Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.

This study explored systemic immune changes in 11 subjects with X-linked retinoschisis (XLRS) in a phase I/IIa adeno-associated virus 8 (AAV8)-RS1 gene therapy trial (ClinicalTrials.gov: NCT02317887). Immune cell proportions and serum analytes were compared to 12 healthy male controls. At pre-dosing baseline the mean CD4/CD8 ratio of XLRS subjects was elevated. CD11c+ myeloid dendritic cells (DCs) and the serum epidermal growth factor (EGF) level were decreased, while CD123+ plasmacytoid DCs and serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α were increased, indicating that the XLRS baseline immune status differs from that of controls. XLRS samples 14 days after AAV8-RS1 administration were compared with the XLRS baseline. Frequency of CD11b+CD11c+ DCc was decreased in 8 of 11 XLRS subjects across all vector doses (1e9-3e11 vector genomes [vg]/eye). CD8+human leukocyte antigen-DR isotype (HLA-DR)+ cytotoxic T cells and CD68+CD80+ macrophages were upregulated in 10 of 11 XLRS subjects, along with increased serum granzyme B in 8 of 11 XLRS subjects and elevated IFN-γ in 9 of 11 XLRS subjects. The six XLRS subjects with ocular inflammation after vector application gave a modestly positive correlation of inflammation score to their respective baseline CD4/CD8 ratios. This exploratory study indicates that XLRS subjects may exhibit a proinflammatory, baseline immune phenotype, and that intravitreal dosing with AAV8-RS1 leads to systemic immune activation with an increase of activated lymphocytes, macrophages, and proinflammatory cytokines.

Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.

This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).

X-Linked Retinoschisis.

X-linked retinoschisis (XLRS) is an inherited vitreoretinal dystrophy causing visual impairment in males starting at a young age with an estimated prevalence of 1:5000 to 1:25,000. The condition was first observed in two affected brothers by Josef Haas in 1898 and is clinically diagnosed by characteristic intraretinal cysts arranged in a petaloid "spoke-wheel" pattern centered in the macula. When clinical electroretinogram (ERG) testing began in the 1960s, XLRS was noted to have a characteristic reduction of the dark-adapted b-wave amplitude despite normal or usually nearly normal a-wave amplitudes, which became known as the "electronegative ERG response" of XLRS disease. The causative gene, RS1, was identified on the X-chromosome in 1997 and led to understanding the molecular and cellular basis of the condition, discerning the structure and function of the retinoschisin protein, and generating XLRS murine models. Along with parallel development of gene delivery vectors suitable for targeting retinal diseases, successful gene augmentation therapy was demonstrated by rescuing the XLRS phenotype in mouse. Two human phase I/II therapeutic XLRS gene augmentation studies were initiated; and although these did not yield definitive improvement in visual function, they gave significant new knowledge and experience, which positions the field for further near-term clinical testing with enhanced, next-generation gene therapy for XLRS patients.

Integrated stem cell signature and cytomolecular risk determination in pediatric acute myeloid leukemia.

Relapsed or refractory pediatric acute myeloid leukemia (AML) is associated with poor outcomes and relapse risk prediction approaches have not changed significantly in decades. To build a robust transcriptional risk prediction model for pediatric AML, we perform RNA-sequencing on 1503 primary diagnostic samples. While a 17 gene leukemia stem cell signature (LSC17) is predictive in our aggregated pediatric study population, LSC17 is no longer predictive within established cytogenetic and molecular (cytomolecular) risk groups. Therefore, we identify distinct LSC signatures on the basis of AML cytomolecular subtypes (LSC47) that were more predictive than LSC17. Based on these findings, we build a robust relapse prediction model within a training cohort and then validate it within independent cohorts. Here, we show that LSC47 increases the predictive power of conventional risk stratification and that applying biomarkers in a manner that is informed by cytomolecular profiling outperforms a uniform biomarker approach.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

"Para-retinal" Vector Administration into the Deep Vitreous Enhances Retinal Transgene Expression.

