Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

Download This Paper Open PDF in Browser Add Paper to My Library Share: Permalink Using these links will ensure access to this page indefinitely Copy URL Immunogenicity of a SARS-CoV-2 DNA Vaccine Formulated With the Fusion-Associated Small Transmembrane Protein Proteolipid Vehicle Delivery System Cell 49 Pages Posted: 1 Nov 2022 Publication Status: Under Review See all articles by Arun RaturiArun RaturiEntos Pharmaceuticals; University of Alberta - Department of OncologyJailal AblackOncoSenXPing WeeEntos PharmaceuticalsPrakash BhandariEntos PharmaceuticalsDouglas W. BrownUniversity of Alberta - Department of OncologyMaryam HejaziUniversity of Alberta - Department of OncologyNichole McMullenDalhousie University - Department of Microbiology & ImmunologyLiliya GrinEntos PharmaceuticalsHector VegaEntos PharmaceuticalsHenry GarciaOisin BiotechnologiesNatasha GovindasamyEntos PharmaceuticalsJitendra KumarEntos PharmaceuticalsPaola Solis AresEntos PharmaceuticalsChandra McAllisterEntos PharmaceuticalsKatia Carmine-SimmenUniversity of Alberta - Department of OncologyPerrin H. BeattyEntos PharmaceuticalsAdam NelsonDalhousie University - Department of Microbiology & ImmunologyEric S. PringleDalhousie University - Department of Microbiology & ImmunologyThornton ThompsonAegis LifeManoj ParmarEntos PharmaceuticalsJennifer GyobaEntos PharmaceuticalsHong JiangAegis LifeBrent JohnstonDalhousie University - Department of Microbiology & ImmunologyCraig McCormickDalhousie University - Department of Microbiology & ImmunologyMary FoleyDalhousie University - Department of Microbiology & ImmunologyMagen Ellen FrancisUniversity of Saskatchewan - Vaccine and Infectious Disease OrganizationBrian AbelAegis LifeAlyson KelvinUniversity of Saskatchewan - Vaccine and Infectious Disease OrganizationRoy DuncanDalhousie University - Department of Microbiology & ImmunologyJohn D. LewisUniversity of Alberta - Department of Oncology More... Abstract DNA vaccines have had limited success in the clinic despite decades of research and development, owing primarily to challenges in delivery. Here we report the generation of DNA vaccine candidates against SARS-CoV-2 utilizing an intracellular nucleic acid delivery platform, FAST-PLV, where plasmid DNA is encapsulated in proteolipid vehicles formulated with a fusion-associated small transmembrane protein and well-tolerated lipids utilizing a scalable microfluidic approach. Selected SARS-CoV2 vaccine candidates comprising full-length SARS-CoV-2 Spike protein combined with two genetic adjuvants (CpG motifs, RIG-I agonist, termed NP-S) elicited potent neutralizing antibody responses in mice and non-human primates at low doses. The NP-S vaccine stimulated significant spike-specific T cell responses including functional cytotoxic T lymphocyte responses. A single dose of NP-S protected hamsters from morbidity following SARS-CoV-2 challenge. The FAST-PLV vaccine platform enables efficient intracellular delivery of DNA payloads and is ideally suited for the development of fridge-stable genetic vaccines against emerging infections.Funding Information: Supported by an operating grant to JDL from the Canadian Institutes of Health Research (CIHR), in partnership with the Institute of Aging and Research Nova Scotia (reference number VR1- 172710), and by operating grants to RD from the CIHR and the Natural Sciences and Engineering Research Council of Canada (NSERC). JDL holds the Frank and Carla Sojonky Chair in Prostate Cancer Research at the University of Alberta, supported by the Alberta Cancer Foundation. RD holds the Killam Chair in Virology at Dalhousie University. DB was funded by fellowships from Prostate Cancer Canada and Mitacs. ESP was funded by postdoctoral fellowships from Mitacs and the Dalhousie Medical Research Foundation. AN was funded by a Mitacs Industrial Studentship. MH was funded by a Mitacs Industrial Postdoctoral Fellowship.Declaration of Interests: The following authors have no competing interests:Nichole McMullen, Katia Carmine-Simmen, Adam Nelson, Eric S. Pringle, Manoj Parmar, Jennifer Gyoba, Brent Johnston, Craig McCormick, Mary Foley, Magen Ellen Francis, Alyson Kelvin, Henry GarciaThe