Ovarian cancer (OVCA) is an aggressive and often recurrent malignancy with limited long-term responses to standard chemotherapy. To address the urgent need for novel, targeted therapies, we evaluated the efficacy of a tumor-tropic neural stem cell (NSC) platform delivering a conditionally replicative adenovirus (CRAd), NSC.CRAd-S-pk7, in preclinical models of advanced OVCA. A single intraperitoneal dose of NSC.CRAd-S-pk7 delivering 3×109 infectious units (IFU) significantly reduced tumor burden and increased median survival by 33% (from 57 to 76 d) in immunocompetent mice, and 48% (from 54 to 80 days) in immunodeficient mice bearing OVCA peritoneal metastases. No observed adverse effects occurred at or below this dose. Optimization of the NSC transduction protocol enabled at least a 10-fold increase in CRAd-S-pk7 viral payload per cell, reducing the therapeutic cell dose by more than an order of magnitude—from 60 million to just 1 million NSCs. Repeated dosing further decreased tumor burden and increased median survival by 60% (from 57 to 91 days) in immunocompetent mice, suggesting a contribution from innate immune activation. These findings establish NSC.CRAd-S-pk7 as a promising oncolytic viro-immunotherapy treatment for advanced OVCA and support its advancement toward clinical translation.

Abstract Although adeno-associated virus (AAV) has enjoyed enormous success as a delivery modality for gene therapy, it suffers from high prevalence of preexisting neutralizing antibodies in human populations, limiting who can receive potentially life-saving treatments. As a novel solution to this issue, we employed SpyTag-SpyCatcher molecular glue technology to facilitate packaging of AAVs inside of recombinant protein vault nanoparticles. Vaults are endogenous particles produced by mammalian cells. We therefore hypothesized that they may shield packaged molecules from neutralizing antibodies. Vaults have previously been utilized to deliver drugs and proteins into cells, but our study represents the first time anyone has packaged an entire virus inside of a vault. We showed that our vaultAAV delivery vehicle transduces cells in the presence of anti-AAV neutralizing serum. VaultAAV is positioned as a new gene therapy delivery platform with potential to overcome the neutralizing antibody problem, expanding the scope of AAV treatments.

Current cancer therapies, including cytotoxic agents, molecularly targeted drugs, and immune-based treatments, are often constrained by therapeutic resistance, systemic toxicity, and inconsistent clinical efficacy. The adeno-associated virus (AAV) vector-based therapy emerges as a promising strategy to address these limitations, owing to its favorable safety profile and high degree of engineerability. AAV-based gene therapy development is expanding beyond the treatment of rare monogenic disorders to encompass chronic diseases and cancer conditions associated with a high disease burden, driven not only by advances in vector technology but also by strategic considerations such as development costs, clinical needs, and commercial viability. Although challenges such as pre-existing host immunity and limited transgene packaging capacity remain, recent advances in biotechnology are actively mitigating these barriers. This review highlights recent progress in AAV-based gene therapy for cancer challenges and discusses future directions for the implementation of AAV vectors as a next-generation therapeutic modality in oncology.

Ovarian cancer (OVCA) is an aggressive and often recurrent malignancy with limited long-term responses to standard chemotherapy. To address the urgent need for novel, targeted therapies, we evaluated the efficacy of a tumor-tropic neural stem cell (NSC) platform delivering a conditionally replicative adenovirus (CRAd), NSC.CRAd-S-pk7, in preclinical models of advanced OVCA. A single intraperitoneal dose of NSC.CRAd-S-pk7 delivering 3 × 109 infectious units (IFUs) significantly reduced tumor burden and increased median survival by 33% (from 57 to 76 days) in immunocompetent mice, and 48% (from 54 to 80 days) in immunodeficient mice bearing OVCA peritoneal metastases. No observed adverse effects occurred at or below this dose. Optimization of the NSC transduction protocol enabled at least a 10-fold increase in CRAd-S-pk7 viral payload per cell, reducing the therapeutic cell dose by more than an order of magnitude-from 60 million to just 1 million NSCs. Repeated dosing further decreased tumor burden and increased median survival by 60% (from 57 to 91 days) in immunocompetent mice, suggesting a contribution from innate immune activation. These findings establish NSC.CRAd-S-pk7 as a promising oncolytic viro-immunotherapy treatment for advanced OVCA and support its advancement toward clinical translation.