Intravitreal administration for human adeno-associated vector (AAV) delivery is easier and less traumatic to ocular tissues than subretinal injection, but it gives limited retinal transduction. AAV vectors are large (about 4,000 kDa) compared with most intraocular drugs, such as ranibizumab (48 kDa), and the large size impedes diffusion to reach the retina from the usual injection site in the anterior/mid-vitreous. Intuitively, a preferred placement for the vector would be deep in the vitreous near the retina, which we term "para-retinal" delivery. We explored the consequences of para-retinal intravitreal delivery in the rabbit eye and in non-human primate (NHP) eye. 1 h after para-retinal administration in the rabbit eye, the vector concentration near the retina remained four times greater than in the anterior vitreous, indicating limited vector diffusion through the gelatinous vitreous matrix. In NHP, para-retinal placement showed greater transduction in the fovea than vector applied in the mid-vitreous. More efficient retinal delivery translates to using lower vector doses, with reduced risk of ocular inflammatory exposure. These results indicate that para-retinal delivery yields more effective vector concentration near the retina, thereby increasing the potential for better retinal transduction in human clinical application.

Trans-Ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Transduction in Large Animal Eye After Intravitreal Vector Administration.

Purpose: Electric micro-current has been shown to enhance penetration and transduction of adeno-associated viral (AAV) vectors in mouse retina after intravitreal administration. We termed this: "electric-current vector mobility (ECVM)." The present study considered whether ECVM could augment retinal transduction efficiency of intravitreal AAV8-CMV-EGFP in normal rabbit and nonhuman primate (NHP) macaque. Potential mechanisms underlying enhanced retinal transduction by ECVM were also studied. Methods: We applied an electric micro-current across the intact eye of normal rabbit and monkey in vivo for a brief period immediately after intravitreal injection of AAV8-CMV-EGFP. Retinal GFP expression was evaluated by fundus imaging in vivo. Retinal immunohistochemistry was performed to assess the distribution of retinal cells transduced by the AAV8-EGFP. Basic fibroblast growth factor (bFGF) was analyzed by quantitative RT-polymerase chain reaction (PCR). Müller glial reactivity and inner limiting membrane (ILM) were examined by the glial fibrillary acidic protein (GFAP) and vimentin staining in mouse retina, respectively. Results: ECVM significantly increased the efficiency of AAV reaching and transducing the rabbit retina following intravitreal injection, with gene expression in inner nuclear layer, ganglion cells, and Müller cells. Similar trend of improvement was observed in the ECVM-treated monkey eye. The electric micro-current upregulated bFGF expression in Müller cells and vimentin showed ILM structural changes in mouse retina. Conclusions: ECVM promotes the transduction efficiency of AAV8-CMV-GFP in normal rabbit and monkey retinas following intravitreal injection. Translational Relevance: This work has potential translational relevance to human ocular gene therapy by increasing retinal expression of therapeutic vectors given by intravitreal administration.

Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets.

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor-related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2- population different from conventional IL-18Rα-ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

Repeat administration of proteins to the eye with a single intraocular injection of an adenovirus vector.

Delivery of therapeutic proteins, such as antiangiogenic proteins, to the eye is a demonstrated method for the control of age-related macular degeneration (AMD). However, one of the key limitations is the requirement for frequent and repeated intraocular injections. In this article, we demonstrate that repeated protein production in the eye can be stimulated from the cytomegalovirus (CMV) promoter without repeat intraocular injections using a small molecule, all-trans retinoic acid (ATRA). ATRA by systemic delivery can stimulate protein production multiple times in the eye. Administration of ATRA resulted in stimulation of gene expression to relevant levels that block abnormal blood vessel growth in an experimental animal model for AMD. These data support the principles of this technological discovery to therapeutic applications for chronic ocular diseases.

Persistent expression of PEDF in the eye using high-capacity adenovectors.

