Mary Y. Hurwitz

Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Response of retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir

PURPOSE To evaluate the feasibility and safety of adenovirus-mediated gene therapy as a treatment for tumor seeds in the vitreous of children with retinoblastoma. PATIENTS AND METHODS An Institutional Biosafety Committee-, Institutional Review Board-, Recombinant DNA Advisory Committee-, and US Food and Drug Administration-approved phase I study used intrapatient dose escalation of adenoviral vector containing a herpes simplex thymidine kinase gene (AdV-TK) followed by systemic administration of ganciclovir to treat bilateral retinoblastoma with vitreous tumor seeding refractory to standard therapies. Vitreous tumor seeds were treated by intravitreous injection of AdV-TK adjacent to disease sites. Each injection was followed by ganciclovir delivered intravenously every 12 hours for 7 days. RESULTS Eight patients with vitreous tumor seeds were enrolled. One patient who was treated with 10(8) viral particles (vp) had resolution of the tumor seeds around the injection site. The seven patients who were treated with doses > or = 10(10) vp had resolution of their vitreous tumor seeds documented by fundoscopy. Toxicity included mild inflammation at 10(10) vp and moderate inflammation, corneal edema, and increased intraocular pressure at 10(11) vp. One patient was free of active vitreous tumor seeds 38 months after therapy. There has been no evidence of extraocular spread of tumor along the needle tract in any patient. CONCLUSION AdV-TK followed by ganciclovir can be administered safely to children with retinoblastoma. Suicide gene therapy may contribute to the treatment of children with retinoblastoma tumor seeds in the vitreous, a resistant complication of retinoblastoma.

Metastatic and Nonmetastatic Models of Retinoblastoma

To generate animal models of retinoblastoma that closely resemble metastatic and nonmetastatic human disease for the purposes of examining tumor biology and developing alternate treatments, human retinoblastoma cell lines were injected into the vitreal cavities of immunodeficient mice. Two reproducible animal models with contrasting biological behaviors analogous to human retinoblastoma have been developed. The Y79 retinoblastoma model demonstrated specific tumor evolution similar to that seen in human invasive and metastatic disease. Y79 retinoblastoma cells formed intraocular tumors that were initially confined to the vitreal cavity. Tumors progressively invaded the retina, subretinal space, choroid, optic nerve head, and anterior chamber of the eye. Tumors progressed into the subarachnoid space and focally invaded the brain. Metastases were detected in the contralateral optic nerve. Large tumors developed extraocular extensions. The histology of the tumors showed a poorly differentiated pattern with high mitotic rate, foci of necrosis, and calcification. The WERI-Rb model more closely resembled nonmetastatic human retinoblastoma. WERI- Rb tumors were localized in the eye with only anterior choroidal invasion at late stages. To examine potential biological differences in vitro, the retinoblastoma cell lines were cocultured with adherent choroid cells or adherent glioma cells which represent the targets of invasive retinoblastoma in vivo. Consistent with the in vivo observations, Y79 cells but not WERI-Rb cells adhere specifically to both the choroidal and the glioma cell lines.

Suicide gene therapy for treatment of retinoblastoma in a murine model.

Children presenting with large retinoblastomas are currently treated by enucleation. As most patients are young children, the long-term repercussions of such surgery are often devastating. Subsequent radiation or chemotherapy, although effective in managing residual tumor, greatly increase the probability of the development of second malignancies later in life. Smaller tumors can sometimes be managed with local cryo- or laser surgery, thus saving the eye. The hypothesis that gene therapy could be used to reduce the tumor size sufficiently to allow local control was tested using a murine model of retinoblastoma. Y79Rb human retinoblastoma cells can be killed in vitro when transduced with an adenoviral vector containing the herpes simplex thymidine kinase gene (AdV-TK) followed by treatment with the prodrug ganciclovir. Intravitreal injections of Y79Rb cells in immunodeficient mice produce an aggressive, metastatic murine model of retinoblastoma. When these murine retinoblastomas were transduced in vivo with AdV-TK and the animals treated with intraocular injections of ganciclovir, 70% showed a complete ablation of detectable tumor. Treated animals had a significant prolongation of progression-free survival as compared with untreated controls. Gene therapy effectively reduced the tumor burden in this murine model of retinoblastoma. Thus gene therapy, in conjunction with local surgical control, may provide an effective alternative to enucleation, systemic chemotherapy, or radiotherapy for treatment of large, nonmetastatic retinoblastomas in children.

