Marco Marzulli

Effective Treatment of an Orthotopic Xenograft Model of Human Glioblastoma Using an EGFR-retargeted Oncolytic Herpes Simplex Virus

Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types.

Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412–1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89–905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124–160 and 341–368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1s amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function.

Loss of Pol32 in Drosophila melanogaster causes chromosome instability and suppresses variegation

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32’s participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32rd1 and pol32rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutantssuppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

520. Arming a Tumor Targeted Oncolytic HSV Vector with MMP9 for Enhanced Distribution and Killing Activity

Early phase human clinical trials using several versions of oncolytic herpes simplex virus type 1 (oHSV) have shown promise in the treatment of glioblastoma multiforme, but efficacy has been limited. Impediments to oHSV therapy include poor virus spread due in part to the tumor extracellular matrix, and insufficient replication in tumor cells as a result of attenuating mutations. Thus, our central goal was to improve oncolytic vector delivery, replication, and spread while maintaining safety and tumor specificity. We had already developed a new class of two stage tumor targeted oHSV combining (i) selective infection through tumor-specific receptors and (ii) selective replication based on differential expression of microRNAs (miRs) in tumor and normal cells. We further modified our vector by arming it with the matrix metalloproteinase 9 (MMP9) as a means to reduce vector trapping in the tumor extracellular matrix. MMP9 degrades collagen type IV, a major component of the extracellular matrix (ECM) and basement membranes of glioblastomas. Here we show that: (i) MMP9 expression improves vector spread in GBM neurospheres in vitro; (ii) MMP9 enhances the oncolytic therapeutic efficacy in animal model of human GBM without compromising vector safety.

111. HSV Vectors Retargeted for Infection of TrkA Receptor-Bearing, Pain-Sensing C-Fibers

Chronic pain represents a major cause of morbidity and effective pain therapy remains a significant unmet medical need. The standard of care relies primarily on systemic drug therapies that do not target the site of pain sensation. These therapies often have limited effectiveness, deleterious side effects, and ,induce tolerance. Herpes simplex viruses (HSV)-based gene therapy vectors offer an attractive alternative to drug therapy since therapeutic genes can be delivered to sensory nerve afferents where pain is arising. Our goal is to develop a transductionally retargeted HSV vector to selectively deliver therapeutic genes to those neurons activated during chronic pain states. NGF/TRKA signaling mediates the pain response associated with inflammatory hyperalgesia and neuropathic pain conditions, making TrkA-expressing cells an important target for chronic pain gene therapy. To obtain a fully retargeted HSV, the virus attachment/entry component glycoprotein D (gD) can be modified to eliminate recognition of its cognate receptors (HVEM and nectin1) and introduce a new ligand into the N-terminus of gD to allow entry through its corresponding cellular receptor. Therefore, we replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp) NGF to create a TrkA targeting protein, gD:ppNGF(Y38), that can still bind nectin1. Virus expressing gD:ppNGF(Y38) was propagated on cells expressing nectin1 and purified virus was shown to enter J1.1-2, nectin1-deficient cells, only when transduced with TrkA receptor (J/TrkA cells). To enhance the propagation of these vectors on complementing cells, we developed genetic selection methods to isolate retargeted virus variants that display enhanced entry and spread on J/TrkA cells. We found that a selected variant (J4H) had acquired mutations in other HSV envelope glycoproteins, including one glycoprotein involved in envelope-cell fusion events (gH) and two that were previously shown to contribute to virus spread (gE and gI). Moreover, we show that the gH mutation alone, when introduced into the parental virus backbone, enhances entry and minimally improves virus spread. We are currently investigating the specific phenotypes of the individual gE and gI mutations. A fully TrkA-retargeted J4H-based virus (J4HΔ38), further modified to completely eliminate nectin1-binding, also displays enhanced entry and spread on J/TrkA cells. We are currently testing the J4HΔ38 virus in primary sensory neurons in culture and in infections of animals in vivo. We suggest that the TrkA-vector retargeting will provide a means for transduction of pain sensing C-fibers to more precisely introduce pain-relieving gene products.

