P53 Gene Therapy Research Articles

From regulation to deregulation of p53 in hematologic malignancies: implications for diagnosis, prognosis and therapy.

The p53 protein, encoded by the TP53 gene, serves as a critical tumor suppressor, playing a vital role in maintaining genomic stability and regulating cellular responses to stress. Dysregulation of p53 is frequently observed in hematological malignancies, significantly impacting disease progression and patient outcomes. This review aims to examine the regulatory mechanisms of p53, the implications of TP53 mutations in various hematological cancers, and emerging therapeutic strategies targeting p53. We conducted a comprehensive literature review to synthesize recent findings related to p53's multifaceted role in hematologic cancers, focusing on its regulatory pathways and therapeutic potential. TP53 mutations in hematological malignancies often lead to treatment resistance and poor prognosis. Current therapeutic strategies, including p53 reactivation and gene therapy, show promise in improving treatment outcomes. Understanding the intricacies of p53 regulation and the consequences of its mutations is essential for developing effective diagnostic and therapeutic strategies in hematological malignancies, ultimately enhancing patient care and survival.

The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.

Evaluation of cyclooxygenase-2, P53, vascular endothelial growth factor, and nitric oxide synthase-2 in angiogenesis and growth of tobacco-related malignancies

Objectives: In India, tobacco consumption is responsible for half of all the cancers in men and a quarter in women. The present study focuses on the expression of cyclooxygenase-2 (COX-2), P53, vascular endothelial growth factor (VEGF), and nitric oxide synthase (NOS) and their relationship with the growth and angiogenesis of tobacco-related malignancies of the oral cavity, esophagus, lungs, and stomach. It further evaluates the carcinogenic action of nicotine and examines whether COX-2 and NOS-2 overexpression is responsible for tumor growth and associated angiogenic VEGF expression via its receptor. Materials and Methods: A cross-sectional study on 140 biopsies, resected specimens of cancer of oral cavity, esophagus, stomach, and lungs, was done. Immunohistochemical evaluation for p53, COX-2, VEGF, and inducible NOS was done. Relevant statistical analysis was applied for the significance of the findings. Statistical Analysis: Relevant statistical analysis was done using SPSS, chi-square and Fisher's exact tests were applied for the significance of the findings. A p-value of < 0.05 was considered as significant value. Results: Immunohistochemical evaluation of pattern of expression of COX-2, NOS-2, VEGF, and p53 was done in both tobacco- and nontobacco-associated cases. The results of the present study revealed an upregulation of COX-2, NOS-2, VEGF, and p53 in all the malignancies. Conclusion: The present results indicated that p53 protein accumulation and increased expression of COX-2, NOS-2, and VEGF might be responsible for carcinogenesis and tumor aggressiveness by enhancing angiogenesis. A possible significant effect of nicotine on COX-2 and P53 expression in tumorigenesis is revealed. These data might have important implications for the therapeutic use of COX-2, NOS-2, and VEGF inhibitors as well as of p53 gene therapy in future anticancer therapeutic strategies in tobacco-related malignancies.

P53-Bad* Fusion Gene Therapy Induces Apoptosis In Vitro and Reduces Zebrafish Tumor Burden in Hepatocellular Carcinoma.

With few curative treatments and a global yearly death rate of over 800,000, hepatocellular carcinoma (HCC) desperately needs new therapies. Although wild-type p53 gene therapy has been shown to be safe in HCC patients, it has not shown enough efficacy to merit approval. This work aims to show how p53 can be re-engineered through fusion to the pro-apoptotic BH3 protein Bcl-2 antagonist of cell death (Bad) to improve anti-HCC activity and potentially lead to a novel HCC therapeutic, p53-Bad*. p53-Bad* is a fusion of p53 and Bad, with two mutations, S112A and S136A. We determined mitochondrial localization of p53-Bad* in liver cancer cell lines with varying p53 mutation statuses via fluorescence microscopy. We defined the apoptotic activity of p53-Bad* in four liver cancer cell lines using flow cytometry. To determine the effects of p53-Bad* in vivo, we generated and analyzed transgenic zebrafish expressing hepatocyte-specific p53-Bad*. p53-Bad* localized to the mitochondria regardless of the p53 mutation status and demonstrated superior apoptotic activity over WT p53 in early, middle, and late apoptosis assays. Tumor burden in zebrafish HCC was reduced by p53-Bad* as measured by the liver-to-body mass ratio and histopathology. p53-Bad* induced significant apoptosis in zebrafish HCC as measured by TUNEL staining but did not induce apoptosis in non-HCC fish. p53-Bad* can induce apoptosis in a panel of liver cancer cell lines with varying p53 mutation statuses and induce apoptosis/reduce HCC tumor burden in vivo in zebrafish. p53-Bad* warrants further investigation as a potential new HCC therapeutic.

