Small molecule targeting of transcription-replication conflict for selective chemotherapy.

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.

Candelilla Wax Extracted by Traditional Method and an Ecofriendly Process: Assessment of Its Chemical, Structural and Thermal Properties.

A comparative study was carried out on the chemical, structural and thermal properties of candelilla wax from four wax-producing communities in Mexico, which was obtained by two extraction processes, the conventional one using sulfuric acid (SA) and an eco-friendly alternative process using citric acid (CA) as the extracting agent. The waxes were analyzed by basic chemistry (acidity, saponification, ester indexes, and others), color, Fourier transform infrared spectroscopy (FTIR), Raman micro-spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), and hardness and brittleness measurements. The waxes obtained by the environmentally friendly process showed differences in their physicochemical properties when compared to waxes from the conventional process. In addition, they showed some improvements, such as lighter shades and harder waxes, suggesting that the new environmentally friendly process is a viable option.

Glioma-targeted delivery of exosome-encapsulated antisense oligonucleotides using neural stem cells.

Tropism of neural stem cells (NSCs) to hypoxic tumor areas provides an opportunity for the drug delivery. Here, we demonstrate that NSCs effectively transport antisense oligonucleotides (ASOs) targeting oncogenic and tolerogenic signal transducer and activator of transcription 3 (STAT3) protein into glioma microenvironment. To enable spontaneous, scavenger receptor-mediated endocytosis by NSCs, we used previously described CpG-STAT3ASO conjugates. Following uptake and endosomal escape, CpG-STAT3ASO colocalized with CD63+ vesicles and later with CD63+CD81+ exosomes. Over 3 days, NSCs secreted exosomes loaded up to 80% with CpG-STAT3ASO. Compared to native NSC exosomes, the CpG-STAT3ASO-loaded exosomes potently stimulated immune activity of human dendritic cells or mouse macrophages, inducing nuclear factor κB (NF-κB) signaling and interleukin-12 (IL-12) production. Using orthotopic GL261 tumors, we confirmed that NSC-mediated delivery improved oligonucleotide transfer from a distant injection site into the glioma microenvironment versus naked oligonucleotides. Correspondingly, the NSC-delivered CpG-STAT3ASO enhanced activation of glioma-associated microglia. Finally, we demonstrated that NSC-mediated CpG-STAT3ASO delivery resulted in enhanced antitumor effects against GL261 glioma in mice. Peritumoral injections of 5 × 105 NSCs loaded ex vivo with CpG-STAT3ASO inhibited subcutaneous tumor growth more effectively than the equivalent amount of oligonucleotide alone. Based on these results, we anticipate that NSCs and NSC-derived exosomes will provide a clinically relevant strategy to improve delivery and safety of oligonucleotide therapeutics for glioma treatment.

Hepatitis B Vaccination in Advanced Chronic Kidney Disease: A Quality Improvement Project at a Veteran Affairs Chronic Kidney Disease Clinic.

Hepatitis B vaccination is recommended in all patients with end-stage kidney disease (ESKD). However, only 50-60% of these patients achieve protective antibody levels if immunized after starting dialysis. Strategies to overcome this low seroconversion rate include a 6-month vaccination schedule starting earlier [chronic kidney disease (CKD) stage 4 and 5] to ensure immunity when patients progress to ESKD. We conducted a quality improvement program to immunize pre-dialysis patients. Patients who were found to have a negative baseline serology with a negative hepatitis B surface antibody level (HBsAb) were offered vaccination on a 6-month schedule (0, 1 and 6 months) with one of two available vaccines within the VA system (Recombivax™ or Engerix™). HBsAb titers were checked 3-4 months later, and titers ≥ 12 mIU/mL were indicative of immunity at VA. Patients who did not seroconvert were offered a repeat schedule of three more doses. We screened 198 patients (187 males and 11 females) with CKD 4 and 5 [glomerular filtration rate (GFR) < 29 mL/min/1.73 m2]. The median age of this cohort was 72 years (range 38-92 years). During the study period of 5 years (2015-2020), 10 patients were excluded since their GFR had improved to more than 30 mL/min/1.73 m2, 24 others had baseline immunity and 2 refused vaccination. The hepatitis B vaccination series was not started on 106 patients. Of the remaining 56, 12 patients progressed to ESKD and started dialysis before completion of the vaccination schedule, 6 expired and 1 did not come to clinic in 2020 due to the pandemic. Of the 37 patients who completed the vaccination schedule, 16 achieved seroconversion with adequate HBsAb titers, 10 did not develop immunity despite a second hepatitis B vaccination series, while 11 did not get a second series. Given the low seroconversion rate, albeit in a small cohort, vaccination should be considered in patients with earlier stages of CKD. Other options include studies on FDA approved vaccines of shorter duration. We plan to increase awareness among nephrologists, patients and nursing staff about the importance of achieving immunity against hepatitis B.