113. HSV Vector Development for Targeted Gene Delivery

Gene therapy often requires selective gene delivery to specific cellular subpopulations to provide the most effective therapeutic outcome and avoid off-target effects. This can be accomplished by viral vector targeting involving replacement of the natural virus-cell interactions that trigger virus entry, with novel cell-specific interactions, thereby permitting delivery of therapeutic transgenes to specific cell types. Strategies for full retargeting of HSV require (i) mutagenesis-mediated virus detargeting from its cognate receptors (HVEM and nectin1) recognized by the virus attachment/entry component glycoprotein D (gD) and (ii) the introduction of new ligands into gD that allow entry through recognition of cognate cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptors GFRa1 or TrkA, we employed the ligands GDNF and NGF, respectively. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRa1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient cells in a GFRa1-dependent manner. Likewise, TrkA-dependent virus entry was observed using ppNGF as a targeting ligand. Although propagation of completely retargeted viruses (mutationally inactivated for both HVEM and nectin1 binding) can be achieved in complementing cell lines that express the target receptor, we observed differences among cell lines in retargeted virus spread. HSV receptor-deficient J1.1-2 and B78H1 cells engineered to express the target receptors allowed virus entry, but only B78H1 based cells displayed observable spread. U2OS cells engineered to express GFRa1 demonstrated the most robust virus entry and spread, but these cells express abundant nectin1, providing strong selective pressure for reversion of the nectin1 binding defect of fully retargeted viruses during propagation. In one approach to overcome this problem, we developed genetic selection methods to isolate retargeted virus variants that both entered and spread on J1.1-2 cells transduced with the target receptor. We found that selected variants had acquired mutations in other envelope glycoproteins, including one glycoprotein involved in envelope-cell fusion events (gH) and two that were previously shown to contribute to the spreading process (gE and gI). We expect that the combination of virus mutants and engineered cell lines for efficient production of stably retargeted HSV gene therapy vectors will allow us to achieve HSV retargeting to a broader range of receptors.

497. HSV Vector-Mediated Expression of a Novel Drug Activated Pain Signalling Therapeutic Abrogates Pain

Gene therapy approaches to treat chronic pain have been limited by short-term duration, the inability to turn off therapeutic gene expression to avoid unwanted side-effects or tolerance. To overcome this, we employed replication-defective herpes simplex virus (rdHSV) vectors expressing the glycine receptor alpha-1 (GlyRa1) subunit to silence pain-signaling neurons in a variety of pain models upon the addition of glyine. However, the therapeutic window was small since centrally occurring GlyR in the spinal cord were activated at higher glycine doses. While this was encouraging, the application of GlyRa1 as an effective therapeutic requires further modifications to avoid off-target effects. It was clear that the most effective use of this method would require targeted GlyRa1 expression in specific neuronal subpopulations to avoid silencing of non-nociceptive neurons, limiting receptor expression to neurons involved in chronic pain signaling. Treatment specificity may be achieved through the use of a GlyRa1 that is altered in a manner to be activated by a specific drug that does not cause activation of the normal receptor. Others have identified a mutant form of the receptor (GlyRa1F207A/A288G or G2M) that no longer responds to glycine but rather responds to a novel ligand, ivermectin, an FDA-approved anti-helmenthes drug that binds to the mutant receptor in the nM range while mM quantities of ivermectin are needed to activate the wt-GlyRa1.We engineered G2M rdHSV vectors under the control of the CMV promoter and only upon addition of 100μM Ivermectin could we silence thermal pain while the wt-GlyRa1 was only activated by 100mM Glycine. We tested the effective dose range in a rat model of post-herpetic neuralgia (PHN) that elicits both thermal and mechanical pain behaviors that last out to 9 weeks. We showed that 100μM-10nM Ivermectin was able to block both mechanical and thermal pain, while only 100mM Glycine was able to abrogate those same behaviors, over a 10,000-fold difference. To further reduce off target effects, we engineered rdHSV vectors expressing G2M using neuronal fiber-specific promoters whose transgene expression will be limited to either large As-fibers (NF200) or C-fibers (TRPV1). Studies in rats showed that TRPV1p driven G2M dramatically reduced thermal pain as expected with this C-fiber specific promoter only upon Ivermectin addition to a greater magnitude than that observed with CMV. This activity could be reversed specifically using resiniferatoxin (RTx), a capsaicin analogue that caused C-fiber retraction. NF200 driven G2M failed to block thermal pain, though mildly effected mechanical pain only after RTx treatment. We are currently testing the transcriptionally targeted G2M vectors in the rat PHN pain model. Together, these results demonstrate the power of employing a therapeutic product that responds preferentially to Ivermectin rather than glycine, thereby providing the only receptor in vector-infected animals that is activated by relatively low doses of this well-tolerated drug that can be administered systemically, orally or even topically.