A Novel P53 Nanomedicine Reduces Immunosuppression and Augments Anti-PD-1 Therapy for Non-Small Cell Lung Cancer in Syngeneic Mouse Models.

Lung cancer is among the most common and lethal cancers and warrants novel therapeutic approaches to improving patient outcomes. Although immune checkpoint inhibitors (ICIs) have demonstrated substantial clinical benefits, most patients remain unresponsive to currently approved ICIs or develop resistance after initial response. Many ongoing clinical studies are investigating combination therapies to address the limited efficacy of ICIs. Here, we have assessed whether p53 gene therapy via a tumor-targeting nanomedicine (termed SGT-53) can augment anti-programmed cell death-1 (PD-1) immunotherapy to expand its use in non-responding patients. Using syngeneic mouse models of lung cancers that are resistant to anti-PD-1, we demonstrate that restoration of normal p53 function potentiates anti-PD-1 to inhibit tumor growth and prolong survival of tumor-bearing animals. Our data indicate that SGT-53 can restore effective immune responses against lung cancer cells by reducing immuno-suppressive cells (M2 macrophages and regulatory T cells) and by downregulating immunosuppressive molecules (e.g., galectin-1, a negative regulator of T cell activation and survival) while increasing activity of cytotoxic T cells. These results suggest that combining SGT-53 with anti-PD-1 immunotherapy could increase the fraction of lung cancer patients that responds to anti-PD-1 therapy and support evaluation of this combination particularly in patients with ICI-resistant lung cancers.

Red fluorescent AIEgens based multifunctional nonviral gene vectors for the efficient combination of gene therapy and photodynamic therapy in anti-cancer

Precise molecular engineering of AIEgens-based cationic delivery systems for high transfection efficiency (TE) and effective photodynamic therapy (PDT) holds a huge potential for cancer treatment. Herein, three amphiphiles (DT-C6/8/12-M) consisting of di(triazole-[12]aneN3) (M) and 1,1-dicyano-2-phenyl-2-(4-diphenylamino)phenyl-ethylene (DT) units have been developed to achieve luminescent tracking, efficient TE, and effective PDT in vitro and in vivo. These compounds exhibited strong aggregated induced emission (AIE) at 630 nm and mega Stokes shifts of up to 160 nm. They were able to bind DNA into nanoparticles with suitable sizes, positive surface potential, and good biocompatibility in the presence of DOPE. Among them, vector DT-C12-M/DOPE with n-dodecyl linker achieved a transfection efficiency as high as 42.3 folds that of Lipo2000 in PC-3 cell lines. DT-C12-M/DOPE exhibited the capability of successful endo/lysosomal escape and rapid nuclear delivery of pDNA, and the gene delivery process was clearly monitored via confocal laser scanning microscopy. Moreover, efficient reactive oxygen species (ROS) generation by DT-C12-M upon light irradiation led to effective PDT in vitro . We further show that combination of p53 gene therapy and PDT dramatically enhanced cancer therapeutic outcome in vivo. This “three birds, one stone” strategy offers a novel and promising approach for real-time tracking of gene delivery and better cancer treatment

Modulation of p53 expression in cancer-associated fibroblasts prevents peritoneal metastasis of gastric cancer

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are associated with the establishment and progression of peritoneal metastasis. This study investigated the efficacy of replicative oncolytic adenovirus-mediated p53 gene therapy (OBP-702) against CAFs and peritoneal metastasis of gastric cancer (GC). Higher CAF expression in the primary tumor was associated with poor prognosis of GC, and higher CAF expression was also observed with peritoneal metastasis in immunohistochemical analysis of clinical samples. And, we found transcriptional alteration of p53 in CAFs relative to normal gastric fibroblasts (NGFs). CAFs increased the secretion of cancer-promoting cytokines, including interleukin-6, and gained resistance to chemotherapy relative to NGFs. OBP-702 showed cytotoxicity to both GC cells and CAFs but not to NGFs. Overexpression of wild-type p53 by OBP-702 infection caused apoptosis and autophagy of CAFs and decreased the secretion of cancer-promoting cytokines by CAFs. Combination therapy using intraperitoneal administration of OBP-702 and paclitaxel synergistically inhibited the tumor growth of peritoneal metastases and decreased CAFs in peritoneal metastases. OBP-702, a replicative oncolytic adenovirus-mediated p53 gene therapy, offers a promising biological therapeutic strategy for peritoneal metastasis, modulating CAFs in addition to achieving tumor lysis.

Tumor suppressor immune gene therapy to reverse immunotherapy resistance

BackgroundWhile immune checkpoint inhibitors are becoming a standard of care for multiple types of cancer, the majority of patients do not respond to this form of immunotherapy. New approaches are required to overcome resistance to immunotherapies.MethodsWe investigated the effects of adenoviral p53 (Ad-p53) gene therapy in combination with immune checkpoint inhibitors and selective IL2 or IL15 CD122/132 agonists in the aggressive B16F10 tumor model resistant to immunotherapies. To assess potential mechanisms of action, pre- and post- Ad-p53 treatment biopsies were evaluated for changes in gene-expression profiles by Nanostring IO 360 assays.ResultsThe substantial synergy of “triplet” Ad-p53 + CD122/132 + anti-PD-1 therapy resulted in potential curative effects associated with the complete tumor remissions of both the primary and contralateral tumors. Interestingly, contralateral tumors, which were not injected with Ad-p53 showed robust abscopal effects resulting in statistically significant decreases in tumor size and increased survival (p < 0.001). None of the monotherapies or doublet treatments induced the complete tumor regressions. Ad-p53 treatment increased interferon, CD8+ T cell, immuno-proteosome antigen presentation, and tumor inflammation gene signatures. Ad-p53 treatment also decreased immune-suppressive TGF-beta, beta-catenin, macrophage, and endothelium gene signatures, which may contribute to enhanced immune checkpoint inhibitor (CPI) efficacy. Unexpectedly, a number of previously unidentified, strongly p53 downregulated genes associated with stromal pathways and IL10 expression identified novel anticancer therapeutic applications.ConclusionsThese results imply the ability of Ad-p53 to induce efficacious local and systemic antitumor immune responses with the potential to reverse resistance to immune checkpoint inhibitor therapy when combined with CD122/132 agonists and immune checkpoint blockade. Our findings further imply that Ad-p53 has multiple complementary immune mechanisms of action, which support future clinical evaluation of triplet Ad-p53, CD122/132 agonist, and immune checkpoint inhibitor combination treatment.

Analysis of Adenoviral p53 Gene Therapy Clinical Trials in Recurrent Head and Neck Squamous Cell Carcinoma.