Despite advances in immunotherapy, the prognosis for patients with glioblastoma (GBM) remains poor. The efficacy of GBM-targeted immunotherapies is limited by the paucity of functional T cells in the tumor microenvironment, a consequence of the local and systemic immunosuppression prevalent in patients with GBM. To overcome these challenges, here we develop a treatment strategy we term "expand and pull," which uses systemic administration of rhIL-7-hyFc, a long-acting recombinant human interleukin-7, to increase peripheral T cell abundance ("expand"), followed by intratumoral oncolytic virus treatment to recruit these cells to the tumor microenvironment ("pull"). We show that rhIL-7-hyFc improves the efficacy of multiple oncolytic viral therapies in syngeneic immuno-resistant mouse models of glioma. Combining rhIL-7-hyFc and Zika virus (ZIKV) increases systemic and intratumoral T cell abundance, improves cytotoxic T cell function, and delays expression of inhibitory checkpoint receptors, resulting in long-term tumor-free survival. We observe similar survival efficacy in experiments using a safer, genetically modified Δ10 3'-UTR ZIKV, as well as the clinically tested oncolytic adenovirus, Delta24-RGD. Collectively, our findings demonstrate that augmentation of both the systemic and local immune responses improves the utility of GBM-targeted immunotherapies.

The emergence of highly pathogenic avian H5N1 influenza viruses in dairy cows and humans has increased the potential for another pandemic. To address this risk, we developed chimpanzee adenoviral (ChAd)-vectored H5 hemagglutinin-targeted vaccines and tested their immunogenicity and efficacy in rodents. Immunization with ChAd-Texas (clade 2.3.4.4b) vaccine in mice elicits neutralizing antibody responses and confers protection against viral infection and mortality upon challenge with a human H5N1 isolate (A/Michigan/90/2024, clade 2.3.4.4b). Intranasal delivery of the ChAd-Texas vaccine elicits mucosal antibody and T cell responses and confers greater protection than intramuscular immunization. In Syrian hamsters, a single intranasal dose of ChAd-Texas vaccine prevents weight loss and reduces airway infection after H5N1 A/Michigan/90/2024 or A/Texas/37/2024 challenge. Importantly, prior seasonal influenza vaccination does not impair antibody responses or protection after intranasal delivery of the ChAd-Texas vaccine. These results support the development of mucosally administered ChAd-Texas HA vaccines as an effective platform for HPAI H5N1 preparedness.

The continued evolution of SARS-CoV-2 variants that evade immunity highlights a need to develop vaccines that elicit variant-specific antibodies and neutralize emerging strains. However, immune imprinting from antecedent SARS-CoV-2 exposure can limit the generation of such antibodies. Here, we evaluate strategies to enhance variant-specific antibody responses in female mice primed with Wuhan-1 spike-based mRNA or chimpanzee adenoviral-vectored (ChAd) vaccines and boosted with Omicron variant-matched vaccines. Altering the intramuscular injection site did not substantially affect variant-specific serum antibody responses. However, increasing booster antigen doses, performing repeated boosters, and administering booster vaccines intranasally enhanced variant-specific responses against the vaccine-matched Omicron strain. Boosting intranasally with a ChAd vaccine encoding the spike protein of Omicron XBB.1.5 elicited stronger XBB.1.5-specific responses in serum, bronchoalveolar lavage fluid, and draining lymph nodes than intramuscular boosting with the same vaccine. Regardless of booster regimen, neutralizing activity against XBB.1.5 was predominantly mediated by antibodies that were non-reactive to Wuhan-1 spike. These findings establish that in mice, intranasal or repeated variant-matched boosting can overcome the effects of imprinting and enhance immunity against SARS-CoV-2 strains.

