Adenovirus-mediated Gene Therapy Research Articles

Angiotensin 1 peptide-conjugated CdSe/ZnS quantum dots for cardiac-specific hydrogen sulfide targeted therapy in myocardial ischemia-reperfusion injury.

Myocardial ischemia/reperfusion (I/R) injury remains a major obstacle in cardiovascular therapies. Hydrogen sulfide (H2S) shows promise for mitigating I/R injury, but conventional delivery methods, such as NaHS injections or adenovirus-mediated CSE gene therapy, face low efficiency and systemic side effects. This study explores the use of angiotensin 1 (AT1) peptide-conjugated CdSe/ZnS quantum dots (QDs) for targeted delivery of cystathionine-γ-lyase (CSE) plasmids to the myocardium, aiming to boost local H2S production and minimize I/R injury. CdSe/ZnS QDs were conjugated with AT1 peptides to create a nanocarrier system capable of delivering the CSE plasmid specifically to the myocardium. In vivo fluorescence imaging confirmed heart-specific accumulation. Myocardial infarct size, cardiac function, cell death, and oxidative stress were evaluated. Endoplasmic reticulum stress and mitophagy markers, including CHOP/GRP78/eIF2α, were analyzed, and the CHOP gene's role was further assessed using an adenovirus vector. The AT1-conjugated nanocarriers significantly increased CSE expression in the myocardium, as confirmed by fluorescence imaging, without affecting other organs. This localized delivery reduced myocardial infarct size, improved cardiac function, and decreased oxidative stress and cell death. Importantly, a reduction in endoplasmic reticulum stress and mitophagy markers was observed, suggesting that cardioprotection was mediated via the CHOP/GRP78/eIF2α signaling pathway. Reintroduction of CHOP using an adenovirus vector reversed these protective effects, confirming the pathway's involvement. This study demonstrates that AT1 peptide-conjugated QDs can effectively deliver CSE plasmids to the heart, providing significant protection against I/R injury through enhanced localized H2S production. This approach offers a promising, targeted, and side-effect-free therapy for myocardial I/R injury, with potential for clinical translation.

Evaluation of anti-vector immune responses to adenovirus-mediated lung gene therapy and modulation by αCD20

Although the last decade has seen tremendous progress in drugs that treat cystic fibrosis (CF) due to mutations that lead to protein misfolding, there are approximately 8%-10% of subjects with mutations that result in no significant CFTR protein expression demonstrating the need for gene editing or gene replacement with inhaled mRNA or vector-based approaches. A limitation for vector-based approaches is the formation of neutralizing humoral responses. Given that αCD20 has been used to manage post-transplant lymphoproliferative disease in CF subjects with lung transplants, we studied the ability of αCD20 to module both T and B cell responses in the lung to one of the most immunogenic vectors, E1-deleted adenovirus serotype 5. We found that αCD20 significantly blocked luminal antibody responses and efficiently permitted re-dosing. αCD20 had more limited impact on the Tcell compartment, but reduced tissue resident memory Tcell responses in bronchoalveolar lavage fluid. Taken together, these pre-clinical studies suggest that αCD20 could be re-purposed for lung gene therapy protocols to permit re-dosing.

Long-Term Follow-Up of Phase I Trial of Oncolytic Adenovirus-Mediated Cytotoxic and Interleukin-12 Gene Therapy for Treatment of Metastatic Pancreatic Cancer.

The long-term follow-up findings of the phase I trial evaluating the efficacy of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy in metastatic pancreatic cancer (mPC) seem very promising. The study employed a replication-competent Adenovector in combination with chemotherapy in a dose-escalation format. The trial demonstrated a clinically meaningful median overall survival (OS) benefit, with patients in the highest dose cohort exhibiting an impressive median OS of 18.4 months. This contrasts starkly with patients receiving lower doses who experienced a median OS of 4.8 and 3.5 months, respectively. Remarkably, subject number 10, who received the highest dose, demonstrated an extraordinary survival of 59.1 months, presenting a compelling case for further exploration. Additionally, this patient displayed complete responses in lung and liver metastases, a rare occurrence in mPC treatment. Statistical analyses supported the observed survival benefit. The unprecedented OS results emphasize the potential of this treatment strategy and pave the way for future investigations into this promising gene therapy approach.

