Gene Editing/Gene Therapies: CHALLENGE TO DEVELOP GMP COMPLIANT MANUFACTURING PROCESS FOR COXSACKIE VIRUS TYPE B3

Background & Aim: Gene therapies has become recognized for its remarkable clinical benefits in a variety of medical applications, in particular recent approval of an Ad vector-based COVID-19 vaccines have attracted recent global attention. Here, we present key considerations for GMP compliant process development for Coxsackie virus type B3 (CVB3), an oncolytic virus designed for clinical trial in triple-negative breast cancer. Methods, Results & Conclusion(s): CVB3 is a non-enveloped, linear single-strand RNA virus with a size of approximately 27-33 um in diameter. From the initial type using the zonal rotor centrifuge to the advanced type using the tangential flow filtration system and ion chromatograph, we considered the points of the design concept in constructing the manufacturing process. The final design system is constructed as a closed and single-use manufacturing system in which all processes from upstream large-scale cell culture to downstream target purification and concentration steps. In brief, HEK293 cell suspension extended in 3L serum-free medium infected with CVB3, up to 3.6 times 10 to 7 of TCID50 /mL before going to downstream steps, made total 150 mL of final products as 8.43 times 10 to 7 of TCID50/mL concentration. Although further quality control challenges remain that is removal of product-related impurities such as human cellular proteins and residual DNA/RNA to increase virus purity, this concept is effectively applicable even for other types of viruses as GMP manufacturing processes, and would be also important for technology transfer to future commercial production.Copyright © 2023 International Society for Cell & Gene Therapy

Deciphering the Relationship between SARS-CoV-2 and Cancer.

Some viruses are known to be associated with the onset of specific cancers. These microorganisms, oncogenic viruses or oncoviruses, can convert normal cells into cancer cells by modulating the central metabolic pathways or hampering genomic integrity mechanisms, consequently inhibiting the apoptotic machinery and/or enhancing cell proliferation. Seven oncogenic viruses are known to promote tumorigenesis in humans: human papillomavirus (HPV), hepatitis B and C viruses (HBV, HCV), Epstein-Barr virus (EBV), human T-cell leukemia virus 1 (HTLV-1), Kaposi sarcoma-associated herpesvirus (KSHV), and Merkel cell polyomavirus (MCPyV). Recent research indicates that SARS-CoV-2 infection and COVID-19 progression may predispose recovered patients to cancer onset and accelerate cancer development. This hypothesis is based on the growing evidence regarding the ability of SARS-CoV-2 to modulate oncogenic pathways, promoting chronic low-grade inflammation and causing tissue damage. Herein, we summarize the main relationships known to date between virus infection and cancer, providing a summary of the proposed biochemical mechanisms behind the cellular transformation. Mechanistically, DNA viruses (such as HPV, HBV, EBV, and MCPyV) encode their virus oncogenes. In contrast, RNA viruses (like HCV, HTLV-1) may encode oncogenes or trigger host oncogenes through cis-/-trans activation leading to different types of cancer. As for SARS-CoV-2, its role as an oncogenic virus seems to occur through the inhibition of oncosuppressors or controlling the metabolic and autophagy pathways in the infected cells. However, these effects could be significant in particular scenarios like those linked to severe COVID-19 or long COVID. On the other hand, looking at the SARS-CoV-2─cancer relationship from an opposite perspective, oncolytic effects and anti-tumor immune response were triggered by SARS-CoV-2 infection in some cases. In summary, our work aims to recall comprehensive attention from the scientific community to elucidate the effects of SARS-CoV-2 and, more in general, ß-coronavirus infection on cancer susceptibility for cancer prevention or supporting therapeutic approaches.

SARS-CoV-2 as an Oncolytic Virus Following Reactivation of the Immune System: A Review.

The effects SARS-CoV-2 inflicts on human physiology, especially in patients who developed COVID-19, can range from flu-like symptoms to death, and although many lives have been lost during the pandemic, others have faced the resolution of aggressive neoplasms that once proclaimed a poor prognosis following traditional treatments. The purpose of this review was to analyze several fortunate case reports and their associated biomolecular pathways to further explore new avenues that might provide oncological treatments in the future of medicine. We included papers that discussed cases in which patients affected by COVID-19 suffered beneficial changes in their cancer status. Multiple mechanisms which elicited a reactivation of the host's immune system included cross-reactivity with viral antigens and downregulation of neoplastic cells. We were able to identify important cases presenting the resolution/remission of different aggressive neoplasms, for which most of the time, standard-of-care treatments offered little to no prospect towards a cure. The intricacy of the defense mechanisms humans have adopted against cancer cells through the millennia are still not well understood, but SARS-CoV-2 has demonstrated that the same ruinous cytokine storm which has taken so many lives can paradoxically be the answer we have been looking for to recalibrate the immunological system to retarget and vanquish malignancies.