following authors are employees/shareholders of Entos Pharmaceuticals and/or Aegis Life: Arun Raturi, Jailal Ablack, Ping Wee, Prakash Bhandari, Douglas Brown, Maryam Hejazi, Liliya Grin, Hector Vega, Natasha Govindasamy, Jitendra Kumar, Paola Solis Ares, Chandra McAllister, Perrin H. Beatty, Thornton Thompson, Hong Jiang, Brian Abel, Roy Duncan, John D. LewisEthics Approval Statement: All rodent studies were carried out according to the Canadian Council on Animal Care (CCAC) guidelines and approved by the University of Alberta Animal Care and Use Committee.All in-life NHP procedures were carried out by Virscio, Inc, under the guidance of the Institutional Animal Care and Use Committee (IACUC) of the St. Kitts Biomedical Research Foundation (SKBRF), St Kitts, West Indies. SKBRF research facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). African green monkeys (Chlorocebus sabaeus) are an invasive species on the island of St. Kitts and were procured locally using approved practices with IACUC oversight. Keywords: CD8+ T cells, cytotoxic T lymphocytes, FAST protein, genetic adjuvants, neutralizing antibodies, plasmid DNA vaccine, proteolipid vehicle, PLV, SARS-CoV-2, Spike protein Suggested Citation: Suggested Citation Raturi, Arun and Ablack, Jailal and Wee, Ping and Bhandari, Prakash and Brown, Douglas W. and Hejazi, Maryam and McMullen, Nichole and Grin, Liliya and Vega, Hector and Garcia, Henry and Govindasamy, Natasha and Kumar, Jitendra and Ares, Paola Solis and McAllister, Chandra and Carmine-Simmen, Katia and Beatty, Perrin H. and Nelson, Adam and Pringle, Eric S. and Thompson, Thornton and Parmar, Manoj and Gyoba, Jennifer and Jiang, Hong and Johnston, Brent and McCormick, Craig and Foley, Mary and Francis, Magen Ellen and Abel, Brian and Kelvin, Alyson and Duncan, Roy and Lewis, John D., Immunogenicity of a SARS-CoV-2 DNA Vaccine Formulated With the Fusion-Associated Small Transmembrane Protein Proteolipid Vehicle Delivery System. Available at SSRN: https://ssrn.com/abstract=4241174 This version of the paper has not been formally peer reviewed. Arun Raturi Entos Pharmaceuticals ( email ) University of Alberta - Department of Oncology ( email ) Jailal Ablack OncoSenX ( email ) Ping Wee Entos Pharmaceuticals ( email ) Prakash Bhandari Entos Pharmaceuticals ( email ) Douglas W. Brown University of Alberta - Department of Oncology ( email ) Maryam Hejazi University of Alberta - Department of Oncology ( email ) Nichole McMullen Dalhousie University - Department of Microbiology & Immunology ( email ) Liliya Grin Entos Pharmaceuticals ( email ) Hector Vega Entos Pharmaceuticals ( email ) Henry Garcia Oisin Biotechnologies ( email ) Natasha Govindasamy Entos Pharmaceuticals ( email ) Jitendra Kumar Entos Pharmaceuticals ( email ) Paola Solis Ares Entos Pharmaceuticals ( email ) Chandra McAllister Entos Pharmaceuticals ( email ) Katia Carmine-Simmen University of Alberta - Department of Oncology ( email ) Perrin H. Beatty Entos Pharmaceuticals ( email ) Adam Nelson Dalhousie University - Department of Microbiology & Immunology ( email ) Eric S. Pringle Dalhousie University - Department of Microbiology & Immunology ( email ) Thornton Thompson Aegis Life ( email ) Manoj Parmar Entos Pharmaceuticals ( email ) Jennifer Gyoba Entos Pharmaceuticals ( email ) Hong Jiang Aegis Life ( email ) Brent Johnston Dalhousie University - Department of Microbiology & Immunology ( email ) Craig McCormick Dalhousie University - Department of Microbiology & Immunology ( email ) Mary Foley Dalhousie University - Department of Microbiology & Immunology ( email ) Magen Ellen Francis University of Saskatchewan - Vaccine and Infectious Disease Organization ( email ) Brian Abel Aegis Life ( email ) Alyson Kelvin University of Saskatchewan - Vaccine and Infectious Disease Organization ( email ) Roy Duncan Dalhousie University - Department of Microbiology & Immunology ( email ) John D. Lewis (Contact Author) University of Alberta - Department of Oncology ( email ) Download This Paper Open PDF in Browser Please enable JavaScript to view the comments powered by Disqus. 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The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.