BackgroundWe conducted an analysis of previous adenoviral p53 (Ad-p53) treatment data in recurrent head and neck squamous cell carcinoma (HNSCC) patients to identify optimal Ad-p53 treatment methods for future clinical trials.MethodsThe analysis involved recurrent HNSCC patients treated with Ad-p53 for whom p53 genotyping and immunohistochemistry tumor biomarker studies had been performed (n = 70). Ad-p53 tumor treatment responses defined by RECIST 1.1 criteria were correlated with Ad-p53 dose and tumor p53 biomarkers. Gene expression profiles induced by Ad-p53 treatment were evaluated using the Nanostring IO 360 panel.ResultsAd-p53 dose based upon the injected tumor volume had a critical effect on tumor responses. All responders had received Ad-p53 doses greater than 7 × 1010 viral particles/cm3 of tumor volume. There was a statistically significant difference in tumor responses between patients treated with greater than 7 × 1010 viral particles/cm3 compared to patients treated at lower Ad-p53 doses (Tumor Response 31% (9/29) for Ad-p53 > 7 × 1010 viral particles/cm3 versus 0% (0/25) for Ad-p53 < 7 × 1010 viral particles/cm3; p = 0.0023). All responders were found to have favorable p53 biomarker profiles defined by less than 20% p53 positive tumor cells by immunohistochemistry (IHC), wild type p53 gene sequence or p53 deletions, truncations, or frame-shift mutations without functional p53 tetramerization domains. Preliminary gene expression profiling results revealed that Ad-p53 treatment increased interferon signaling, decreased TGF-beta and beta-catenin signaling resulting in an increased CD8+ T cell signature which are associated with increased responses to immune checkpoint blockade.ConclusionsOur findings have important implications for future p53 targeted cancer treatments and identify fundamental principles to guide Ad-p53 gene therapy. We discovered that previous Ad-p53 clinical trials were negatively impacted by the inclusion of patients with unfavorable p53 biomarker profiles and by under dosing of Ad-p53 treatment. Future Ad-p53 clinical trials should have favorable p53 biomarker profiles inclusion criteria and Ad-p53 dosing above 7 × 1010 viral particles/cm3 of injected tumor volume. Preliminary gene expression profiling identified p53 mechanisms of action associated with responses to immune checkpoint blockade supporting evaluation of Ad-p53 in combination with immune checkpoint inhibitors.

Synthesis of RNA-based gene regulatory devices for redirecting cellular signaling events mediated by p53.

Rationale: The p53 gene is a well-known tumor suppressor, and its mutation often contributes to the occurrence and development of tumors. Due to the diversity and complexity of p53 mutations, there is still no effective p53 gene therapy. In this study, we designed and constructed an aptazyme switch that could effectively sense cellular wild-type p53 protein and regulate downstream gene function flexibly. The application of this artificial device in combination with Cre-LoxP and dCas9-VP64 tools achieved a precisely targeted killing effect on tumor cells.Methods: The affinity of the aptamer to p53 protein was verified by SPR. p53 aptazyme and gene circuits were chemically synthesized. The function of the gene circuit was detected by cell proliferation assay, apoptosis assay and Western blot. The nude mouse transplantation tumor experiment was used to evaluate the inhibitory effect of gene circuits on tumor cells in vivo.Results: The results of the SPR experiment showed that the p53 aptamer RNA sequence had a robust binding effect with p53 protein. The p53 aptazyme could efficiently sense wild-type p53 protein and initiate self-cleavage in cells. The Cre-p53 aptazyme gene circuit and dCas9-VP64/sgRNA mediated gene circuit designed based on p53 aptazyme significantly inhibited the growth and promoted the apoptosis of wild-type p53-deficient cancer cells in vitro. In addition, the gene circuits also had a significant inhibitory effect on tumors in vivo.Conclusion: The study developed a novel and efficient ribozyme switch for p53-specific recognition and provided a modular strategy for aptazyme binding to cellular proteins. In addition, the p53 aptazyme successfully inhibited tumor growth through a combined application with other synthetic biological tools, providing a new perspective for cancer therapy.

P53-Bad: A Novel Tumor Suppressor/Proapoptotic Factor Hybrid Directed to the Mitochondria for Ovarian Cancer Gene Therapy.

Clinical trials involving p53 gene therapy for ovarian cancer failed due to the dominant negative inhibition of wild-type p53 and multiple genetic aberrations in ovarian cancer. To overcome this problem, we have designed a more potent chimeric gene fusion, called p53-Bad, that combines p53 with the mitochondrial pro-apoptotic factor Bad. Unlike wild-type p53, which acts as a nuclear transcription factor, this novel p53-Bad construct has multiple unique mechanisms of action including a direct and rapid apoptotic effect at the mitochondria. The mitochondrial localization, transcription activity, and apoptotic activity of the constructs were tested. The results suggest that p53 can be effectively targeted to the mitochondria by controlling the phosphorylation of pro-apoptotic Bad, which can only localize to the mitochondria when Ser-112 and Ser-136 of Bad are unphosphorylated. By introducing S112A and S136A mutations, p53-Bad fusion cannot be phosphorylated at these two sites and always localizes to the mitochondria. p53-Bad constructs also have superior activity over p53 and Bad alone. The apoptotic activity is consistent in many ovarian cancer cell lines regardless of the endogenous p53 status. Both p53 and the BH3 domain of Bad contribute to the superior activity of p53-Bad. Our data suggests that p53-Bad fusions are capable of inducing apoptosis and should be further pursued for gene therapy for ovarian cancer.