Multiple Treatment Cycles of Neural Stem Cell Delivered Oncolytic Adenovirus for the Treatment of Glioblastoma.

Tumor tropic neural stem cells (NSCs) can improve the anti-tumor efficacy of oncovirotherapy agents by protecting them from rapid clearance by the immune system and delivering them to multiple distant tumor sites. We recently completed a first-in-human trial assessing the safety of a single intracerebral dose of NSC-delivered CRAd-Survivin-pk7 (NSC.CRAd-S-pk7) combined with radiation and chemotherapy in newly diagnosed high-grade glioma patients. The maximum feasible dose was determined to be 150 million NSC.CRAd-Sp-k7 (1.875 × 1011 viral particles). Higher doses were not assessed due to volume limitations for intracerebral administration and the inability to further concentrate the study agent. It is possible that therapeutic efficacy could be maximized by administering even higher doses. Here, we report IND-enabling studies in which an improvement in treatment efficacy is achieved in immunocompetent mice by administering multiple treatment cycles intracerebrally. The results imply that pre-existing immunity does not preclude therapeutic benefits attainable by administering multiple rounds of an oncolytic adenovirus directly into the brain.

Large, Anionic Liposomes Enable Targeted Intraperitoneal Delivery of a TLR 7/8 Agonist To Repolarize Ovarian Tumors' Microenvironment.

Ovarian cancer is the most lethal gynecological malignancy in the United States. Current standard of treatment includes surgical debulking and chemotherapy, such as cisplatin and paclitaxel. However, the patients' response rate for chemotherapy in ovarian cancer is not optimal, and they often develop chemoresistance and suffer from side effects. Current clinical trials make extensive use of immune checkpoint blockade (ICB) as a novel cancer immunotherapeutic strategy against ovarian tumors. However, the response rates for ICB antibodies remain limited to 10-20% of treated ovarian cancer patients despite the success of this approach in melanoma, renal, head and neck, and nonsmall cell lung cancers. This lack of efficacy is often attributed to the "cold" immune status of ovarian tumors, as these tumors often have a low number of tumor-infiltrating lymphocytes (TILs) but a high number of suppressive immune cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), or regulatory T cells (Tregs). Repolarizing TAMs could be a promising strategy to reshape the tumor immune microenvironment and promote antitumor activity when combined with ICBs. Toll-like receptor (TLR) 7 and 8 agonists, such as imiquimod and resiquimod, are potent immunostimulatory molecules with potential to repolarize macrophages. However, these small molecules have poor pharmacokinetic profiles and can induce severe side effects when administered systemically. Previously, our group demonstrated that various large, anionic nanomaterials (silica, PLGA, and polystyrene) specifically target TAMs when administered intraperitoneally (IP) to ovarian tumor-bearing mice. In the present study, we demonstrate that large, anionic liposomes administered IP also efficiently localize to TAMs and can be used to target the delivery of resiquimod. Resiquimod delivered in this targeted fashion promoted activation of M1 macrophages and T cell infiltration, while reducing the percentage of Tregs in the tumor microenvironment. Finally, liposome-formulated resiquimod significantly enhanced the efficacy of PD1 blockade against syngeneic ovarian tumors. We anticipate that further optimization of our liposomal delivery strategy can generate a clinically relevant strategy for more effective and safer immunotherapy for ovarian cancer patients.

Allogeneic human neural stem cells for improved therapeutic delivery to peritoneal ovarian cancer.

BACKGROUND: Immortalized, clonal HB1.F3.CD 21 human neural stem/progenitor cells (NSCs), loaded with therapeutic cargo prior to intraperitoneal (IP) injection, have been shown to improve the delivery and efficacy of therapeutic agents in pre-clinical models of stage III ovarian cancer. In previous studies, the distribution and efficacy of the NSC-delivered cargo has been examined; however, the fate of the NSCs has not yet been explored. METHODS: To monitor NSC tropism, we used an unconventional method of quantifying endocytosed gold nanorods to overcome the weaknesses of existing cell-tracking technologies. RESULTS: Here, we report efficient tumor tropism of HB1.F3.CD 21 NSCs, showing that they primarily distribute to the tumor stroma surrounding individual tumor foci within 3 h after injection, reaching up to 95% of IP metastases without localizing to healthy tissue. Furthermore, we demonstrate that these NSCs are non-tumorigenic and non-immunogenic within the peritoneal setting. CONCLUSIONS: Their efficient tropism, combined with their promising clinical safety features and potential for cost-effective scale-up, positions this NSC line as a practical, off-the-shelf platform to improve the delivery of a myriad of peritoneal cancer therapeutics.

