Person:

Martuza, Robert

Background: Oncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. Results: We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i) two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii) the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV) efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39 mutant). Conclusion: Our Flip-Flop HSV-BAC system enables rapid generation of HSV vectors carrying transgene inserts. By introducing a tumor-specific-promoter-driven ICP4 cassette into pM24-BAC using this system, one should be able to generate transcriptionally-targeted oncolytic HSV vectors. We believe this system will greatly facilitate the screening of a plethora of clinically useful tumor-specific promoters in the context of oncolytic HSV vectors.

Lymphangioleiomyomatosis (LAM) is a rare pulmonary condition often presenting with spontaneous pneumothorax. Imaging or biopsy confirm the diagnosis. Published case reports describe the anesthetic management of patients with LAM undergoing brief procedures. No reports describe the anesthetic management for lengthy neurosurgical procedures. We describe anesthetic management for craniotomy in a patient with LAM. Clinical Features. A woman presented with 2 spontaneous left pneumothoraces. She received a diagnosis of LAM by imaging. She did well after pleurodesis. Hearing loss and tinnitus led to brain imaging demonstrating a large left cerebello-pontine angle mass. She presented for elective craniotomy to remove the mass while preserving cranial nerve function. Our technique for general endotracheal anesthesia aimed to reduce the likelihood of another pneumothorax while providing good surgical conditions and permitting neuromonitoring. Conclusion:. We demonstrate the successful anesthetic management of a patient with LAM undergoing a lengthy suboccipital craniotomy for a posterior fossa mass.

Oncolytic herpes simplex virus-1 (oHSV)–based vectors selectively replicate in tumor cells causing direct killing, ie., oncolysis, while sparing normal cells. oHSV’sare promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We hypothesized that combining the direct oncolytic and antiangiogenic activities of the IL-12 secreting NV1042 oHSV with microtubule disrupting agents (MDA’s) would be an effective means to enhance antitumor efficacy. Vinblastine (VB) was identified among several MDA’s screened that displayed consistent and potent cytotoxic killing of both prostate cancer and endothelial cell lines. In matrigel tube forming assays, VB was found to be highly effective at inhibiting tube formation of HUVEC cells. The combination of VB with NV1023 (the parental virus lacking IL-12) or NV1042 showed additive or synergistic activity against prostate cancer cell lines and was not due to increased oHSV replication by VB. In athymic mice bearing CWR22 prostate tumors, VB in combination with NV1042 was superior to the combination of VB plus NV1023 in reducing tumor burden, appeared to be nontoxic and resulted in a statistically significant diminution in the number of CD31+ cells as compared to other treatment groups. In human organotypic cultures using surgical samples from radical prostatectomies, both NV1023 and NV1042 were localized specifically to the epithelial cells of prostatic glands but not to the surrounding stroma. These data highlight the therapeutic advantage of combining the dual-acting anti-tumor and anti-angiogenic activities of oHSV’s and MDA’s.

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance, yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements, and are sufficient to fully reprogram differentiated GBM cells to ‘induced’ TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies novel therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies.

Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single cell RNA-seq to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.