Vaccinia-based oncolytic immunotherapy Pexastimogene Devacirepvec in patients with advanced hepatocellular carcinoma after sorafenib failure: a randomized multicenter Phase IIb trial (TRAVERSE).

Pexastimogene devacirepvec (Pexa-Vec) is a vaccinia virus-based oncolytic immunotherapy designed to preferentially replicate in and destroy tumor cells while stimulating anti-tumor immunity by expressing GM-CSF. An earlier randomized Phase IIa trial in predominantly sorafenib-naïve hepatocellular carcinoma (HCC) demonstrated an overall survival (OS) benefit. This randomized, open-label Phase IIb trial investigated whether Pexa-Vec plus Best Supportive Care (BSC) improved OS over BSC alone in HCC patients who failed sorafenib therapy (TRAVERSE). 129 patients were randomly assigned 2:1 to Pexa-Vec plus BSC vs. BSC alone. Pexa-Vec was given as a single intravenous (IV) infusion followed by up to 5 IT injections. The primary endpoint was OS. Secondary endpoints included overall response rate (RR), time to progression (TTP) and safety. A high drop-out rate in the control arm (63%) confounded assessment of response-based endpoints. Median OS (ITT) for Pexa-Vec plus BSC vs. BSC alone was 4.2 and 4.4 months, respectively (HR, 1.19, 95% CI: 0.78-1.80; p = .428). There was no difference between the two treatment arms in RR or TTP. Pexa-Vec was generally well-tolerated. The most frequent Grade 3 included pyrexia (8%) and hypotension (8%). Induction of immune responses to vaccinia antigens and HCC associated antigens were observed. Despite a tolerable safety profile and induction of T cell responses, Pexa-Vec did not improve OS as second-line therapy after sorafenib failure. The true potential of oncolytic viruses may lie in the treatment of patients with earlier disease stages which should be addressed in future studies. ClinicalTrials.gov: NCT01387555.

Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes.

Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP) and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) or transforming 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs) and the interaction with the autologous cytotoxic T lymphocyte (CTL) clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1), markers of immunogenic cell death (ICD), could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse melanoma cells and induce additional immunostimulatory effects to promote antitumor immune response. Further investigation in vivo is needed to consolidate the data.

Targeting of adenovirus vectors carrying a tumor cell-specific peptide: in vitro and in vivo studies.

Previously, we have identified a tumor cell-specific peptide, HEW, by panning of phage display libraries on the human colorectal cancer cell line WiDr. In this report we demonstrate that this peptide can modify the infection properties of adenovirus vectors. Increased infectivity of replication-deficient adenovirus 5 vectors in WiDr cells was observed upon genetic insertion of the HEW peptide in the HI loop of the fiber knob. Moreover, whereas the coxsackie and adenovirus receptor (CAR)-ablating fiber mutation S408E abolished apparent infection in CAR-positive WiDr cells, the insertion of HEW completely restored infectivity toward these cells in vitro. To assess whether the de- and re-targeted infection profile was maintained in vivo, the fiber-modified adenovirus vectors were injected intratumorally or intravenously in WiDr tumor-bearing Swiss nu/nu mice. No significant differences in efficiency of infection could be observed suggesting alternative viral uptake mechanisms in vivo. Next, we have included the fiber shaft mutation S(*) in our studies, which was described to confer a de-targeted phenotype in vivo. Reduced gene transfer due to the S(*) mutation both in vitro and in vivo could be confirmed. Insertion of HEW in the HI knob loop of shaft-mutated fiber, however, did not rescue infectivity in target cells neither in vitro nor in vivo. We demonstrate the efficient ligand-mediated re-targeting of adenoviral vector infection to the human cancer cell line WiDr. The lack of apparent re-targeting in the in vivo situation is described.

Augmented trabeculectomy in paediatric glaucoma.

AIMS: To report the experience with trabeculectomy augmented with mitomycin C and 5-fluorouracil for the treatment of paediatric glaucoma. METHODS: Retrospective, interventional case series design was used. The sample included 17 children (29 eyes) with primary (19 eyes) or secondary (10 eyes) glaucoma who were treated with augmented trabeculectomy as the primary procedure between 1990 and 2002. Data were collected on age and family history, preoperative and end of follow up intraocular pressure, cup/disc ratio (evaluated by drawing), visual acuity, complications, and post-surgery treatment. RESULTS: Patient age at surgery ranged from 1 month to 8 years; most patients (n = 14, 82.3%) were aged less than 1 year (range 1 month-8 months, mean 3.95 (SD 2.56) months); three patients (17.7%) were aged 3, 5, and 8 years. The duration of follow up was 3-120 months (mean 46 months). Intraocular pressure significantly improved from 21 mm Hg to 60 mm Hg (mean 33.1 (10) mm Hg) before surgery to 6-26 mm Hg (mean 17.1 (6) mm Hg) after, (p <0.0001). There was no significant change in cup/disc ratio: 0.1-0.8 (mean 0.42 (0.26)) before and 0.1-1.0 (mean 0.511 (0.27)) after (p = 0.45). In 22 eyes (75.8%), intraocular pressure was controlled at less than 20 mm Hg and the cup/disc ratio remained stable or improved. The life table success rate for intraocular pressure control remained stable at 86% at the 12, 24, and 36 months and after 48 months decreased to 53%. There was no significant difference in the life table results between primary and secondary glaucoma. 14 eyes (48.2%) had a visual acuity better than 20/120 by the end of follow up. Repeated surgery was necessary in eight eyes (27.5%), and additional antiglaucoma treatment in 13 (44.8%). Complications included retinal detachment 1 year after surgery, choroidal detachment, and blebitis (one eye each). CONCLUSIONS: Augmented trabeculectomy with mitomycin C and 5-fluorouracil may serve as the primary procedure in a selected group of paediatric patients with glaucoma.

Pupillary block following posterior chamber intraocular lens implantation in adults.

BACKGROUND: Pupillary block rarely occurs after cataract extraction with posterior chamber intraocular lens implantation. METHODS: A series of six patients (seven eyes) treated for pupillary block after posterior chamber intraocular lens implantation between 1990 and 2001 is described; in one eye, the attack occurred after phacoemulsification. RESULTS: The interval between pupillary block development and the cataract surgery ranged from 1 day to 5 years. In all eyes, treatment consisted of neodymium-YAG laser peripheral iridotomy. In four eyes, the laser peripheral iridotomy relieved the block (one procedure in two; two to three procedures in two). One patient was also treated with YAG capsulotomy, and two patients needed additional surgical intervention. CONCLUSION: Despite the rarity of the complication of pupillary block after posterior chamber intraocular lens implantation, physicians should be aware of the sometimes difficult course of recovery after treatment.

Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector.

Gene transfer with 'gutted' vectors is associated with persistent transgene expression and absence of hepatotoxicity, but the requirement of helper viruses hampers efficient production and leads to contamination of viral batches with these helper-viruses. In the present study, gene transfer with a helper-virus independent E(1)/E(3)/E(4)-deleted adenoviral vector induced persistent expression of human apo A-I (200 +/- 16 mg/dl at day 35, 190 +/- 15 mg/dl at 4 months, 170 +/- 16 mg/dl at 6 months) and stable transgene DNA levels (3.5 +/- 0.60 at day 35, 3.3 +/- 0.39 at 4 months, 3.1 +/- 0.47 mg/dl at 6 months) in C57BL/6 mice in the absence of significant toxicity. The vector contained the 1.5 kb human alpha(1)-antitrypsin promoter in front of the genomic human apo A-I sequence and four copies of the human apo E enhancer (hAAT.gA-I.4xapoE) and was deleted in E(1), E(3) and E(4). Reintroduction of E(4) ORF 3 and E(4) ORF 4 in the viral backbone caused a more than four-fold decline of transgene DNA between day 35 and 4 months after transfer both in wild-type and in C57BL/6 SCID and C57BL/6 Rag-1(-/-) mice, indicating that the effect of E(4) ORF 3 and E(4) ORF 4 is independent of a cellular immune response against viral epitopes. Co-injection of an E(1)-deleted vector containing no expression cassette and the E(1)/E(3)/E(4)-deleted vector containing the hAAT.gA-I.4xapoE expression cassette indicated that E(4) gene products destabilize transgene DNA in trans. Gene transfer with an E(1)/E(3)/E(4)-deleted vector containing only E(4) ORF 3 and the hAAT.gA-I.4xapoE expression cassette was associated with transgene DNA decline, but not with hepatotoxicity, indicating that transgene DNA persistence and hepatotoxicity are dissociated processes. After transfer with E(1)/E(3)/E(4)-deleted vectors containing expression cassettes with a different promoter or a different position of the apo E enhancers, transgene DNA levels were less stable than after transfer with the vector containing hAAT.gA-I.4xapoE, indicating that the expression cassette is an important determinant of episomal stability. In conclusion, gene transfer with an E(1)/E(3)/E(4)-deleted vector containing the hAAT.gA-I.4xapoE expression cassette induces persistent expression of human apo A-I in the absence of hepatotoxicity. Transgene DNA turnover is independent of an adaptive cellular immune response against viral epitopes and of hepatotoxicity. E(1)/E(3)/E(4)-deleted vectors containing transgenes under control of the hAAT promoter in combination with four copies of the human apo E enhancer may be suitable for hepatocyte-specific overexpression of transgenes after gene transfer. doi:10.1038/sj.gt.3301824

Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death.

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.

Mitomycin C and 5-fluorouracil antimetabolite therapy for pediatric glaucoma filtration surgery.

PURPOSE: To evaluate the efficacy and safety of augmented trabeculectomy with 5-fluorouracil (5-FU) and mitomycin C (MMC) compared to 5-FU only for the treatment of pediatric glaucoma. PATIENTS AND METHODS: In a prospective randomized clinical trial, 8 children (12 eyes) with pediatric glaucoma, either congenital or secondary to: lens aspiration, Sturge-Weber syndrome, or steroids underwent augmented trabeculectomy. Six patients (8 eyes) underwent augmented trabeculectomy with 5-FU plus MMC and 2 patients (4 eyes) underwent augmented trabeculectomy with 5-FU only. MAIN OUTCOME MEASURES: Between-group comparison of postoperative parameters: change in intraocular pressure (IOP), dependence on antiglaucoma medication, number of 5-FU injections, cup-disc ratio, corneal diameter, drug-induced complications. RESULTS: In the 5-FU/MMC group, 7/8 eyes showed good control of postoperative IOP (9-16 mm Hg), which was independent of antiglaucoma therapy; only 2 injections of 5-FU were needed. By contrast, in the 5-FU group, no control of the postoperative IOP (21-23 mm Hg) was achieved in 4/4 eyes, and these patients remained dependent on antiglaucoma medication; up to 6 injections of 5-FU were used. There was no deterioration in the cup-disc ratio or the corneal diameter in either group. Results were maintained on follow-up (23-27 months). No significant drug-induced complications were noted. CONCLUSION: Augmented trabeculectomy with adjunctive 5-FU/MMC may be an option for the control of pediatric glaucoma in patients with a poor surgical prognosis.

Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products.

Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.

Differential influence of the E4 adenoviral genes on viral and cellular promoters.

BACKGROUND: Strong and stable transgene expression is fundamental to the success of recombinant adenovirus vectors in human gene therapy. However, control of transgene expression is a complex process, involving both viral and cellular factors. In this study, the influence of the E4 adenoviral region on the activity of various promoters was investigated in vitro and in vivo. METHODS: Pairs of isogenic E1o and E1oE4o vectors were generated and compared. Levels of transgene expression were determined by Northern blot, ELISA and FACS analysis. Initiation of transcription was studied by nuclear run-on assays. RESULTS: Similar to the viral CMV and RSV promoters, the activity of the ubiquitous cellular PGK promoter required the presence of the E4 genes in vitro and in vivo. In contrast, transgene expression from selected liver- and tumor-specific promoters did not require E4 functions. CONCLUSION: Together with the reported low liver toxicity of E1oE4o vectors, the independence of E4 of liver-specific promoters renders such vectors interesting alternatives to the use of gutless vectors.

Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3.

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022-2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

Gene transfer into canine myoblasts.

We have developed and characterized cultures of healthy and dystrophic canine myoblasts for the evaluation of various gene transfer protocols. The number of desmin-positive myoblasts was elevated (>>80%) in cultures of myoblasts obtained from different muscle territories, the diaphragm muscle giving rise to the purest cultures. Myoblasts from dogs turned out to be a very convenient source of well transfectable and transducible cells. Transfection with plasmid DNA allowed efficient transgene expression (50% of beta-galactosidase positive cells and about 375 ng luciferase/mg protein after transfection with a calcium phosphate-precipitated plasmid). Infection with high concentrations of adenoviral and retroviral vectors allowed transgene (beta-galactosidase or mini-dystrophin) detection in about 75 to 90% of the canine cells. Therefore, primary dog myoblast cultures represent a useful in vitro model for viral and non-viral gene delivery, as well as for functional evaluation and cell grafting with applications in genetic diseases, vaccination or production of circulating therapeutic proteins.

Intravitreal gas injection for hypotony after cataract surgery.

PURPOSE: To evaluate the efficacy and safety of intravitreal perfluoropropane gas injection to treat hypotony after cataract surgery. SETTING: The ophthalmology department of a major tertiary medical center. METHODS: After uneventful cataract extraction, 5 patients with hypotony due to iridocyclitis, choroidal detachment, and serous retinal detachment were treated with an intravitreal injection of 1.0 cc of perfluoropropane gas. RESULTS: The hypotony, choroidal detachment, and exudative retinal detachment resolved in all 5 patients, and visual acuity improved. No complications were observed. CONCLUSION: Intravitreal gas injection can be used to treat hypotony after cataract surgery in selected patients.

In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

A comparative study of two dose regimens of latanoprost in patients with elevated intraocular pressure.

OBJECTIVE: The purpose of the study was to determine whether latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinor PGF2a-isopropyl ester), a new prostaglandin analogue that has been found effective in reducing intraocular pressure (IOP) in humans, is equally effective at lower concentrations than those currently employed. DESIGN AND PARTICIPANTS: Fifty patients with glaucoma or ocular hypertension were treated in a randomized, crossover, double-masked fashion with 1 drop of latanoprost (50 microg/ml once daily and 15 microg/ml twice daily) in the affected eye(s) for 3 weeks on each concentration. Tonometry was obtained at 8:00, 13:00, and 17:00 hours at baseline (untreated) and after 3 weeks on each concentration. Placebo (a buffer solution of latanoprost eye drop) was administered for complete masking of the study. RESULTS: Mean baseline (untreated) diurnal IOP for the entire sample was 24.7 mmHg. Intraocular pressure was reduced by 6.1 mmHg with latanoprost 15 microg/ml twice daily, and by 7.5 mmHg with 50 microg/ml once daily. Results with both regimens were significant (P < 0.001 each, Student's t-test). However, the 50 microg/ml dose was significantly more effective than the 15 microg/ml dose, with a difference of 1.4 mmHg (P < 0.001, ANOVA). Both dose regimens were well tolerated, with little, predominantly mild, ocular discomfort. The higher dose did not cause more hyperemia at 3 weeks than the lower one, i.e., the lower dose yielded a slightly higher score (1.8 mm) on the visual analogue scale (P < 0.29, ANOVA). CONCLUSIONS: Latanoprost administered at a concentration of 50 microg/ml once daily effectively reduces IOP in patients with elevated IOP. Administration of a lower concentration (15 microg/ml) twice daily is less effective, but still significant.

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response.

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Clinical dose-regimen studies with latanoprost, a new ocular hypotensive PGF2 alpha analogue.

This review summarizes recent short-term clinical studies evaluating the ocular hypotensive efficacy of different dose-regimens of latanoprost. When tested in ocular hypertensive and glaucoma patients concomitantly treated with timolol, 0.006% latanoprost given only in the evening, was found to be more effective than the same concentration given in the morning and evening. In patients with open angle, pseudoexfoliation and normal tension glaucoma not receiving other treatment, once-daily 0.005% latanoprost monotherapy was more effective than twice-daily 0.0015% latanoprost treatment. No significant differences were found in conjunctival hyperemia, sensory irritation or blood-aqueous barrier permeability between these two treatment regimens. Although the ocular hypotensive efficacy of once-daily application of the lower concentration (0.0015%) latanoprost was not investigated, we would conclude, based on the studies reviewed here, that at a concentration of 0.005%, once-a-day dosing of latanoprost is highly effective in significantly reducing intraocular pressure, causing only minimal, clinically acceptable short-term ocular side effects.