Safe and efficient nucleic acid delivery to targeted cell populations remains a challenge in the fields of cell and gene therapy. Toward this end, we attempted to utilize the "DogTag-DogCatcher" system to target adenoviral vectors. "DogTag" is a short peptide that forms a spontaneous isopeptide bond upon mixing with its partner protein, "DogCatcher." We genetically incorporated the DogTag peptide into the protein responsible for initial binding of the virus to its target cell, the fiber. This allowed permanent linking of DogCatcher-fused single-domain or single-chain antibodies at the fiber. This modification allowed simple, effective, and exclusive targeting of the vector to cells bound by the linked antibody. This enhanced gene transfer into primary B and T cells by up to 60-fold in vitro and 2- to 3-fold in vivo in mice without other alterations to vector tropism. Although the system's in vivo performance is currently suboptimal and additional engineering is needed prior to further use, these studies form the basis of a novel method for targeting adenovirus that can be combined with additional well-characterized adenovirus modifications toward applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

Safe and efficient nucleic acid delivery to targeted cell populations remains a significant unmet need in the fields of cell and gene therapy. Towards this end, we pursued Adenoviral vectors genetically modified with the "DogTag" molecular glue peptide, which forms a spontaneous covalent bond with its partner protein, "DogCatcher". Genetic fusion of DogCatcher to single-domain or single-chain antibodies allowed covalent tethering of the antibody at defined locales on the vector capsid. This modification allowed simple, effective and exclusive targeting of the vector to cells bound by the linked antibody. This dramatically enhanced gene transfer into primary B and T cells in vitro and in vivo in mice. These studies form the basis of a novel method for targeting Adenovirus that is functional in stringent in vivo contexts and can be combined with additional well characterized Adenovirus modifications towards applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

Chikungunya virus (CHIKV) is a pathogenic arthritogenic alphavirus responsible for large-scale human epidemics for which a vaccine was recently approved for use. Mayaro virus (MAYV) is a related emerging alphavirus with epidemic potential with circulation overlap potential with CHIKV. We previously reported the ability of a non-replicating human adenovirus (AdV)-vectored vaccine expressing the MAYV structural polyprotein to protect against disease in mice following challenge with MAYV, CHIKV and UNAV. Herein, we evaluated mouse immunity and protective efficacy for an AdV-CHIKV full structural polyprotein vaccine in combination with heterologous AdV-MAYV prime/boost regimens versus vaccine coadministration. Heterologous prime/boost regimens skewed immunity toward the prime vaccine antigen but allowed for a boost of cross-neutralizing antibodies, while vaccine co-administration elicited robust, balanced responses capable of boosting. All immunization strategies protected against disease from homologous virus infection, but reciprocal protective immunity differences were revealed upon challenge with heterologous viruses. In vivo passive transfer experiments reproduced the inequity in reciprocal cross-protection after heterologous MAYV challenge. We detected in vitro antibody-dependent enhancement of MAYV replication, suggesting a potential mechanism for the lack of cross-protection. Our findings provide important insights into rational alphavirus vaccine design that may have important implications for the evolving alphavirus vaccine landscape.

Adeno-associated virus (AAV) has found immense success as a delivery system for gene therapy, yet the small 4.7 kb packaging capacity of the AAV sharply limits the scope of its application. In addition, high doses of AAV are frequently required to facilitate therapeutic effects, leading to acute toxicity issues. While dual and triple AAV approaches have been developed to mitigate the packaging capacity problem, these necessitate even higher doses to ensure that co-infection occurs at sufficient frequency. To address these challenges, we herein describe a novel delivery system consisting of adenovirus (Ad) covalently linked to multiple adeno-associated virus (AAV) capsids as a new way of more efficiently co-infecting cells with lower overall amounts of AAVs. We utilize the DogTag-DogCatcher (DgT-DgC) molecular glue system to construct our AdAAVs and we demonstrate that these hybrid virus complexes achieve enhanced co-transduction of cultured cells. This technology may eventually broaden the utility of AAV gene delivery by providing an alternative to dual or triple AAV which can be employed at lower dose while reaching higher co-transduction efficiency. ### Competing Interest Statement The authors have declared no competing interest.