Phage-Derived Oncolytic Viruses with 3C from Seneca Valley Virus for Targeted Therapy of Cervical Cancer

Cancer gene therapy based on various gene delivery vectors has some potential but also has obvious disadvantages. In this study, a new M13 phage-based oncolytic virus is constructed that carried the RGD peptides to target tumor cells and the 3C gene of Seneca Valley virus (SVV) preceded by a eukaryotic initial transcriptional region (ITR) to transcribe an oncolytic protein to kill tumor cells. Recombinant virus particles of 1200 nm in length are obtained in large quantities by transfecting the recombinant M13 phage plasmid into the host BL2738 and are investigated in vitro in tumor cells and in vivo in tumor-bearing mice to evaluate their antitumor effect. The experiments using Hela cells confirm that the engineered M13 phage can target and enter Hela cells, and express the SVV 3C protein, resulting in apoptosis of target cells by upregulating the expression of caspase 3. Furthermore, the results of experiments in vivo also show that the recombinant phage significantly inhibits the enhanced tumor volume in nude mice compared to the control groups. The M13 phage may be engineered to fuse with a variety of oncolytic proteins to inhibit the growth of tumor cells in the future, providing a promising phage-based targeted oncolytic reagent.

Phase I study of VSV-GP (BI 1831169) as monotherapy or combined with ezabenlimab in advanced and refractory solid tumors.

Patients with advanced, recurrent or metastatic cancer have poor prognosis despite treatment advancements. Vesicular stomatitis virus (VSV)-glycoprotein (GP; BI 1831169) is a chimeric VSV with its neurotropic glycoprotein G replaced by the non-neurotropic GP of the lymphocytic choriomeningitis virus. This live, recombinant oncolytic virus has demonstrated preclinical efficacy as a viral-based immunotherapy due to its interferon-dependent tumor specificity, potent oncolysis and stimulation of antitumor immune activity. Co-administration of the immune checkpoint inhibitor, ezabenlimab (BI 754091), alongside VSV-GP may synergistically enhance antitumor immune activity. Here, we describe the rationale and design of the first-in-human, phase I, dose-escalation study of VSV-GP alone and in combination with the immune checkpoint inhibitor ezabenlimab in patients with advanced, metastatic or relapsed and refractory solid tumors (NCT05155332).

There is a need to develop new treatments for people living with cancer. Immunotherapy is a type of medicine that works by helping the body's natural defenses, known as the immune system, to destroy cancer cells. There are different types of immunotherapies such as oncolytic viruses (OVs) and immune checkpoint inhibitors (ICIs). OVs are viruses that may help destroy cancer cells while leaving normal cells unharmed. They work by replicating within cancer cells; this causes them to burst and release more of the virus which then infects nearby cancer cells and activates the body's immune system. ICIs may be able to work together with OVs to amplify this effect. Vesicular stomatitis virus (VSV)-glycoprotein (GP) is a type of OV that has been shown to effectively destroy cancer cells in animal studies. This first-in-human study will investigate VSV-GP on its own and in combination with an ICI called ezabenlimab for the treatment of late-stage cancer or cancer that has spread to multiple parts of the body. Here, we describe the background and design of this study in progress which aims to find out if VSV-GP alone or in combination with ezabenlimab is effective against cancer, the suitable dose and if any side effects occur. Trial Registration Number: NCT05155332 (ClinicalTrials.gov).

Correlation of SARS­CoV­2 to cancer: Carcinogenic or anticancer? (Review).

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) is highly infectious and pathogenic. Among patients with severe SARS­CoV­2­caused by corona virus disease 2019 (COVID­19), those complicated with malignant tumor are vulnerable to COVID­19 due to compromised immune function caused by tumor depletion, malnutrition and anti­tumor treatment. Cancer is closely related to the risk of severe illness and mortality in patients with COVID­19. SARS­CoV­2 could promote tumor progression and stimulate metabolism switching in tumor cells to initiate tumor metabolic modes with higher productivity efficiency, such as glycolysis, for facilitating the massive replication of SARS­CoV­2. However, it has been shown that infection with SARS­CoV­2 leads to a delay in tumor progression of patients with natural killer cell (NK cell) lymphoma and Hodgkin's lymphoma, while SARS­CoV­2 elicited anti­tumor immune response may exert a potential oncolytic role in lymphoma patients. The present review briefly summarized potential carcinogenicity and oncolytic characteristics of SARS­CoV­2 as well as strategies to protect patients with cancer during the COVID­19 pandemic.

Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy.

Gene therapy is currently in the public spotlight. Several gene therapy products, including oncolytic virus (OV), which predominantly replicates in and kills cancer cells, and COVID-19 vaccines have recently been commercialized. Recombinant adenoviruses, including replication-defective adenoviral vector and conditionally replicating adenovirus (CRA; oncolytic adenovirus), have been extensively studied and used in clinical trials for cancer and vaccines. Here, we review the biology of wild-type adenoviruses, the methodological principle for constructing recombinant adenoviruses, therapeutic applications of recombinant adenoviruses, and new technologies in pluripotent stem cell (PSC)-based regenerative medicine. Moreover, this article describes the technology platform for efficient construction of diverse "CRAs that can specifically target tumors with multiple factors" (m-CRAs). This technology allows for modification of four parts in the adenoviral E1 region and the subsequent insertion of a therapeutic gene and promoter to enhance cancer-specific viral replication (i.e., safety) as well as therapeutic effects. The screening study using the m-CRA technology successfully identified survivin-responsive m-CRA (Surv.m-CRA) as among the best m-CRAs, and clinical trials of Surv.m-CRA are underway for patients with cancer. This article also describes new recombinant adenovirus-based technologies for solving issues in PSC-based regenerative medicine.