Extensively necrotic retinoblastoma is associated with high-risk prognostic factors

CONTEXT Retinoblastoma is the most common malignant intraocular tumor in children. It has been shown that adjuvant therapy following enucleation in patients with high-risk histopathologic features significantly decreases the mortality. We describe the association of extensive necrosis of tumor and intraocular structures with 2 of the major risk factors: optic nerve invasion and choroidal invasion. This may alert the pathologist who makes the observation of extensive necrosis to carefully search for histologic features associated with adverse outcome. OBJECTIVE To determine whether extensively necrotic retinoblastoma is associated with high-risk histologic prognostic factors for metastatic disease and patient survival. DESIGN Retrospective case series. Forty-three eyes of 43 patients with retinoblastoma who underwent enucleation between 1990 and 2001 were evaluated. Medical records, histopathology specimens, pathology reports, and clinical photographs were reviewed. Tumors were designated as exhibiting extensive necrosis if more than 95% of tumor cells and intraocular tissues were necrotic. The main outcome measure was the association of extensive tumor necrosis with 3 high-risk histopathologic features: extraocular extension, optic nerve invasion, or choroidal invasion. Metastatic disease, patient survival, and associations with pathologic findings were also analyzed. RESULTS Optic nerve head invasion (P < .001), post-lamina-cribrosal invasion (P < .001), and choroidal invasion by tumor (P = .004) were observed more frequently in eyes with extensive necrosis compared with eyes without extensive necrosis. Two of the 11 patients with extensively necrotic intraocular retinoblastoma died from metastatic disease (P = .06). None of the 32 patients without extensive necrosis developed metastatic disease or died. CONCLUSIONS Extensive ocular tissue and tumor necrosis is associated with histologic high-risk prognostic factors for tumor metastasis and mortality.

A single intravenous injection of oncolytic picornavirus SVV-001 eliminates medulloblastomas in primary tumor-based orthotopic xenograft mouse models

Difficulties of drug delivery across the blood-brain barrier (BBB) and failure to eliminate cancer stem cells (CSCs) are believed to be the major causes of tumor recurrences in children with medulloblastoma (MB). Seneca Valley virus-001 (SVV-001) is a naturally occurring oncolytic picornavirus that can be systemically administered. Here, we report its antitumor activities against MB cells in a panel of 10 primary tumor-based orthotopic xenograft mouse models. We found that SVV-001 killed the primary cultured xenograft cells, infected and replicated in tumor cells expressing CSC surface marker CD133, and eliminated tumor cells capable of forming neurospheres in vitro in 5 of the 10 xenograft models. We confirmed that SVV-001 could pass through BBB in vivo. A single i.v. injection of SVV-001 in 2 anaplastic MB models led to widespread infection of the preformed intracerebellar (ICb) xenografts, resulting in significant increase in survival (2.2-5.9-fold) in both models and complete elimination of ICb xenografts in 8 of the 10 long-term survivors. Mechanistically, we showed that the intracellular replication of SVV-001 is mediated through a subverted autophagy that is different from the bona fide autophagic process induced by rapamycin. Our data suggest that SVV-001 is well suited for MB treatment. This work expands the current views in the oncolytic therapy field regarding the utility of oncolytic viruses in simultaneous targeting of stem and nonstem tumor cells.