Background & AimOncolytic viro-immunotherapy is a promising cancer treatment that uses replication-competent viruses to induce tumor cell lysis and stimulate an anti-tumor immune response. Clinical trial success has been limited by rapid immune-mediated inactivation of the free viral vectors, and poor viral distribution at metastatic tumor sites. Neural stem cells (NSCs) are an ideal delivery vehicle to selectively target OVs to tumor sites due to their 1) inherent tumor-topism, 2) ability to protect the OV from immune clearance, 3) enabling viral amplification en route to tumor sites - resulting in increased viral payload and distribution to tumors. They may also increase stimulation of an anti-tumor immune response due to their tumor associated antigens. We have established a GMP MCB of an immortalized, stable NSC line (HB1.F3.CD21) that has demonstrated clinical safety and tumor tropism. We have also established a GMP MVSS of a conditionally replication competent adenovirus (CRAd-S-pk7) with two genetic modifications: 1) a polylysine fiber knob addition to increase tumor infectivity, and 2) insertion of a survivin promoter to drive E1A transcription. A first-in-human phase I clinical trial of single dose NSC.CRAd-S-pk7 as an adjunct to SOC in newly diagnosed glioma patients demonstrated safety and therapeutic promise (Lancet Onc 2021). A multiple dose study in recurrent glioma patients is ongoing at COH (IND 19532). More recently, we observed impressive targeting and penetration of intraperitoneal (IP) ovarian cancer metastases after IP NSC administration. Translational studies for Stage III ovarian cancer are ongoing, following pre-IND review by the FDA, with our goal of initiating a phase I clinical trial in 2025.Methods, Results & ConclusionBiodistribution, efficacy and safety studies are conducted in human xenograft and immunocompetent syngeneic murine models of IP ovarian cancer. We are optimizing dosing regimens, OV infection of NSCs, and freeze-thaw protocols, assessing anti-tumor activity, mechanism of action and toxicity. Results. A high percentage of IP CRAd-S-pk7 NSCs localize to ovarian mets, increasing long-term survival vs. free virus. We identify the LOAEL dose, demonstrating negligible toxicity, and confirm CRAd-S-pk7 NSC treatment increases T-cell infiltration into the tumor micro-environment (improves CD8+/FoxP3 ratio). Conclusions. Use of NSC as a vehicle to deliver OVs to metastatic tumor sites can overcome the current hurdles limiting clinical efficacy of free OVs.

A mucosal route of vaccination could prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at the site of infection and limit transmission. We compared protection against heterologous XBB.1.16 challenge in nonhuman primates (NHPs) ~5 months following intramuscular boosting with bivalent mRNA encoding WA1 and BA.5 spike proteins or mucosal boosting with a WA1-BA.5 bivalent chimpanzee adenoviral-vectored vaccine delivered by intranasal or aerosol device. NHPs boosted by either mucosal route had minimal virus replication in the nose and lungs, respectively. By contrast, protection by intramuscular mRNA was limited to the lower airways. The mucosally delivered vaccine elicited durable airway IgG and IgA responses and, unlike the intramuscular mRNA vaccine, induced spike-specific B cells in the lungs. IgG, IgA and T cell responses correlated with protection in the lungs, whereas mucosal IgA alone correlated with upper airway protection. This study highlights differential mucosal and serum correlates of protection and how mucosal vaccines can durably prevent infection against SARS-CoV-2.

Chronic sleep deprivation has become more prevalent in the modernized society. Insufficient sleep syndrome is one of the most common primary sleep disorders in clinical practice; however, it can go easily unrecognized by general practitioners. Consequences of chronic sleep insufficiency involves multiple organ systems; including, cardiovascular, endocrine, immune systems and results in neurocognitive, behavioral, psychiatric consequences.

A nasally delivered chimpanzee adenoviral-vectored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (ChAd-SARS-CoV-2-S) is currently used in India (iNCOVACC). Here, we update this vaccine by creating ChAd-SARS-CoV-2-BA.5-S, which encodes a prefusion-stabilized BA.5 spike protein. Whereas serum neutralizing antibody responses induced by monovalent or bivalent adenoviral vaccines were poor against the antigenically distant XBB.1.5 strain and insufficient to protect in passive transfer experiments, mucosal antibody and cross-reactive memory T cell responses were robust, and protection was evident against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in mice and hamsters. However, depletion of memory CD8 + T cells before XBB.1.5 challenge resulted in loss of protection against upper and lower respiratory tract infection. Thus, nasally delivered vaccines stimulate mucosal immunity against emerging SARS-CoV-2 strains, and cross-reactive memory CD8 + T cells mediate protection against lung infection by antigenically distant strains in the setting of low serum levels of cross-reactive neutralizing antibodies.

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized ex vivo modification of the cells. Direct in vivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of ex vivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both ex vivo and in vivo. We found that unmodified human adenovirus C5 was capable of infecting B cells in vivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection in vivo.

Supplementary Data from Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures