZT treatment. Nuclear extracts from butyric acid-induced K562 cells treated with an IC50 concentration of AZT exhibited a decrease in GATA-1 and NFE-2 binding by approximately 30% and 35%. In contrast, 2',3'-dideoxycytidine which inhibits cell growth without affecting hemoglobin synthesis, had no effect on binding of GATA-1 and NFE-2 factors. Northern blot analysis revealed a 25% decrease by AZT in GATA-1 mRNA steady-state levels at 24 h and this inhibitory effect was maintained until 72 h after drug addition. A similar decrease in NFE-2 mRNA steady-state levels was observed at 72 h after AZT treatment. This study suggests that AZT inhibition of erythroid differentiation is subsequent to a decrease of nuclear factors gene expression which affect their DNA binding.

Synthesis and evaluation of the anti-HIV activity of aza and deaza analogues of isoddA and their phosphates as prodrugs.

Some aza and deaza analogues of the anti-HIV agent 2',3'-dideoxy-3'-oxoadenosine (isoddA) (8-aza-, 8-aza-1-deaza, 8-aza-3-deaza-, 1-deaza-, and 3-deaza-isoddA) were synthesized and found inactive against HIV in vitro. The hypothesis that the inactivity of these isonucleosides might be due to their poor affinity for cellular nucleoside kinases was checked by the synthesis of a series of 5'-[bis(2,2,2-trichloroethyl) phosphate] triesters and 5'-phenyl phosphoramidate derivatives which, acting as membrane soluble prodrugs, could release the free phosphate form inside the cell. The 5'-(phenylmethoxy)alaninyl phosphate derived from 8-aza-isoddA was found active against HIV-1 and HIV-2 with a potency similar to that of isoddA, while the anti-HIV potency of 5'-(phenylmethoxy)alaninyl phosphate of isoddA proved remarkably higher than that of isoddA, in particular against HIV-2, being similar to that of AZT. Further evidence that 8-aza-isoddA could behave as anti-HIV agent, provided that it is activated as phosphate, was obtained by the synthesis of its 5'-triphosphate derivative, which proved to be an active inhibitor of HIV-1 recombinant reverse transcriptase.