Systemic and local immunosuppression in glioblastoma and its prognostic significance.

The effectiveness of tumor therapy, especially immunotherapy and oncolytic virotherapy, critically depends on the activity of the host immune cells. However, various local and systemic mechanisms of immunosuppression operate in cancer patients. Tumor-associated immunosuppression involves deregulation of many components of immunity, including a decrease in the number of T lymphocytes (lymphopenia), an increase in the levels or ratios of circulating and tumor-infiltrating immunosuppressive subsets [e.g., macrophages, microglia, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs)], as well as defective functions of subsets of antigen-presenting, helper and effector immune cell due to altered expression of various soluble and membrane proteins (receptors, costimulatory molecules, and cytokines). In this review, we specifically focus on data from patients with glioblastoma/glioma before standard chemoradiotherapy. We discuss glioblastoma-related immunosuppression at baseline and the prognostic significance of different subsets of circulating and tumor-infiltrating immune cells (lymphocytes, CD4+ and CD8+ T cells, Tregs, natural killer (NK) cells, neutrophils, macrophages, MDSCs, and dendritic cells), including neutrophil-to-lymphocyte ratio (NLR), focus on the immune landscape and prognostic significance of isocitrate dehydrogenase (IDH)-mutant gliomas, proneural, classical and mesenchymal molecular subtypes, and highlight the features of immune surveillance in the brain. All attempts to identify a reliable prognostic immune marker in glioblastoma tissue have led to contradictory results, which can be explained, among other things, by the unprecedented level of spatial heterogeneity of the immune infiltrate and the significant phenotypic diversity and (dys)functional states of immune subpopulations. High NLR is one of the most repeatedly confirmed independent prognostic factors for shorter overall survival in patients with glioblastoma and carcinoma, and its combination with other markers of the immune response or systemic inflammation significantly improves the accuracy of prediction; however, more prospective studies are needed to confirm the prognostic/predictive power of NLR. We call for the inclusion of dynamic assessment of NLR and other blood inflammatory markers (e.g., absolute/total lymphocyte count, platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, systemic immune-inflammation index, and systemic immune response index) in all neuro-oncology studies for rigorous evaluation and comparison of their individual and combinatorial prognostic/predictive significance and relative superiority.

The need for paradigm shift: prognostic significance and implications of standard therapy-related systemic immunosuppression in glioblastoma for immunotherapy and oncolytic virotherapy.

Despite significant advances in our knowledge regarding the genetics and molecular biology of gliomas over the past two decades and hundreds of clinical trials, no effective therapeutic approach has been identified for adult patients with newly diagnosed glioblastoma, and overall survival remains dismal. Great hopes are now placed on combination immunotherapy. In clinical trials, immunotherapeutics are generally tested after standard therapy (radiation, temozolomide, and steroid dexamethasone) or concurrently with temozolomide and/or steroids. Only a minor subset of patients with progressive/recurrent glioblastoma have benefited from immunotherapies. In this review, we comprehensively discuss standard therapy-related systemic immunosuppression and lymphopenia, their prognostic significance, and the implications for immunotherapy/oncolytic virotherapy. The effectiveness of immunotherapy and oncolytic virotherapy (viro-immunotherapy) critically depends on the activity of the host immune cells. The absolute counts, ratios, and functional states of different circulating and tumor-infiltrating immune cell subsets determine the net immune fitness of patients with cancer and may have various effects on tumor progression, therapeutic response, and survival outcomes. Although different immunosuppressive mechanisms operate in patients with glioblastoma/gliomas at presentation, the immunological competence of patients may be significantly compromised by standard therapy, exacerbating tumor-related systemic immunosuppression. Standard therapy affects diverse immune cell subsets, including dendritic, CD4+, CD8+, natural killer (NK), NKT, macrophage, neutrophil, and myeloid-derived suppressor cell (MDSC). Systemic immunosuppression and lymphopenia limit the immune system's ability to target glioblastoma. Changes in the standard therapy are required to increase the success of immunotherapies. Steroid use, high neutrophil-to-lymphocyte ratio (NLR), and low post-treatment total lymphocyte count (TLC) are significant prognostic factors for shorter survival in patients with glioblastoma in retrospective studies; however, these clinically relevant variables are rarely reported and correlated with response and survival in immunotherapy studies (e.g., immune checkpoint inhibitors, vaccines, and oncolytic viruses). Our analysis should help in the development of a more rational clinical trial design and decision-making regarding the treatment to potentially improve the efficacy of immunotherapy or oncolytic virotherapy.

Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing ß-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to ß-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a "Trojan horse".

In Vivo Tracking for Oncolytic Adenovirus Interactions with Liver Cells.