Ocular neovascularization, the growth of abnormal blood vessels in the eye, is a factor shared by the most common blinding diseases in developed countries. Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic and neuroprotective protein that is normally produced in the eye. When delivered via an adenovector, PEDF can block the growth of new blood vessels and trigger the selective regression of abnormal vessels in animal models of ocular disease. Because of the absence of adenoviral genes, high-capacity (HC) adenovectors offer the potential for persistent transgene expression and enhanced tolerability. We have assessed the durability of PEDF expression and the induction of ocular inflammation following delivery of a PEDF-expressing HC adenovector compared to earlier generation vectors. The HC vector mediated prolonged PEDF expression in tissue-cultured pigmented epithelial cells and when delivered by intravitreal injection into the mouse eye. Delivery of first-generation adenovectors resulted in a dose-dependent increase in cytokine/chemokine gene expression, which correlated with the infiltration of inflammatory cells in the eye. In comparison, the levels of inflammatory gene expression and the intraocular infiltrate were substantially reduced following delivery of the HC vector. These results support the development of the HC adenovector gene delivery system for ocular disease.

Identification and Analyses of Extra-Cranial and Cranial Rhabdoid Tumor Molecular Subgroups Reveal Tumors with Cytotoxic T Cell Infiltration.

Extra-cranial malignant rhabdoid tumors (MRTs) and cranial atypical teratoid RTs (ATRTs) are heterogeneous pediatric cancers driven primarily by SMARCB1 loss. To understand the genome-wide molecular relationships between MRTs and ATRTs, we analyze multi-omics data from 140 MRTs and 161 ATRTs. We detect similarities between the MYC subgroup of ATRTs (ATRT-MYC) and extra-cranial MRTs, including global DNA hypomethylation and overexpression of HOX genes and genes involved in mesenchymal development, distinguishing them from other ATRT subgroups that express neural-like features. We identify five DNA methylation subgroups associated with anatomical sites and SMARCB1 mutation patterns. Groups 1, 3, and 4 exhibit cytotoxic T cell infiltration and expression of immune checkpoint regulators, consistent with a potential role for immunotherapy in rhabdoid tumor patients.

Alternate serotype adenovector provides long-term therapeutic gene expression in the eye.

PURPOSE: To determine whether the duration of transgene expression from an alternate adenovector serotype, Ad35, can provide advantages over an Ad5 serotype vector following a single intravitreal (IVT) administration. METHODS: To assess the transgene expression profile, mice received one IVT injection of Ad5- or Ad35-based vectors expressing green fluorescent protein (GFP), luciferase or pigment epithelium-derived factor (PEDF). At specified time points following vector administration, eyes were monitored for GFP expression, or eyes were harvested and assayed for adenovector genomes, luciferase activity or PEDF levels. Ad35-based vector in vivo biologic activity was investigated using a mouse model of laser-induced choroidal neovascularization (CNV). On Day 0, mice received one IVT injection of Ad5.PEDF or Ad35.PEDF (HI-RGD) followed by laser-induced CNV on Day 28. Fourteen days later, animals were perfused with fluorescein-labeled dextran and CNV lesion size quantitated in choroidal flat mounts. RESULTS: These studies demonstrate that following a single IVT adenovector administration: 1) gene expression is prolonged following administration of an Ad35 compared to an Ad5-based vector; 2) the amount of vector genomes in the eye remain constant out to 60 days post injection of both Ad5 and Ad35-based vectors; and 3) an Ad35.PEDF (HI-RGD) vector inhibits CNV in a mouse model at 42 days post injection. CONCLUSIONS: These studies show that transgene and genome levels are prolonged in the eye following 1 IVT injection of an Ad35-based vector. Moreover, therapeutic gene levels from 1 IVT administration of Ad35.PEDF (HI-RGD) vector block abnormal blood vessel growth in a laser-induced CNV mouse model.

Trans-scleral delivery of antiangiogenic proteins.