Complete response in a frail patient with high-grade B-cell lymphoma to only one cycle of R-CHOP or to prolonged COVID-19?

BACKGROUND: COVID-19 infection increases mortality in hematological malignancies. In a large meta-analysis, patients aged 60 years and older had a significantly higher risk of death than patients under 60 years of age [1]. Furthermore, a high risk of death and reduced survival in patients receiving B cell depletion therapy with prolonged COVID-19 infection was reported in a recent study [2]. High-grade B-cell lymphomas are classified as morphologically aggressive lymphomas with the presence of a high mitotic index and Ki-67 proliferation rates. They demonstrate aggressive behavior clinically as well as morphologically, and COVID-19 infection is an important factor that increases mortality in these patients. Herein, we present an elderly patient with a diagnosis of high-grade B-cell lymphoma, in whom a complete response was observed after prolonged COVID-19 infection. CASE SUMMARY: An 81-year-old female patient received her first cycle of R-CHOP (rituximab, cyclophosphamide, vincristine, and prednisolone) treatment after being diagnosed with high- grade B-cell lymphoma. After being discharged from the hospital, the patient was referred to the emergency department with complaints of fever and fatigue when she came for the second cycle of chemotherapy. Her COVID-19 PCR test was found positive. She was admitted to the infectious diseases service and favipiravir treatment was started. On the 24th day of hospitalization, it was decided to perform interim FDG-PET/CT (Fluorodeoxyglucose - Positron Emission Tomography/Computed Tomography) scan at a time that her PCR (Polymerase Chain Reaction) test was still positive. A complete metabolic response was detected in her imaging. On the 26th day, the PCR test became negative and the patient was transferred to the oncology service and received the second cycle of R-CHOP treatment. CONCLUSION: Our case emphasizes that antitumor effect could be seen in a patient with SARS-CoV-2 infection and a hematologic malignancy. It also highlights being alert to prolonged COVID-19 infection in patients receiving B-cell depletion therapy.

Real-time PCR assay as a simple and efficient tool for viral stability study.

Aim: For oncolytic virus trials, regulatory agencies often require pharmaceutical industry to evaluate risks of released viruses from patients to environment. This study was to establish a real-time PCR method to assess viral shedding and viral stability in human urine. Results/methodology: Herein, we describe an incubation of viral drug product in human urine and use of real-time PCR as a simple, efficient and high throughput assay to assess the level and stability of a nonenveloped and single stranded RNA virus. The viral stability issue is critical to the collection, transport, storage and testing of clinical samples. Discussion/conclusion: In summary, this simple method provides useful viral stability information at various temperatures and detergents. A similar approach may apply to other RNA viruses (including SARS-CoV-2).

New viral vectors for infectious diseases and cancer.

Since the discovery in 1796 by Edward Jenner of vaccinia virus as a way to prevent and finally eradicate smallpox, the concept of using a virus to fight another virus has evolved into the current approaches of viral vectored genetic vaccines. In recent years, key improvements to the vaccinia virus leading to a safer version (Modified Vaccinia Ankara, MVA) and the discovery that some viruses can be used as carriers of heterologous genes encoding for pathological antigens of other infectious agents (the concept of 'viral vectors') has spurred a new wave of clinical research potentially providing for a solution for the long sought after vaccines against major diseases such as HIV, TB, RSV and Malaria, or emerging infectious diseases including those caused by filoviruses and coronaviruses. The unique ability of some of these viral vectors to stimulate the cellular arm of the immune response and, most importantly, T lymphocytes with cell killing activity, has also reawakened the interest toward developing therapeutic vaccines against chronic infectious diseases and cancer. To this end, existing vectors such as those based on Adenoviruses have been improved in immunogenicity and efficacy. Along the same line, new vectors that exploit viruses such as Vesicular Stomatitis Virus (VSV), Measles Virus (MV), Lymphocytic choriomeningitis virus (LCMV), cytomegalovirus (CMV), and Herpes Simplex Virus (HSV), have emerged. Furthermore, technological progress toward modifying their genome to render some of these vectors incompetent for replication has increased confidence toward their use in infant and elderly populations. Lastly, their production process being the same for every product has made viral vectored vaccines the technology of choice for rapid development of vaccines against emerging diseases and for 'personalised' cancer vaccines where there is an absolute need to reduce time to the patient from months to weeks or days. Here we review the recent developments in viral vector technologies, focusing on novel vectors based on primate derived Adenoviruses and Poxviruses, Rhabdoviruses, Paramixoviruses, Arenaviruses and Herpesviruses. We describe the rationale for, immunologic mechanisms involved in, and design of viral vectored gene vaccines under development and discuss the potential utility of these novel genetic vaccine approaches in eliciting protection against infectious diseases and cancer.