Hepatotoxicity remains an as yet unsolved problem for adenovirus (Ad) cancer therapy. The toxic effects originate both from rapid Kupffer cell (KCs) death (early phase) and hepatocyte transduction (late phase). Several host factors and capsid components are known to contribute to hepatotoxicity, however, the complex interplay between Ad and liver cells is not fully understood. Here, by using intravital microscopy, we aimed to follow the infection and immune response in mouse liver from the first minutes up to 72 h post intravenous injection of three Ads carrying delta-24 modification (Ad5-RGD, Ad5/3, and Ad5/35). At 15-30 min following the infusion of Ad5-RGD and Ad5/3 (but not Ad5/35), the virus-bound macrophages demonstrated signs of zeiosis: the formation of long-extended protrusions and dynamic membrane blebbing with the virus release into the blood in the membrane-associated vesicles. Although real-time imaging revealed interactions between the neutrophils and virus-bound KCs within minutes after treatment, and long-term contacts of CD8+ T cells with transduced hepatocytes at 24-72 h, depletion of neutrophils and CD8+ T cells affected neither rate nor dynamics of liver infection. Ad5-RGD failed to complete replicative cycle in hepatocytes, and transduced cells remained impermeable for propidium iodide, with a small fraction undergoing spontaneous apoptosis. In Ad5-RGD-immune mice, the virus neither killed KCs nor transduced hepatocytes, while in the setting of hepatic regeneration, Ad5-RGD enhanced liver transduction. The clinical and biochemical signs of hepatotoxicity correlated well with KC death, but not hepatocyte transduction. Real-time in vivo tracking for dynamic interactions between virus and host cells provides a better understanding of mechanisms underlying Ad-related hepatotoxicity.

Superior infectivity of the fiber chimeric oncolytic adenoviruses Ad5/35 and Ad5/3 over Ad5-delta-24-RGD in primary glioma cultures.

Ad5-delta-24-RGD is currently the most clinically advanced recombinant adenovirus (rAd) for glioma therapy. We constructed a panel of fiber-modified rAds (Ad5RGD, Ad5/3, Ad5/35, Ad5/3RGD, and Ad5/35RGD, all harboring the delta-24 modification) and compared their infectivity, replication, reproduction, and cytolytic efficacy in human and rodent glioma cell lines and short-term cultures from primary gliomas. In human cells, both Ad5/35-delta-24 and Ad5/3-delta-24 displayed superior infectivity and cytolytic efficacy over Ad5-delta-24-RGD, while Ad5/3-delta-24-RGD and Ad5/35-delta-24-RGD did not show further improvements in efficacy. The expression of the adenoviral receptors/coreceptors CAR, DSG2, and CD46 and the integrins αVß3/αVß5 did not predict the relative cytolytic efficacy of the fiber-modified rAds. The cytotoxicity of the fiber-modified rAds in human primary normal cultures of different origins and in primary glioma cultures was comparable, indicating that the delta-24 modification did not confer tumor cell selectivity. We also revealed that CT-2A and GL261 glioma cells might be used as murine cell models for the fiber chimeric rAds in vitro and in vivo. In GL261 tumor-bearing mice, Ad5/35-delta-24, armed with the immune costimulator OX40L as the E2A/DBP-p2A-mOX40L fusion, produced long-term survivors, which were able to reject tumor cells upon rechallenge. Our data underscore the potential of local Ad5/35-delta-24-based immunovirotherapy for glioblastoma treatment.

Study of the Therapeutic Efficiency of Transduced Olfactory Ensheathing Cells in Spinal Cord Cysts.

Posttraumatic spinal cord cysts are difficult to treat with medication and surgery. Gene-cell therapy is a promising area of treatment for such patients. However, optimal gene-cell construct for this therapy has not been developed. We investigated the therapeutic efficiency of human olfactory ensheathing cells (OECs) transduced by adenoviral vector encoding the mature form of brain-derived neurotrophic factor (mBDNF) in spinal cord cysts. The adenoviral vectors Ad5/35-CAG-mBDNF and Ad5/35-CAG-Fluc were constructed. Spinal cysts were modeled in female Wistar rats. We selected animals at the early and intermediate stages of recovery with scores to 13 according to the Basso, Beattie and Bresnahan (BBB) scale. The efficiency of therapy was evaluated by BBB tests. No cytotoxicity was detected using the Resazurin/AlamarBlue assay for both vectors at multiplicity of infection (MOIs) of 1, 5, and 25. There was an increase in the proliferation of cells treated with Ad5/35-CAG-mBDNF at MOIs of 5 and 25. The hind limb mobility after the transplantation of Ad5/35-CAG-mBDNF- and Ad5/35-CAG-Fluc-transduced human OECs and nontransduced OECs had approximately the same tendency to improve. Cyst reduction was observed with the transplantation of all the samples. Although Ad5/35-CAG-mBDNF-transduced OECs had high BDNF expression levels in vitro, these cells lacked positive effect in vivo because they did not exhibit significant effect concerning functional test when comparing the groups that received the same numbers of OECs. The therapeutic efficiency of transduced OECs appears to be due to the cell component. The autological and tissue-specific human OECs are promising for the personalized cell therapy. It is extremely important to test new gene-cell constructs based on these cells for further clinical use.

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation.

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

Recombinant Adenoviruses for Delivery of Therapeutics Following Spinal Cord Injury.

The regeneration of nerve tissue after spinal cord injury is a complex and poorly understood process. Medication and surgery are not very effective treatments for patients with spinal cord injuries. Gene therapy is a popular approach for the treatment of such patients. The delivery of therapeutic genes is carried out in a variety of ways, such as direct injection of therapeutic vectors at the site of injury, retrograde delivery of vectors, and ex vivo therapy using various cells. Recombinant adenoviruses are often used as vectors for gene transfer. This review discusses the advantages, limitations and prospects of adenovectors in spinal cord injury therapy.