625. Arming a Tumor Targeted Oncolytic Herpes Simplex Virus Type 1 With Matrix Metalloproteinase 9 for Enhanced Vector Distribution and Killing Activity

Early phase human clinical trials using several versions of oncolytic herpes simplex virus type 1 (oHSV) have shown promise in the treatment of GBM, but efficacy has been limited. Impediments to oHSV therapy include poor virus spread due in part to the tumor extracellular matrix, and insufficient replication in tumor cells as a result of attenuating mutations. Thus, the central goal of this project is to improve oncolytic vector delivery, replication and spread while maintaining safety and tumor specificity. We have already developed a new class of two stage tumor targeted oHSV combining (i) selective infection through tumor-specific receptors and (ii) selective replication based on differential expression of microRNAs (miRs) in tumor and normal cells. We further modify our vector by arming with the matrix metalloproteinase 9 (MMP9) as a means to reduce vector trapping in the tumor extracellular matrix. Here we show that MMP9 expression (i) enhances Oncolytic vector spreading in GBM neruospheres in vitro and (ii) improves tumor killing in a xenogeneic model of primary human GBM with significant long-term survival (≥50%) comparable to the control.

Abstract 3546: Arming a tumor targeted oncolytic herpes simplex sirus type 1 with matrix metalloproteinase 9 for enhanced vector distribution and killing activity

Early phase human clinical trials using several versions of oncolytic herpes simplex virus type 1 (oHSV) have shown promise in the treatment of GBM, but efficacy has been limited. Impediments to oHSV therapy include poor virus spread due in part to the tumor extracellular matrix, and insufficient replication in tumor cells as a result of attenuating mutations. Thus, the central goal of this project is to improve oncolytic vector delivery, replication and spread while maintaining safety and tumor specificity. We have already developed a new class of two stage tumor targeted oHSV combining (i) selective infection through tumor-specific receptors and (ii) selective replication based on differential expression of microRNAs (miRs) in tumor and normal cells. We further modify our vector by arming with the matrix metalloproteinase 9 (MMP9) as a means to reduce vector trapping in the tumor extracellular matrix. Here we show that MMP9 expression (i) enhances Oncolytic vector spreading in GBM neruospheres in vitro and (ii) improves tumor killing in a xenogeneic model of primary human GBM with significant long-term survival (≥50%) comparable to the control. Citation Format: Aofei Li, Marco Marzulli, Mingdi Zhang, William Goins, Balveen Kaur, Chelsea Bolyard, Nduka Amankulor, Daniela Leronni, Paola Sette, Justus Cohen, Joseph Glorioso, Paola Grandi. Arming a tumor targeted oncolytic herpes simplex sirus type 1 with matrix metalloproteinase 9 for enhanced vector distribution and killing activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3546. doi:10.1158/1538-7445.AM2015-3546

Abstract 703: Highly selective HSV virotherapy for glioblastoma

Glioblastoma Multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSV) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals. Although proven safe for treatment of GBM in patients, oHSVs efficacy has been limited, a consequence of poor intra-tumoral virus replication and spread. To counter these limitations, we have developed oHSVs whose selective replication in GBM cells does not rely on defective genes. This was accomplished by (i) full retargeting of oHSV to utilize the epidermal growth factor receptor (EGFR) for infection of human GBM tumor cells and (ii) further vector engineering to modify the essential HSV immediate early gene (ICP4) for sensitivity to repression by the microRNA mir-124. Mir-124 is highly expressed in neurons but virtually absent in GBM and highly conserved among different species. The mir-124-regulated vector was unable to replicate in nude mice following intracranial inoculation supporting vector safety and was shown to be effective in the treatment of human GBM in nude mice. To enhance vector intra-tumor vector spread, our EGFR retargeted-mir-124 controlled vector was further modified by vector arming with the matrix metalloproteinase gene encoding MMP9. MMP9 degrades collagen type IV, a major component of the extracellular matrix (ECM) and basement membranes of glioblastomas but absent in normal brain tissue. Studies are ongoing to determine whether MMP9 expression enhances vector spread in GBM neurospheres and as a therapeutic agent for enhanced treatment of human GBM in animals Citation Format: Aofei Li, Marco Marzulli, Lucia Mazzacurati, Hiroaki Uchida, Justus Cohen, Joseph Glorioso, Paola Grandi. Highly selective HSV virotherapy for glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2014-703