Novel Chimeric Poxvirus CF17 Improves Survival in a Murine Model of Intraperitoneal Ovarian Cancer Metastasis.

Despite improvements in surgical techniques and chemotherapy, ovarian cancer remains the most lethal gynecologic cancer. Thus, there is an urgent need for more effective therapeutics, particularly for chemo-resistant peritoneal ovarian cancer metastases. Oncolytic virotherapy represents an innovative treatment paradigm; however, for oncolytic viruses tested from the last generation of genetically engineered viruses, the therapeutic benefits have been modest. To overcome these limitations, we generated a chimeric poxvirus, CF17, through the chimerization of nine species of orthopoxviruses. Compared with its parental viruses, CF17 has demonstrated superior oncolytic characteristics. Here, we report the oncolytic potential of CF17 in ovarian cancer. Replication of CF17 and its resulting cytotoxicity were observed at multiplicities of infection (MOIs) as low as 0.001 in human and mouse cancer cell lines in vitro. Furthermore, CF17 exerted potent antitumor effects in a syngeneic mouse model of ovarian cancer at doses as low as 6 × 106 plaque-forming units. Together, these data merit further investigation of the potential use of this novel chimeric poxvirus as an effective treatment for aggressive intraperitoneal ovarian cancer.

Specific targeting of ovarian tumor-associated macrophages by large, anionic nanoparticles.

Immunotherapy is emerging as one of the most effective methods for treating many cancers. However, immunotherapy can still introduce significant off-target toxicity, and methods are sought to enable targeted immunotherapy at tumor sites. Here, we show that relatively large (>100-nm) anionic nanoparticles administered intraperitoneally (i.p.) selectively accumulate in tumor-associated macrophages (TAMs). In a mouse model of metastatic ovarian cancer, fluorescently labeled silica, poly(lactic-co-glycolic acid), and polystyrene nanoparticles administered i.p. were all found to selectively accumulate in TAMs. Quantifying silica particle uptake indicated that >80% of the injected dose was in TAMs. Particles that were smaller than 100 nm or cationic or administered intravenously (i.v.) showed no TAM targeting. Moreover, this phenomenon is likely to occur in humans because when freshly excised human surgical samples were treated with the fluorescent silica nanoparticles no interaction with healthy tissue was seen but selective uptake by TAMs was seen in 13 different patient samples. Ovarian cancer is a deadly disease that afflicts ∼22,000 women per year in the United States, and the presence of immunosuppressive TAMs at tumors is correlated with decreased survival. The ability to selectively target TAMs opens the door to targeted immunotherapy for ovarian cancer.

Quantitative Evaluation of Intraventricular Delivery of Therapeutic Neural Stem Cells to Orthotopic Glioma.

Neural stem cells (NSCs) are inherently tumor-tropic, which allows them to migrate through normal tissue and selectively localize to invasive tumor sites in the brain. We have engineered a clonal, immortalized allogeneic NSC line (HB1.F3.CD21; CD-NSCs) that maintains its stem-like properties, a normal karyotype and is HLA Class II negative. It is genetically and functionally stable over time and multiple passages, and has demonstrated safety in phase I glioma trials. These properties enable the production of an "off-the-shelf" therapy that can be readily available for patient treatment. There are multiple factors contributing to stem cell tumor-tropism, and much remains to be elucidated. The route of NSC delivery and the distribution of NSCs at tumor sites are key factors in the development of effective cell-based therapies. Stem cells can be engineered to deliver and/or produce many different therapeutic agents, including prodrug activating enzymes (which locally convert systemically administered prodrugs to active chemotherapeutic agents); oncolytic viruses; tumor-targeted antibodies; therapeutic nanoparticles; and extracellular vesicles that contain therapeutic oligonucleotides. By targeting these therapeutics selectively to tumor foci, we aim to minimize toxicity to normal tissues and maximize therapeutic benefits. In this manuscript, we demonstrate that NSCs administered via intracerebral/ventricular (IVEN) routes can migrate efficiently toward single or multiple tumor foci. IVEN delivery will enable repeat administrations for patients through an Ommaya reservoir, potentially resulting in improved therapeutic outcomes. In our preclinical studies using various glioma lines, we have quantified NSC migration and distribution in mouse brains and have found robust migration of our clinically relevant HB1.F3.CD21 NSC line toward invasive tumor foci, irrespective of their origin. These results establish proof-of-concept and demonstrate the potential of developing a multitude of therapeutic options using modified NSCs.

Silica Coated Paclitaxel Nanocrystals Enable Neural Stem Cell Loading For Treatment of Ovarian Cancer.

Ovarian cancer is commonly diagnosed only after it has metastasized to the abdominal cavity (stage III). While the current standard of care of intraperitoneal (IP) administration of cisplatin and paclitaxel (PTX) combination chemotherapy has benefit, patient 5-year survival rates are low and have not significantly improved in the past decade. The ability to target chemotherapy selectively to ovarian tumors while sparing normal tissue would improve efficacy and decrease toxicities. We have previously shown that cisplatin-loaded nanoparticles (NPs) loaded within neural stem cells (NSCs) are selectively delivered to ovarian tumors in the abdominal cavity following IP injection, with no evidence of localization to normal tissue. Here we extended the capabilities of this system to also include PTX delivery. NPs that will be loaded into NSCs must contain a high amount of drug by weight but constrain the release of the drug such that the NSCs are viable after loading and can successfully migrate to tumors. We developed silica coated PTX nanocrystals (Si[PTX-NC]) meeting these requirements. Si[PTX-NC] were more effective than uncoated PTX-NC or Abraxane for loading NSCs with PTX. NSCs loaded with Si[PTX-NC] maintained their migratory ability and, for low dose PTX, were more effective than free PTX-NC or Si[PTX-NC] at killing ovarian tumors in vivo. This work demonstrates that NSC/NP delivery is a platform technology amenable to delivering different therapeutics and enables the pursuit of NSC/NP targeted delivery of the entire preferred chemotherapy regimen for ovarian cancer. It also describes efficient silica coating chemistry for PTX nanocrystals that may have applications beyond our focus on NSC transport.

Bcl-2 Overexpression Improves Survival and Efficacy of Neural Stem Cell-Mediated Enzyme Prodrug Therapy.

Tumor-tropic neural stem cells (NSCs) can be engineered to localize gene therapies to invasive brain tumors. However, like other stem cell-based therapies, survival of therapeutic NSCs after transplantation is currently suboptimal. One approach to prolonging cell survival is to transiently overexpress an antiapoptotic protein within the cells prior to transplantation. Here, we investigate the utility and safety of this approach using a clinically tested, v-myc immortalized, human NSC line engineered to contain the suicide gene, cytosine deaminase (CD-NSCs). We demonstrate that both adenoviral- and minicircle-driven expression of the antiapoptotic protein Bcl-2 can partially rescue CD-NSCs from transplant-associated insults. We further demonstrate that the improved CD-NSC survival afforded by transient Bcl-2 overexpression results in decreased tumor burden in an orthotopic xenograft glioma mouse model following administrations of intracerebral CD-NSCs and systemic prodrug. Importantly, no evidence of CD-NSC transformation was observed upon transient overexpression of Bcl-2. This research highlights a critical need to develop clinically relevant strategies to improve survival of therapeutic stem cell posttransplantation. We demonstrate for the first time in this disease setting that improving CD-NSC survival using Bcl-2 overexpression can significantly improve therapeutic outcomes.

Intraperitoneal Administration of Neural Stem Cell-Nanoparticle Conjugates Targets Chemotherapy to Ovarian Tumors.

Ovarian cancer is particularly aggressive once it has metastasized to the abdominal cavity (stage III). Intraperitoneal (IP) as compared to intravenous (IV) administration of chemotherapy improves survival for stage III ovarian cancer, demonstrating that concentrating chemotherapy at tumor sites has therapeutic benefit; unfortunately, IP therapy also increases toxic side effects, thus preventing its completion in many patients. The ability to target chemotherapy selectively to ovarian tumors while sparing normal tissue would improve efficacy and decrease toxicities. We have previously shown that tumor-tropic neural stem cells (NSCs) dramatically improve the intratumoral distribution of nanoparticles (NPs) when given intracerebrally near an orthotopic brain tumor or into a flank xenograft tumor. Here, we show that NPs either conjugated to the surface of NSCs or loaded within the cells are selectively delivered to and distributed within ovarian tumors in the abdominal cavity following IP injection, with no evidence of localization to normal tissue. IP administration is significantly more effective than IV administration, and NPs carried by NSCs show substantially deeper penetration into tumors than free NPs. The NSCs and NPs target and localize to ovarian tumors within 1 h of administration. Pt-loaded silica NPs (SiNP[Pt]) were developed that can be transported in NSCs, and it was found that the NSC delivery of SiNP[Pt] (NSC-SiNP[Pt]) results in higher levels of Pt in tumors as compared to free drug or SiNP[Pt]. To the best of our knowledge, this work represents the first demonstration that cells given IP can target the delivery of drug-loaded NPs.