Treatment of invasive retinoblastoma in a murine model using an oncolytic picornavirus

Retinoblastoma, the most common intraocular malignancy of childhood, metastasizes by initial invasion of the choroid and the optic nerve. There is no effective treatment for metastatic retinoblastoma, especially when the central nervous system (CNS) is involved, and prevention of this complication is a treatment priority. Seneca Valley Virus (SVV-001) is a conditionally replication-competent picornavirus that is not pathogenic to normal human cells but can kill human retinoblastoma cells in vitro with an IC(50) of <1 viral particle (vp) per cell. A xenograft murine model of metastatic retinoblastoma was used to examine the therapeutic potential of SVV-001. Histopathologic analysis of ocular and brain tissues after a single tail vein injection of SVV-001 (1 x 10(13) vp/kg) showed effective treatment of choroid and ocular nerve tumor invasion (1 of 20 animals with invasive disease in the treated group versus 7 of 20 animals with invasive disease in the control group; P = 0.017) and prevention of CNS metastasis (0 of 20 animals with CNS metastatic disease in the treated group versus 4 of 20 animals with CNS disease in the control group; P = 0.036). There were no observed adverse events due to the virus in any of the treated animals. SVV-001 may be effective as a treatment of locally invasive and metastatic retinoblastoma.

Intravenous injection of oncolytic picornavirus SVV-001 prolongs animal survival in a panel of primary tumor–based orthotopic xenograft mouse models of pediatric glioma

BACKGROUND Seneca Valley virus (SVV-001) is a nonpathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. We examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas. METHODS In vitro antitumor activities were examined in primary cultures, preformed neurospheres, and self-renewing glioma cells derived from 6 patient tumor orthotopic xenograft mouse models (1 anaplastic astrocytoma and 5 GBM). In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids. RESULTS SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, preformed neurospheres, and self-renewing stemlike single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 × 10(12) viral particles/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < .05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < .01). CONCLUSION SVV-001 possesses strong antitumor activity against pediatric malignant gliomas and utilizes α2,3-linked and α2,6-linked sialic acids as mediators of tumor cell infection. Our findings support the consideration of SVV-001 for clinical trials in children with malignant glioma.

The Mammalian Pineal Expresses the Cone but Not the Rod Cyclic GMP Phosphodiesterase

Abstract: To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion‐exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS‐1) and to calmodulin‐phosphodiesterase complexes (ACC). Profiles were assayed with 10 µmol/L [3H]cyclic GMP in the presence of calcium‐calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS‐1, which corresponded to the activity of the cone but, not to the rod in bovine retina. ROS‐1 immunoadsorbed ∼80% of the activity in the 60‐day‐old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of ∼87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase‐specific antiserum. The specific activity of the cone‐like phosphodiesterase in 10‐day‐old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.

Immune consequences of intraocular administration of modified adenoviral vectors.

To investigate the immune consequences of intraocular administration of modified adenoviral vectors, C57BL/6 normal and retinal degeneration C57BL/6 (rd/rd) mice were immunized with subcutaneous, subretinal, vitreal, or anterior chamber injections of replication-deficient adenovirus (AdV) containing the Escherichia coli beta-galactosidase gene (AdV-LacZ). Fourteen days after the initial inoculation, the animals were immune challenged with an injection of AdV-LacZ in the right ear pinna. Antigen-induced delayed type hypersensitivity (DTH) was measured by determining relative ear swelling. Normal C57BL/6 mice immunized with subretinal, vitreal, or anterior chamber injections did not demonstrate a DTH response. The rd/rd C57BL/6 mice injected in the anterior chamber with the viral construct also did not respond with DTH in a manner similar to normal mice responding to intraocular injection and subsequent challenge. However, the rd/rd C57BL/6 mice immunized by the subretinal or vitreal route did respond to immune challenge with a DTH response. Histologic examination of the eyes showed a lack of infiltration by inflammatory cells. Although these results suggest that the potential for immune consequences is reduced when modified adenoviral vectors are used in the normal ocular environment, these vectors used in the vitreal cavity of rd/rd animals may induce a systemic response to the vectors.