The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase, which allows the study of phase-specific cellular events. While interphase cells can be easily distinguished from mitotic cells by examining their chromosome morphology, it is much more difficult to separate and distinguish the interphases from each other. Here, we describe drug-derived protocols for synchronizing HeLa cells to various interphases of the cell cycle: G1 phase, S phase, and G2 phase. G1 phase synchronization is achieved through serum starvation, S phase synchronization is achieved through a double thymidine block, and G2 phase synchronization is achieved through the release of the double thymidine block followed by roscovitine treatment. Successful synchronization can be assessed using flow cytometry to examine the DNA content and Western blot to examine the expression of various cyclins.

Abstract While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative as patients will eventually become resistant. In this study, we sought to develop an effective suicide gene therapy approach for solid tumors that specifically exploits their unique transcriptional activation state. The tumor suppressor p53 is frequently mutated or dysregulated in cancer, and as a result the upstream signaling pathways activating p53 transcription are strongly upregulated. RNA-seq analysis has demonstrated that p53 transcription is significantly upregulated in almost all forms of cancer. Additionally, HCT116 cells lacking functional p53 display a 6-fold increase in p53 promoter activity when compared to its wild type p53 parent cell line. To exploit this, we have developed a Fusogenix FAST-LNP formulation to deliver a p53-driven inducible suicide gene, iCasp9, to solid tumors and destroy them upon activation with small molecule dimerizer, Rapamycin. To establish a proof-of-concept, plasmid encoding iCasp9 and luciferase under control of the p53 promoter was constructed and evaluated in a panel of cancer cell lines. While LNPs administered without Rapamycin or Rapamycin administered alone had no impact on cell viability, we observed greater than 90% apoptotic cell death when both were employed in a wide range of cancer cell lines with p53 deletions or mutations, as measured using cell viability assays, imaging assays, as well as Annexin V and TUNEL flow cytometry. Induction of iCasp9 protein expression and caspase-mediated apoptosis was confirmed using Western blot. No cell death was observed in cells with intact p53 such as human umbilical vein endothelial cells or the fibroblast cell line IMR-90. Next, we assessed the efficacy of FAST-LNPs containing p53-iCasp9 in xenograft PC-3 and H1299 models of human prostate cancer and lung cancer respectively. In some experiments, tumors were implanted subcutaneously in the flanks of 30 mice and allowed to grow to 500 mm3 before treatment by intravenous doses of 100 µg LNP twice per week during continuous low dosing of Rapamycin. We observed a rapid and dramatic reduction in tumor volume averaging 87% over the following 48 hours, with durable response. Tumors in control mice continued to grow exponentially. Overall survival of mice was extended 250% in the PC-3 cohort and 300% in the H1299 cohort. Optimization of number and concentration of LNP doses should allow for long term control of both localized and systemic disease. In conclusion, we describe a novel LNP gene therapy approach for the treatment of cancer with high selectivity for tumors with dysregulated p53 transcriptional activation. This approach has the potential to provide a highly efficacious alternative to current therapies for localized and advanced solid tumors. Citation Format: Douglas Wilson Brown, Arun Raturi, Prakash Bhandari, Deborah Sosnowski, Liliya Grin, Ping Wee, Hector Vega, Jennifer Gyoba, Maryam Hejazi, Jailal Ablack, Matthew Scholz, John D. Lewis. Selective ablation of solid tumors using a p53-targeted FAST-LNP gene therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4069.