PURPOSE: In this study, we investigated the penetration of various proteins into the mouse eye after a periocular injection of the protein or an adenoviral vector (Ad) expressing the protein. METHODS: At several time points after the injection, the retina, retinal pigmented epithelium/choroid, and sclera were dissected and enzyme-linked immunosorbent assays were performed. RESULTS: After a periocular injection of AdsFlt-1.10, AdTGFbeta.10, or AdPEDF.11, choroidal levels of pigment epithelium-derived factor (PEDF) and transforming growth factor-beta (TGF-beta) were not significantly different from scleral levels, and choroidal levels of sFlt-1 (soluble Flt-1 or soluble VEGF receptor 1) were only moderately reduced from scleral levels, indicating that each of these proteins penetrate the sclera well. In contrast, retinal levels of each of the three proteins were low compared to choroidal levels, suggesting poor penetration into the retina. Levels of PEDF in the choroid peaked 2 h after a periocular injection of PEDF protein and returned to baseline between 6 and 24 h, and peak levels in the retina were 8.6% of peak choroidal levels. Levels of green fluorescent protein, a protein unlikely to have any binding sites in mouse tissues, peaked in the choroid 2 h after the periocular injection and were undetectable by 4 h, while peak levels in the retina were 64.3% of peak choroidal levels. CONCLUSIONS: These data suggest that size and binding characteristics of proteins are likely to influence their ability to penetrate the eye from the periocular space, but in general, proteins as large as 50-75 kDa penetrate well into the choroid, but not into the retina. Periocular injections are feasible for the treatment of choroidal neovascularization with proteins or vectors that express them, but additional investigations are needed before they can be considered for treatment of retinal diseases.

Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: results of a phase I clinical trial.

Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) were given a single intravitreous injection of an E1-, partial E3-, E4-deleted adenoviral vector expressing human pigment epithelium- derived factor (AdPEDF.11). Doses ranging from 10(6) to 10(9.5) particle units (PU) were investigated. There were no serious adverse events related to AdPEDF.11 and no dose-limiting toxicities. Signs of mild, transient intraocular inflammation occurred in 25% of patients, but there was no severe inflammation. Six patients experienced increased intraocular pressure that was easily controlled by topical medication. All adenoviral cultures were negative. At 3 and 6 months after injection, 55 and 50%, respectively, of patients treated with 10(6)-10(7.5) PU and 94 and 71% of patients treated with 10(8)-10(9.5) PU had no change or improvement in lesion size from baseline. The median increase in lesion size at 6 and 12 months was 0.5 and 1.0 disk areas in the low-dose group compared with 0 and 0 disk areas in the high-dose group. These data suggest the possibility of antiangiogenic activity that may last for several months after a single intravitreous injection of doses greater than 10(8) PU of AdPEDF.11. This study provides evidence that adenoviral vector-mediated ocular gene transfer is a viable approach for the treatment of ocular disorders and that further studies investigating the efficacy of AdPEDF.11 in patients with neovascular AMD should be performed.

Repeated administration of adenovector in the eye results in efficient gene delivery.

PURPOSE: To determine whether repeat administration of an adenovector (Ad) into the eye results in efficient gene delivery and to test whether transgenes can be expressed from an adenovector expression system in the presence of preexisting, neutralizing anti-Ad antibodies. METHODS: To assess the efficiency of repeated gene delivery of an adenovector expression system, C57Bl/6 mice received one, two, or three injections (intravitreal [IVT] or periocular [PO]) of AdNull.11D (empty cassette) at 2-week intervals, followed by a single AdLuciferase (AdL.11D) IVT or PO injection. Mice were killed approximately 24 hours after AdL.11D injection and the eyes were enucleated and stored until assayed. Serum samples were also analyzed to determine whether repeated IVT or PO injections lead to induction of neutralizing antibodies directed against an adenovector delivery system. To determine whether preexisting neutralizing anti-Ad antibodies would block transgene expression, mice were preimmunized with one, two, or three intramuscular (IM) injection(s) of AdNull.11D (1 x 10(9) particle units [pu]). Fourteen days later, when systemic anti-Ad antibody titers were expected to exist, mice were given a single AdL.11D injection (IVT or PO) and killed, and the eyes and serum collected. RESULTS: These studies show that multiple injections at 2-week intervals with adenovectors (IM, IVT, or PO) did not prevent transgene expression in the eye. Moreover, measurement of neutralizing anti-Ad antibody titers revealed that measurable anti-Ad antibody titers in mice did not ablate transgene expression. CONCLUSIONS: These studies suggest that transgene expression after repeated adenovector administration into the eye is feasible and repeated injections, whether given IVT or PO, do not lead to an immediate increase in neutralizing anti-Ad antibody titers. Moreover, preimmunization of mice by systemic exposure to adenovector, does not block transgene expression in the eye. These studies indicate that repeat administration of adenovectors (IVT and PO) into the eye can be considered in designing future clinical trials and that the pre-existence of neutralizing anti-Ad antibodies probably does not mitigate activity.

Early production of human neutrophils and platelets posttransplant is severely compromised by growth factor exposure.

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34(+) CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells.

Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

Periocular gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization in a human-sized eye.

Gene transfer provides a potential way to achieve sustained delivery of therapeutic proteins to the eye. Studies in rodents have suggested that periocular injection of adenoviral vectors containing expression cassettes for antiangiogenic proteins results in high intraocular levels of the proteins and suppression of choroidal neovascularization (CNV). However, the differences in size and scleral thickness between mouse and human eyes make it difficult to ascertain if periocular gene transfer is a feasible approach for treating human choroidal diseases. To address this issue, we tested the effect of periocular injection of an expression cassette for pigment epithelium-derived factor (PEDF) packaged in adenoviral vector (AdPEDF.11) in a CNV model in pigs, which have eyes that are very similar to humans in size and scleral thickness. Periocular injection of beta-galactosidase (AdLacZ.11) resulted in prominent transduction of periocular tissues, as was seen in mice. Periocular injection of AdPEDF.11 caused increased levels of PEDF in the choroid and significantly reduced the amount of CNV at rupture sites in Bruch's membrane. These data suggest that periocular gene transfer may be feasible for treatment of human choroidal diseases.

Laser photocoagulation and, to a lesser extent, photodynamic therapy target and enhance adenovirus vector-mediated gene transfer in the rat retina.

PURPOSE: To evaluate the transduction efficiency and localization of a reporter gene after intravitreous injection of adenovirus vector in laser photocoagulation (PC)- and photodynamic therapy (PDT)-treated eyes. METHODS: Adult Lewis rats received fundus PC, fundus PDT, or no treatment. Intravitreous injection of an adenovirus vector containing the construct expressing beta-galactosidase (AdlacZ.11D) was performed in each group. All eyes were then enucleated for histochemistry and processed for quantitative image analysis. RESULTS: In eyes with no treatment, there was moderate to intense staining for lacZ in the anterior segment, but little in the retina. In eyes treated with PC and PDT, there was significantly more LacZ staining in the retina. The increased staining corresponded closely with the sites treated with PC and PDT. Gene transduction in PC-treated eyes was enhanced and extended to at least 135 days after virus delivery, but not extended in PDT-treated eyes. Gene transfer and expression were targeted and enhanced at the site of laser burns, at all doses tested (3 x 10(5) to 3 x 10(9) particles per eye). CONCLUSIONS: Compared with untreated eyes, eyes treated with PC and to a lesser extent PDT, manifest increased transduction efficiency, in areas of the retina that are targeted by laser treatment. This finding suggests a new and promising strategy for the treatment of retinochoroidal neovascularization. Adenovirus gene therapy in combination with PC or PDT would have the advantage of increased transduction efficiency; increased duration of transgene expression; targeted delivery; and, potentially, a lower effective dose of virus.