Adenovirus-mediated Sirt1 and Tgfbr2 gene therapy improves fertility in natural ovarian aging and doxorubicin-induced premature ovarian insufficiency mice

Premature ovarian insufficiency (POI) may lead to early menopause, fertility loss and birth defects without effective treatment. Here, we explored the efficacy and safety of gene therapy to rescue ovarian function in both natural ovarian aging and doxorubicin (Dox) induced POI mice. Sirt1 and Tgfbr2 were screened out and identified as key genes in both murine and human ovarian tissues. Then, adenovirus (AdV) was selected as a suited carrier for ovarian infection after comparison of multiple viral vectors. In both two models, murine fertility was significantly improved after AdV-Sirt1 and AdV-Tgfbr2 invention individually or in combination, without obvious side effects to themselves and their offspring. Compared with the control group, the successful pregnancy rate in the 9-month-old-AdV-Sirt1 group increased by 60 % (67 % vs 42 %). Meanwhile, the pregnancy rate in the AdV-Tgfbr2 + Dox group increased by 85 % (55.6 % vs 20 %). The biological process of ovarian follicle development and fibrosis was rescued. Our work demonstrated that AdV-Sirt1 and AdV-Tgfbr2 therapy alleviates natural ovarian aging and Dox-associated POI, which may be potentially applicable for female fertility protection.

TMIC-70. A TRANSCRIPTIONALLY ACTIVE EPIGENETIC IMPRINTING OF THE JAK/STAT PATHWAY MEDIATED BY H3.3-G34 MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG) INDUCES AN IMMUNOREACTIVE TME

Abstract Pediatric high-grade gliomas (pHGGs) stand as the primary cause of cancer-associated fatalities among children. Sixteen percent of pHGGs found in children and young adults present Gly34Arg/Val substitutions within the histone H3.3 (H3.3-G34R/V). Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we showed that this mutation leads to downregulation of the DNA repair pathways and enhanced susceptibility to DNA damage. DNA damage response (DDR) inhibitors in vivo, further increased genetic instability, and activation of the cGAS-STING pathway, which induces the release of immune-stimulatory cytokines. We carried out an epigenetic analysis using ChIP-seq in our GEMM, and our findings indicate that this mutation triggers transcriptionally active chromatin states within genes linked to immune reactivity, particularly JAK-STAT signaling pathway genes. Correspondingly, G34-mutant pHGG cells release immune-activating cytokines that reprogram the immune cells within the tumor microenvironment (TME). Myeloid derived suppressor cells from G34-mutant pHGG are phenotypically less immunosuppressive than the MDCS form histone wild type pHGG. Moreover, G34-mutant pHGG TME exhibit higher frequency of CD8 T cells. Based on these findings, we assessed the efficacy of an adenoviral-mediated immunostimulatory gene therapy (GT). This therapy mediates expression of Flt3L within pHGG TME, which aids dendritic cell recruitment. Thymidine kinase (TK), converts ganciclovir (GSV) into a cytotoxic compound, leading to pHGG cells’ death. GT resulted in 60% of long-term survival in mice harboring G34R pHGG. The animals that survived due to the GT did not develop tumors after a rechallenge with G34R pHGG cells, demonstrating that the treatment induces antitumoral immunological memory. In conclusion, our findings suggest that G34-mutant pHGG patients could benefit from combined therapies that target genetic instability and stimulate the immune system. Funded by NIH/NINDS.

Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

Neovascular age-related macular degeneration AMD (nAMD) is characterized by choroidal neovascularization (CNV) and could lead to irreversible blindness. However, anti-vascular endothelial growth factor (VEGF) therapy has limited efficacy. Therefore, we generated a chimpanzee adenoviral vector (AdC68-PFC) containing three genes, pigment endothelial-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble forms of CD59 (sCD59), to treat nAMD. The results showed that AdC68-PFC mediated a strong onset of PEDF, sFlt-1, and sCD59 expression both invivo and invitro. AdC68-PFC showed preventive and therapeutic effects following intravitreal (IVT) injection in the laser-induced CNV model and very low-density lipoprotein receptor-deficient (Vldlr-/-) mouse model. Invitro assessment indicated that AdC68-PFC had a strong inhibitory effect on endothelial cells. Importantly, the safety test showed no evidence of invivo toxicity of adenovirus in murine eyes. Our findings suggest that AdC68-PFC may be a long-acting and safe gene therapy vector for future nAMD treatments.

Safety and biodistribution of XC001 (encoberminogene rezmadenovec) gene therapy in rats: a potential therapy for cardiovascular diseases.