Narrowing the field: cancer-specific promoters for mitochondrially-targeted p53-BH3 fusion gene therapy in ovarian cancer

BackgroundDespite years of research, the treatment options and mortality rate for ovarian cancer remain relatively stagnant. Resistance to chemotherapy and high heterogeneity in mutations contribute to ovarian cancer’s lethality, including many mutations in tumor suppressor p53. Though wild type p53 gene therapy clinical trials failed in ovarian cancer, mitochondrially-targeted p53 fusion constructs, including a fusion with pro-apoptotic protein Bad, have shown much higher apoptotic potential than wild type p53 in vitro. Due to the inherent toxicities of mitochondrial apoptosis, cancer-specificity for the p53 fusion constructs must be developed. Cancer-specific promoters such as hTERT, hTC, Brms1, and Ran have shown promise in ovarian cancer.ResultsOf five different lengths of hTERT promoter, the − 279/+ 5 length relative to the transcription start site showed the highest activity across a panel of ovarian cancer cells. In addition to − 279/+ 5, promoters hTC (an hTERT/CMV promoter hybrid), Brms1, and Ran were tested as drivers of mitochondrially-targeted p53-Bad and p53-Bad* fusion gene therapy constructs. p53-Bad* displayed cancer-specific killing in all ovarian cancer cell lines when driven by hTC, − 279/+ 5, or Brms1.ConclusionsCancer-specific promoters hTC, − 279/+ 5, and Brms1 all display promise in driving p53-Bad* gene therapy for treatment of ovarian cancer and should be moved forward into in vivo studies. -279/+ 5 displays lower expression levels in fewer cells, but greater cancer specificity, rendering it most useful for gene therapeutics with high toxicity to normal cells. hTC and Brms1 show higher transfection and expression levels with some cancer specificity, making them ideal for lowering toxicity in order to increase dose without as much of a reduction in the number of cancer cells expressing the gene construct. Having a variety of promoters available means that patient genetic testing can aid in choosing a promoter, thereby increasing cancer-specificity and giving patients with ovarian cancer a greater chance at survival.

Combination of cabazitaxel and p53 gene therapy abolishes prostate carcinoma tumor growth.

For patients with metastatic prostate cancer, the 5-year survival rate of 31% points to a need for novel therapies and improvement of existing modalities. We propose that p53 gene therapy and chemotherapy, when combined, will provide superior tumor cell killing for the treatment of prostate carcinoma. To this end, we have developed the AdRGD-PGp53 vector which offers autoregulated expression of p53, resulting in enhanced tumor cell killing in vitro and in vivo. Here, we combined AdRGD-PGp53 along with the chemotherapy drugs used in the clinical treatment of prostate carcinoma, mitoxantrone, docetaxel, or cabazitaxel. Our results indicate that all drugs increase phosphorylation of p53, leading to improved induction of p53 targets. In vitro experiments reveal that AdRGD-PGp53 sensitizes prostate cancer cells to each of the drugs tested, conferring increased levels of cell death. In a xenograft mouse model of in situ gene therapy, AdRGD-PGp53 treatment, when combined with cabazitaxel, drastically reduced tumor progression and increased survival rates to 100%. Strikingly, we used a sub-therapeutic dose of cabazitaxel thus avoiding leukopenia, yet still showed potent anti-tumor effects when combined with AdRGD-PGp53 in this mouse model. The AdRGD-PGp53 approach warrants further development for its application in gene therapy of prostate carcinoma.

Construction of the pEGFP-N1-p53/MAR Vector and Its Effect on HEP3B Cell Morphology

Cancer always presents a big problem that endangers human health. In recent years, the use of gene therapy in cancer research has significantly increased. This study aimed to construct a non-viral, wild-type, recombinant eukaryotic expression vector, pEGFP-N1-p53/MAR and verify its mechanism of action in cancer cells in vitro. This investigation provides a novel strategy for p53 gene therapy via regulation of the matrix attachment region (MAR), potentially laying a foundation for the establishment of an anticancer protein bioreactor. The p53 gene was cloned from human peripheral blood and the MAR gene was amplified from chicken liver tissue. The recombinant eukaryotic expression vector pEGFP-N1-p53/MAR was constructed using an E. coli self-replication system. LipofectamineTM 2000 was used as the transfection agent to deliver the plasmid into the human hepatic carcinoma (HEP3B) cell line. We divided the groups as follows: negative control cells without plasmid transfection, vehicle control cells transfected with the PEGFP-N1 vector, and experimental cells transfected with the pEGFP-N1-p53/MAR vector. Cells in each well of the vehicle control and experimental groups were transfected with 1.6 μg of plasmid and 3 μL of liposome. The cellular morphology of each group was analysed using green fluorescence microscopy at 12, 24, 36 and 48 h. Then, statistical analysis of the apoptosis rates among the three groups was performed using SPSS. The ultrastructures of the cells were observed via transmission electron microscopy after transfection for 24 h. Morphological analysis showed that the cells of the experimental group were shrunken and reduced in size and their intercellular connections had disappeared. Additionally, the apoptosis rate in the experimental group was significantly higher than that in the control groups and the cellular microstructure showed that heterochromatin and apoptotic bodies were found in the experimental group. In conclusion, compared with the control groups, the pEGFP-N1-p53/MAR plasmid can effectively promote Hep3B cell apoptosis in vitro.

Effect of exogenous p51a gene on the growth and chemo sensitivity of human lung adenocarcinoma cell lines

Background The dysfunction of p53-mediated apoptosis is the key to tumorigenesis, so most gene therapy programs concentrate on improving the expressing level of wild-type p53 in tumour cells. However, the p53 gene therapy has not yielded satisfactory results in tumours with normal p53 function. A new member of p53 gene family-p63, has provided new hopes. TAp63γ (p51A) resembles p53 the most, thus it might become a new promising therapeutic gene of tumours. Methods We designed the primer pairs of p51A and amplified the p51A cDNA sequence from human skeletal muscle poly A + RNA to construct recombinant plasmid. It was then transfected into human lung adenocarcinoma cell lines A549 and NCI-H1299. RT-PCR, Western blot, MTT, flow cytometry and colony formation assay were used to analyse the growth and chemosensitivity of tumour cells. Results The recombinant plasmid was constructed and transfected into tumour cells successfully. After transfection, p51A mRNA, P51A protein and P21 protein level raised significantly. Cell proliferation capacity and colony formation rate decreased while cell apoptosis rate and chemosensitivity to cisplatin and adriamycin increased significantly. Conclusions Exogenous p51A gene can increase its expression in A549 and NCI-H1299 cells, suppress cell growth and induce cell apoptosis. Moreover, it can also cooperate with chemotherapy and reduce the dose and side-effect. p51A gene can suppress tumours in spite of p53 status and p21 gene might be involved. It might become a new promising therapeutic gene of tumours, which will make up for the limitation of p53 gene therapy.

Combination with SGT-53 overcomes tumor resistance to a checkpoint inhibitor

ABSTRACTThe tumor suppressor p53 responds to genotoxic and oncogenic stresses by inducing cell cycle arrest and apoptosis. Recent studies suggest that p53 also participates in the regulation of cellular immune responses. Here, we have investigated the potential of p53 gene therapy to augment immune checkpoint inhibition by combining an anti-programmed cell death protein 1 (PD1) antibody with SGT-53, our investigational nanomedicine carrying a plasmid encoding human wild-type p53. In three syngeneic mouse tumor models examined including a breast cancer, a non-small cell lung carcinoma, and a glioblastoma, SGT-53 sensitized otherwise refractory tumors to anti-PD1 antibody. The involvement of p53 in enhancing anti-PD1 immunotherapy appears to be multifaceted, since SGT-53 treatment increased tumor immunogenicity, enhanced both innate and adaptive immune responses, and reduced tumor-induced immunosuppression in a 4T1 breast tumor model. In addition, SGT-53 alleviates a fatal xenogeneic hypersensitivity associated with the anti-PD1 antibody in this model. Our data suggest that restoring p53 function by SGT-53 is able to boost anti-tumor immunity to augment anti-PD1 therapy by sensitizing tumors otherwise insensitive to anti-PD1 immunotherapy while reducing immune-related adverse events.

P53 molecular approach to diagnosis and treatment of esophageal squamous cell carcinoma.

We reviewed our research concerning p53 molecules in esophageal squamous cell carcinoma by focusing on the p53 molecular diagnosis and treatment of esophageal squamous cell carcinoma. First, we developed diagnostic tools to analyze serum p53 autoantibodies to detect esophageal squamous cell carcinoma. Positive rate was around 25% to 30% in all patients and around 20% even in stage I patients. Presence of serum p53 antibodies was significantly associated with overexpression of p53 protein in tumor cells. Seropositive patients were more likely than seronegative patients to be resistant to chemotherapy. Monitoring of the titer of serum p53 autoantibodies was useful in predicting patients at high risk of recurrence and/or treatment response. Second, using Ad5CMV‐p53 for 10 patients with advanced esophageal squamous cell carcinoma, we carried out a phase I/II study of adenoviral‐mediated p53 gene therapy. Although no complete response was observed, local tumor was stabilized in nine patients. No serious adverse events related to Ad5CMV‐p53 were observed in these patients. One patient survived for over 5 years after the start of p53 gene therapy. Intratumoral injection of Ad5CMV‐p53 is therefore safe, feasible, and biologically active when given in multiple doses to patients with esophageal squamous cell carcinoma. Our observations from these clinical studies indicate that p53 is a useful molecular target both in the diagnosis and in the treatment of esophageal squamous cell carcinoma.

Far-red light-mediated programmable anti-cancer gene delivery in cooperation with photodynamic therapy

Effective anti-cancer therapy is hurdled by the complicated extracellular and intracellular barriers, and thus a smart gene vector that can enable programmable gene delivery is highly demanded. Photo-manipulation of gene delivery processes features spatial and temporal precision, while majority of current strategies utilizes short-wavelength UV/visible light with poor tissue penetration or high-power-density near-infrared (NIR) light that would cause undesired heat damage. Herein, an ROS-degradable polycation was designed and co-delivered with a photosensitizer (PS), thus realizing photo-programmable gene delivery using far-red light (661 nm) at low optical power density (down to 5 mW cm−2). Thioketal-crosslinked polyethylenimine (TK-PEI) was synthesized to condense p53 gene to form nanocomplexes (NCs), and hyaluronic acid (HA) modified with pheophytin a (Pha) was coated onto NCs to enhance their colloidal stability and enable cancer cell targeting. Short-time (8-min) light irradiation produced non-lethal amount of ROS to disrupt the endosomal membranes and facilitate p53 gene release via degradation of TK-PEI, which collectively enhanced p53 expression levels toward anti-cancer gene therapy. Long-time (30-min) light irradiation at the post-transfection state generated lethal amount of ROS, which cooperatively killed cancer cells to strengthen p53 gene therapy. To the best of our knowledge, this study represents the first example of an “one stone, three birds” approach to realize cooperative anti-cancer gene therapy using low-power-density, long-wavelength visible light as a single stimulus.

Abstract B26: AdCDKN2aIRESp53: A bicistronic adenoviral vector for glioblastoma gene therapy

Abstract One of the hallmarks of tumor cells is the ability to ignore cell death signals, including those that initiate the apoptotic pathway. This ability may stem from the functional loss of p53 and/or CDKN2a activities, a common feature of glioblastoma (GBM), one of the most lethal tumors. In fact, p53 is lost in 50% of cases while loss of CDKN2a is seen in 30% of GBMs. Restoration of the p53 and Rb/CDKN2a pathways should aid in the apoptotic cell response and cooperate to form an effective therapeutic strategy. For example, pharmacologic and gene transfer approaches that target p53 are potent anticancer therapeutics thanks to their strong proapoptotic action as well as their role in responsiveness to chemo- and radiotherapy, among other attributes. p53 gene therapy has been applied clinically and promising results have been achieved for the treatment of GBM. However, p53 may not be sufficient to bring about cell death, requiring additional factors in order to avoid arrest or senescence, responses that have been shown to worsen response to therapy. In order to accomplish the simultaneous transfer of the CDKN2a and p53 genes, we have constructed the bicistronic adenovirus AdCDKN2aIRESp53 that we expect will overcome the barriers to cell death. In parallel, we are also interested in noninvasive and real-time technology that permits monitoring of caspase 3 activity as a reporter of the proapoptotic efficacy of AdCDKN2aIRESp53. In this study, we have used the lentivirus LvGFP-DEVD-ssGluc, developed by the Thannous laboratory. Upon apoptosis, the DEVD peptide is cleaved in response to caspase 3 activation, freeing ssGluc that is secreted and can then be detected in the culture medium. The human GBM cell lines U87 and T98G were transduced with LvGFP-DEVD-ssGluc, followed by sorting until the population of GFP-positive cells reached a purity greater than 95%. Before initiating the gene therapy assays, we determined the transduction parameters, aiming to maximize gene transfer while minimizing toxicity. For the U87 or T98G cell lines, we found that a MOI (multiplicity of infection) of 10 or 30, respectively, resulted in nearly 100% LacZ-positive cells without signs of toxicity. The U87 and T98G cells were transduced with AdLacZ, AdCDKN2a, Adp53, or AdCDKN2aIRESp53 and incubated for 24 h, 48 h, and 72 h before MTT staining. At the latest time point, the bicistronic vector resulted in a 50% decrease in U87 cell staining, while AdCDKN2a or Adp53 reduced staining by 21% or 8%, respectively. In the case of T98G, the bicistronic vector conferred an astonishing 93.5% reduction in staining, yet the transfer of CDKN2a or p53 alone conferred only a 30% decrease in staining in either case. Currently, the use of the caspase reporter system is being explored. While additional experiments are required, we already see a striking difference between the cell lines. U87 (p53wt, CDKN2a-negative) was much less susceptible to the treatment involving the bicistronic AdCDKN2aIRESp53 vector than the T98G (p53-mutant, CDKN2a-negative) cells. We speculate that the p53 status may contribute to this finding. Even so, the use of either p53 or CDKN2a gene transfer alone was relatively ineffective in either cell line as compared to the treatment with the bicistronic vector. This result is consistent with our hypothesis that the remediation of two genes will be better than one. With this promising result, we plan to use this strategy to screen the proapoptotic potential of AdCDKN2aIRESp53 in several cell lines derived from a variety of tumor types. Citation Format: Daniel Vieira C. Oliveira, Eugenia Costanzi-Strauss, Bryan E. Strauss. AdCDKN2aIRESp53: A bicistronic adenoviral vector for glioblastoma gene therapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B26.

Surgery combined with adenoviral p53 gene therapy for treatment of non-small cell lung cancer: a phase II study.

ObjectiveTo assess the efficacy of radical surgery combined with recombinant adenoviral human p53 (rAd-p53) gene therapy in treatment of resectable non-small cell lung cancer.MethodA total of 163 patients with resectable NSCLC meeting the inclusion criteria were randomly assigned to two groups: radical surgery alone (S) and radical surgery plus surgical wound surface injection of 2 x 1012 rAd-p53 units (SP). All patients were followed up for at least 3 years for efficacy and safety. Study endpoints were loco-regional recurrence or distant metastasis (Rec-Met) rate as primary endpoints, and progression free survival (PFS), overall survival (OS) and safety assessments as secondary endpoints.ResultsRecurrence or metastasis (Rec/Met) after surgery were 24/82 (29.27%) in SP group and 37/81 (45.68%) in S group. The difference in the Rec/Met rate was statistically significant (p = 0.0304) by chi-square test. The hazard ratios after adjusting of age and disease stage (S vs. SP) of PFS and OS are 1.772 (95% CI, 1.102 to 2.848) and 2.047 (95% CI, 1.109 to 3.377), respectively. The 3 years PFS and OS for SP vs. S were 71.9% vs. 46.9%, and 88.4% vs. 67.0%, respectively. Differences in PFS and OS between two treatment groups were significant with the p values of 0.0165 and 0.0191, respectively, using Log-Rank test.ConclusionsThe wound surface injection of rAd-p53 showed efficacious effects in preventing recurrence or metastasis and improving PFS and OS after a radical surgery in patients with NSCLC.