Approaches to eliminate senescent cells in vivo using transgenic mouse models have demonstrated significant improvements in lifespan, reduction in cancer incidence, and amelioration of age-related degeneration. These approaches require, however, that the organism be genetically engineered from the embryo and/or repeatedly dosed for the organism’s lifespan, making them challenging to implement in humans using current technologies. To overcome these limitations, we developed a clinically viable senolytic gene therapy consisting of a suicide gene, inducible caspase 9 (iCasp9), under control of the early senescence and tumor suppressive p53 promoter or the late senescence p16Ink4a promoter. In vitro, this gene therapy selectively activates in senescent cells and induces caspase-9-dependent apoptosis. When formulated in the FAST-PLV platform and administered systemically to aged mice, the burden of senescent cells was significantly reduced in various tissues, leading to a 123% increase in post-treatment survival for animals given a combination of p16 and p53 targeted senolytic gene therapies. Treated mice showed significantly reduced frailty, increased physical function, and improved heart health. Gross necropsy indicated a 3-fold reduced tumor incidence. In summary, we demonstrate a novel and redosable senolytic genetic medicine approach that improves healthspan by targeting senescent cells based on their transcriptional activity.

Genetic medicines hold great promise despite limitations in the tolerability and immunogenicity of current delivery platforms. To address this, we have developed a proteolipid vehicle (PLV) incorporating fusion-associated small transmembrane (FAST) proteins from fusogenic orthoreoviruses into a non-toxic lipid formulation. A chimeric FAST protein with enhanced membrane fusion activity was identified. Lipid formulations incorporating the chimeric FAST protein were optimized for high nucleic acid encapsulation, charge neutralization, and improved tolerability in vitro and in vivo. FAST-PLVs administered systemically in mouse and non-human primate models demonstrated broad biodistribution and expression of mRNA and plasmid DNA. FAST-PLVs showed low immunogenicity and maintained activity upon repeat dosing. We utilized FAST-PLVs to deliver a pDNA follistatin gene therapy that increased circulating levels of follistatin, resulting in significantly increased muscle mass and grip strength. The biodistribution, activity and safety profile of FAST-PLVs make them a promising platform for redosable gene therapies and genetic medicines.

Our understanding of the mechanism of cell fate transition during the direct reprogramming of fibroblasts into various central nervous system (CNS) neural cell types has been limited by the lack of a comprehensive analysis on generated cells, independently and in comparison with other CNS neural cell types. Here, we applied an integrative approach on 18 independent high throughput expression data sets to gain insight into the regulation of the transcriptome during the conversion of fibroblasts into induced neural stem cells, induced neurons (iNs), induced astrocytes, and induced oligodendrocyte progenitor cells (iOPCs). We found common down-regulated genes to be mostly related to fibroblast-specific functions, and suggest their potential as markers for screening of the silencing of the fibroblast-specific program. For example, Tagln was significantly down-regulated across all considered data sets. In addition, we identified specific profiles of up-regulated genes for each CNS neural cell types, which could be potential markers for maturation and efficiency screenings. Furthermore, we identified the main TFs involved in the regulation of the gene expression program during direct reprogramming. For example, in the generation of iNs from fibroblasts, the Rest TF was the main regulator of this reprogramming. In summary, our computational approach for meta-analyzing independent expression data sets provides significant details regarding the molecular mechanisms underlying the regulation of the gene expression program, and also suggests potentially useful candidate genes for screening down-regulation of fibroblast gene expression profile, maturation, and efficiency, as well as candidate TFs for increasing the efficiency of the reprogramming process.

The overactivation of epidermal growth factor (EGF) receptor (EGFR) is implicated in various cancers. Endocytosis plays an important role in EGFR-mediated cell signaling. We previously found that EGFR endocytosis during mitosis is mediated differently from interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. By transfecting HeLa, MCF-7, and 293T cells with CBL siRNA or dominant-negative 70z-CBL, we found that at high EGF doses, CBL is required for EGFR endocytosis in mitotic cells, but not in interphase cells. In addition, the endocytosis of mutant EGFR Y1045F-YFP (mutation at the direct CBL binding site) is strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL is completely inhibited in mitosis. Moreover, EGF induces stronger ubiquitination of mitotic EGFR than interphase EGFR, and mitotic EGFR is trafficked to lysosomes for degradation. Furthermore, we showed that, different from interphase, low doses of EGF still stimulate EGFR endocytosis by non-clathrin mediated endocytosis (NCE) in mitosis. Contrary to interphase, CBL and the CBL-binding regions of EGFR are required for mitotic EGFR endocytosis at low doses. This is due to the mitotic ubiquitination of the EGFR even at low EGF doses. We conclude that mitotic EGFR endocytosis exclusively proceeds through CBL-mediated NCE.