Adenovirus-mediated gene therapy holds promise for the treatment of cardiovascular diseases such as refractory angina. However, potential concerns around immunogenicity and vector dissemination from the target injected tissue require evaluation. This study was undertaken to evaluate the safety and biodistribution of XC001, a replication-deficient adenovirus serotype 5 vector expressing multiple isoforms of human vascular endothelial growth factor (VEGF), following direct administration into normal rat myocardium. Animals received the buffer formulation or increasing doses of XC001 (1 × 107, 2.5 × 108 or 2.5 × 109 viral particles). Based on in-life parameters (general health, body weights, clinical pathology, serum cardiac troponin I, plasma VEGF, and gross necropsy), there were no findings of clinical concern. On Day 8, intramyocardial administration of XC001 was associated with dose-related, left ventricular myocardial inflammation at injection sites, resolving by Day 30. XC001 DNA was not detected in blood at any time but was present at Day 8 around the site of injection and to a much lesser extent in the spleen, liver, and lungs, persisting at low levels in the heart and spleen until at least Day 91. These findings demonstrate that intramyocardial injection of XC001 is supported for use in human studies.

Adenovirus-Based Gene Therapy for Bone Regeneration: A Comparative Analysis of In Vivo and Ex Vivo BMP2 Gene Delivery.

Adenovirus-mediated gene therapy is a promising tool in bone regenerative medicine. In this work, gene-activated matrices (GAMs) composed of (1) polylactide granules (PLA), which serve as a depot for genetic constructs or matrices for cell attachment, (2) a PRP-based fibrin clot, which is a source of growth factors and a binding gel, and (3) a BMP2 gene providing osteoinductive properties were studied. The study aims to compare the effectiveness of in vivo and ex vivo gene therapy based on adenoviral constructs with the BMP2 gene, PLA particles, and a fibrin clot for bone defect healing. GAMs with Ad-BMP2 and MSC(Ad-BMP2) show osteoinductive properties both in vitro and in vivo. However, MSCs incubated with GAMs containing transduced cells showed a more significant increase in osteopontin gene expression, protein production, Alpl activity, and matrix mineralization. Implantation of the studied matrices into critical-size calvarial defects after 56 days promotes the formation of young bone. The efficiency of neoosteogenesis and the volume fraction of newly formed bone tissue are higher with PLA/PRP-MSC(Ad-BMP2) implantation (33%) than PLA/PRP-Ad-BMP2 (28%). Thus, ex vivo adenoviral gene therapy with the BMP2 gene has proven to be a more effective approach than the in vivo delivery of gene constructs for bone regeneration.

Abstract CT196: A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer

Abstract Introduction: Men with locally recurrent prostate cancer, after definitive radiotherapy, have few therapeutic options. Oncolytic adenovirus-mediated cytotoxic gene therapy is an investigational cancer therapy. Delivery of the suicide gene to the tumor is by direct intratumoral or systemic injection of a viral vector containing the suicide gene. Our approach incorporates yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK), to confer sensitivity to 5-fluorocytosine (5-FC) and Valganciclovir (vGCV), respectively. The pro-drugs are converted into active drugs that inhibit DNA damage repair. Here we report the safety of oncolytic adenovirus-mediated suicide gene therapy that incorporates an interleukin-12 (IL12) gene for treatment of recurrent prostate cancer. Methods: In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL12) was injected into tumors of 15 subjects with recurrent prostate cancer (T1c-T2) at escalating doses (1 × 1010, 3 × 1010, 1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-FC and vGCV for 7 days. The study endpoint was toxicity through day 30. Experimental endpoints included measurements of serum IL12, interferon gamma (IFNγ), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells (PBMC) and proliferation markers were analyzed by flow cytometry. Results and Conclusions: Fifteen patients received Ad5-yCD/mutTKSR39rep-hIL-12 and oral 5-FC and vGCV. Approximately 92% of the 115 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39rep-hIL-12 DNA was detected in the blood of only two patients. Elevated serum IL12, IFNγ, and CXCL10 levels were detected in 57%, 93%, and 79% of subjects, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39rep-hIL-12 administration in cohort 5. The study did not detect a significant difference in the PSA doubling time (PSADT) between pre and post treatment by paired Wilcoxon rank test (p=0.17). There was no correlation between adenoviral dose and PSADT in each cohort separately or pooled (cohorts1-3 and cohorts 4-5). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39 rep-hIL-12) was well tolerated when administered locally to prostate tumors. Citation Format: Shyam Nyati, Hans Stricker, Kenneth L. Barton, Piln Li, Mohamed Elshaikh, Haythem Ali, Stephen L. Brown, Clara Hwang, James Peabody, Svend O. Freytag, Benjamin Movsas, Farzan Siddiqui. A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT196.

Engineered Oncolytic Adenoviruses: An Emerging Approach for Cancer Therapy.

Cancer is among the major leading causes of mortality globally, and chemotherapy is currently one of the most effective cancer therapies. Unfortunately, chemotherapy is invariably accompanied by dose-dependent cytotoxic side effects. Recently, genetically engineered adenoviruses emerged as an alternative gene therapy approach targeting cancers. This review focuses on the characteristics of genetically modified adenovirus and oncology clinical studies using adenovirus-mediated gene therapy strategies. In addition, modulation of the tumor biology and the tumor microenvironment as well as the immunological responses associated with adenovirus-mediate cancer therapy are discussed.

Adiponectin gene therapy prevents islet loss after transplantation

Significant pancreatic islet dysfunction and loss shortly after transplantation to the liver limit the widespread implementation of this procedure in the clinic. Nonimmune factors such as reactive oxygen species and inflammation have been considered as the primary driving force for graft failure. The adipokine adiponectin plays potent roles against inflammation and oxidative stress. Previous studies have demonstrated that systemic administration of adiponectin significantly prevented islet loss and enhanced islet function at post‐transplantation period. In vitro studies indicate that adiponectin protects islets from hypoxia/reoxygenation injury, oxidative stress as well as TNF‐α‐induced injury. By applying adenovirus mediated transfection, we now engineered islet cells to express exogenous adiponectin gene prior to islet transplantation. Adenovirus‐mediated adiponectin transfer to a syngeneic suboptimal islet graft transplanted under kidney capsule markedly prevented inflammation, preserved islet graft mass and improved islet transplant outcomes. These results suggest that adenovirus‐mediated adiponectin gene therapy would be a beneficial clinical engineering approach for islet preservation in islet transplantation.

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.

In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9

AbstractGenetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.

Long-term safety and efficacy of intramyocardial adenovirus-mediated VEGF-DΔNΔC gene therapy: eight-year follow-up of phase 1 KAT301 study

Abstract Backgound/Introduction In phase I KAT301 trial, adenovirus-mediated intramyocardial vascular endothelial growth factor-DΔNΔC (VEGF-D) gene therapy (GT) resulted in a significant improvement in myocardial perfusion reserve and relieved angina at 1-year follow-up without major safety concerns. Purpose Our objective was to investigate the long-term safety and efficacy of AdVEGF-D GT. A total of 30 patients (24 VEGF-D and 6 randomized and blinded controls) participated in KAT301 trial. Methods The mean follow-up time was 8.2 years (range 6.3–10.4 years). Patients were interviewed for the current severity of symptoms (Canadian Cardiovascular Society class, CCS) and perceived benefit from GT. Medical records were reviewed to assess the incidence of major cardiovascular adverse events (MACEs) and other predefined endpoints including cancer. Results MACE occurred in 15 patients in VEGF-D group and in five patients in control group (21.5 vs. 24.9 per 100 patient-years; hazard ratio 0.90; 95% confidence interval 0.09–9.32; P=0.95). Mortality and comorbidity were similar between the groups. Angina symptoms (CCS) were less severe compared to baseline in VEGF-D group (1.9 vs. 2.9; P=0.006) but not in control group (2.2 vs. 2.6; P=0.414). Conclusion(s) Our study indicates that intramyocardial AdVEGF-D GT is safe in the long-term. In addition, the relief of symptoms remained significant during the follow-up. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Kuopio University Hospital Heart Center Figure 1. The incidence of MACEFigure 2. CCS class

Long-term safety and efficacy of intramyocardial adenovirus-mediated VEGF-DΔNΔC gene therapy eight-year follow-up of phase I KAT301 study

In phase I KAT301 trial, intramyocardial adenovirus-mediated vascular endothelial growth factor -DΔNΔC (AdVEGF-D) gene therapy (GT) resulted in a significant improvement in myocardial perfusion reserve and relieved symptoms in refractory angina patients at 1-year follow-up without major safety concerns. We investigated the long-term safety and efficacy of AdVEGF-D GT. 30 patients (24 in VEGF-D group and 6 blinded, randomized controls) were followed for 8.2 years (range 6.3–10.4 years). Patients were interviewed for the current severity of symptoms (Canadian Cardiovascular Society class, CCS) and perceived benefit from GT. Medical records were reviewed to assess the incidence of major cardiovascular adverse event (MACE) and other predefined safety endpoints. MACE occurred in 15 patients in VEGF-D group and in five patients in control group (21.5 vs. 24.9 per 100 patient-years; hazard ratio 0.97; 95% confidence interval 0.36–2.63; P = 0.95). Mortality and new-onset comorbidity were similar between the groups. Angina symptoms (CCS) were less severe compared to baseline in VEGF-D group (1.9 vs. 2.9; P = 0.006) but not in control group (2.2 vs. 2.6; P = 0.414). Our study indicates that intramyocardial AdVEGF-D GT is safe in the long-term. In addition, the relief of symptoms remained significant during the follow-up.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying IL-24/endostatin enhances glioma therapy.

Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.

Safe and low-dose but therapeutically effective adenovirus-mediated hepatocyte growth factor gene therapy for type 1 diabetes in mice

AimsHepatocyte growth factor (HGF) is a multifunctional cytokine that plays important roles in pancreatic physiology. Approvals of gene therapy drugs have highlighted gene therapy as an innovative new drug modality, but the very recent reports of deaths in clinical trials have provided a warning that high-dose gene therapy can cause dangerous liver toxicity. The present study aimed to develop a safe and low-dose but therapeutically effective adenovirus-mediated HGF gene therapy for streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. Main methodsA single intravenous injection of a low dose (3 × 108 plaque forming units) of adenoviral vector expressing the HGF gene under the transcriptional control of a strong promoter, i.e., the cytomegalovirus immediate-early enhancer and a modified chicken β-actin promoter (Ad.CA-HGF), was given to T1D mice. Key findingsLow-dose HGF gene therapy significantly attenuated the elevation of blood glucose concentrations at the acute phase of T1D, and this effect persisted for several weeks. Temporal upregulation of plasma insulin at the acute phase was maintained at a normal level in Ad.CA-HGF-treated mice, suggesting that the therapeutic mechanism may involve protection of the remaining β-cells by HGF. Liver enzymes in plasma were not elevated in any of the mice, including the Ad.CA-HGF-treated animals, all of which looked healthy, suggesting the absence of lethal adverse effects observed in patients receiving high intravenous doses of viral vectors. SignificanceA low dose of intravenous Ad-mediated HGF gene therapy is clinically feasible and safe, and thus represents a new therapeutic strategy for treating T1D.

Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer

Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently.Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety.Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs.Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.

Phase I trial of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for the treatment of metastatic pancreatic cancer

The safety of oncolytic adenovirus-mediated suicide and interleukin-12 (IL12) gene therapy was evaluated in metastatic pancreatic cancer patients. In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL12) was injected into tumors of 12 subjects with metastatic pancreatic cancer (T2N0M1-T4N1M1) at escalating doses (1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-fluorocytosine (5-FC) therapy for 7 days followed by chemotherapy (FOLFIRINOX or gemcitabine/albumin-bound paclitaxel) starting 21 days after adenovirus injection. The study endpoint was toxicity through day 21. Experimental endpoints included measurements of serum IL12, interferon gamma (IFNG), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells and proliferation markers were analyzed by flow cytometry. Twelve patients received Ad5-yCD/mutTKSR39rep-hIL-12 and oral 5-FC. Approximately 94% of the 121 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39rep-hIL-12 DNA was detected in the blood of two patients. Elevated serum IL12, IFNG, and CXCL10 levels were detected in 42%, 75%, and 92% of subjects, respectively. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39rep-hIL-12 administration. The median survival of patients in the third cohort is 18.1 (range, 3.5–20.0) months. The study maximum tolerated dose (MTD) was not reached.

Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation.

Immune evasion is an important cancer hallmark and the understanding of its mechanisms has generated successful therapeutic approaches. Induction of immunogenic cell death (ICD) is expected to attract immune cell populations that promote innate and adaptive immune responses. Here, we present a critical advance for our adenovirus-mediated gene therapy approach, where the combined p14ARF and human interferon-β (IFNβ) gene transfer to human melanoma cells led to oncolysis, ICD and subsequent activation of immune cells. Our results indicate that IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. In situ gene therapy of s.c. SK-MEL-147 tumors in Nod-Scid mice revealed inhibition of tumor growth and increased survival in response to IFNβ alone or in combination with p14ARF. Emission of critical markers of ICD (exposition of calreticulin, secretion of ATP and IFNβ) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. Co-culture of previously transduced SK-MEL-147 cells with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors resulted in increased levels of activation markers HLA-DR, CD80, and CD86. Activated Mo-DCs were able to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. Preliminary data showed that T cells primed by Mo-DCs activated with p14ARF+IFNβ-transduced SK-MEL-147 cells were able to induce the loss of viability of fresh non-transduced SK-MEL-147 cells, suggesting the induction of a specific cytotoxic population that recognized and killed SK-MEL-147 cells. Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.