The determination of infectious titers of samples containing virus particles is critical for clinical gene therapy applications. Currently available methods that determine the infectivity of viruses/viral vectors are time-consuming. In this study, we developed cell culture-based infection assays that rapidly detect wide ranges of DNA/RNA viruses/viral vectors. Viruses analyzed in this study included Adenovirus type 5, Adenovirus type 41, Vaccinia virus, Measles virus, Coxsackie virus B5, Respiratory Syncytial Virus, and Adenovirus-, Adeno-associated virus-, Retrovirus-, and Lentivirus-based vectors. Susceptible cells were infected for 3 h with samples containing infectious viruses/viral vectors. In developed assays, differences in Cq values (∆Cq) were measured between cells incubated with infectious virus for 3 h and the two controls, including 0 h and heat-inactivated controls. Controls were included to detect background signals. An infection assay with a ∆Cq greater than 3.3 was considered positive. The reproducible and repeatable infection assays were functioning based on virus entry into the cells. For all tested viruses/viral vectors, ∆Cq above 3.3 was detected between cells incubated with infectious virus for 3 h and at least one control, indicating the positive functioning of the assay. The 0 h and heat-inactivated controls that were crucial for the estimation of the background, were successfully applied to all viruses/viral vectors tested in this study. We have developed a rapid cell-culture-based assay for detection of infectious viruses. The assay could be applied to all tested viruses/viral vectors, and has the potential to become a valuable tool in clinical virology and infectious virus diagnostics.

Gene therapy–based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle has been reported to generate potent, short-term pacemaker function in various animal models. Here, we investigated the impact of adeno-associated virus–mediated (AAV-mediated), long-term expression of TBX18 and compared the outcomes with those of the pacemaker ion channel hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 2 (Hcn2). Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, nonfibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete atrioventricular block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activity nor augmented Hcn2-mediated pacing. In conclusion, at functionally nonfibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.

Helper-dependent adenoviral vectors (HDAdVs), which lack all viral coding sequences, enable the delivery of up to 35 kilobases (kb) of therapeutic DNA into target cells. This large packaging capacity facilitates the transfer of complex, large, or even multiple transgenes. Despite the technical complexity of their production, HDAdVs have been extensively evaluated in numerous preclinical studies, and several clinical trials have recently been initiated. This review provides a comprehensive, state-of-the-art overview of recent advances in the HDAdVs technology. It summarizes key strategies for vector development, including capsid engineering and improvements in production methodologies. Furthermore, it reviews preclinical in vivo studies with a focus on vector design, target indications, and therapeutic outcomes. Most preclinical applications have focused on targeting the liver and hematopoietic stem cells; however, additional organ systems and vaccination approaches based on HDAdVs are also being actively explored. In addition to preclinical progress, three recently initiated clinical trials using HDAdVs targeting knee osteoarthritis, chronic granulomatous disease, and solid tumors are highlighted. Nevertheless, several challenges must be addressed to enable broader clinical translation of HDAdVs. These include the complexity of large-scale manufacturing, achieving efficient and specific vector targeting, and overcoming host immune responses. In conclusion, HDAdV-based vector systems hold considerable promise for the treatment of a wide range of diseases. Continued technological advancements, including the development of next-generation adenoviral platforms and the integration of precision gene therapy approaches, are likely to further enhance their potential as versatile biotherapeutic tools.

With an increasing number of human adenoviruses identified, the selection of potential therapeutic vectors broadens. For safety reasons, achieving cell-specific gene delivery is crucial to minimize off-target effects. Therefore, it is essential to gain a systematic understanding of adenovirus receptor-usage. Our aim is to establish a human-originated in vitro model for comparative analysis of human adenoviruses receptor usage. Based on our previous work of human CD46 and coxsackievirus and adenovirus receptor (CAR) knockout cell lines, we generated desmoglein 2 (DSG2) knockout cell lines using genome-engineering technology based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9). All together, we established a panel of cell lines that carry a single, double, or triple knockout of the three major human adenovirus receptors: CAR, CD46 and DSG2. Notably, cell proliferation speed was affected by the CAR-knockout, but not the DSG2-or CD46-knockouts. In addition, the spheroid formation ability was sharply reduced in CAR- or DSG2-knockout cells, but not the CD46-knockout cells. With this receptor-knockout model, we confirmed the receptor usage of nine species B adenoviruses. Furthermore, adenovirus vectors containing a previously identified DSG2-binding affinity-enhanced mutation showed DSG2-dependent cell entry within this cell model, which indicates that they are de-targeted from CD46 — the ubiquitously expressed receptor on all nucleated cells. Collectively, our findings show that the adenovirus major receptor knockout cell lines can serve as an in vitro model to help select adenovirus types suitable for individual applications and to better understand adenovirus infection biology.

Hypoxia is a hallmark of solid tumors and represents a major barrier for effective cancer therapies, including oncolytic virotherapy. While adenoviruses are widely studied as oncolytic agents, the impact of tumor-associated hypoxia on viral infection and spatial spread remains incompletely understood. Here, we investigated how oxygen availability influences adenovirus infection in two-dimensional (2D) cultures and three-dimensional (3D) tumor spheroids. We confirmed that cell lines commonly used in adenovirus research (HEK293A, A549), as well as KP4 pancreatic cancer cells, exhibited a physiological response to hypoxia. In KP4 monolayers, hypoxia strongly reduced adenoviral protein production. To model oxygen gradients found in solid tumors, we established stable KP4 spheroids and performed spatial analysis of HAdV5_GFP infection. When virus was added during spheroid formation and hypoxia development, infection was largely restricted to the well-oxygenated outer rim. In contrast, inoculation of virus under normoxia prior to spheroid formation resulted in a more uniform distribution of infected cells throughout the spheroid. Together, our findings demonstrate that hypoxia not only suppresses adenoviral replication in cell culture but also shapes the spatial pattern of infection in 3D tumor models, highlighting the importance of hypoxia-relevant 3D systems in preclinical evaluation of oncolytic adenoviruses.

Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle reportedly generated potent short-term pacemaker function in various animal models. Here, we investigated the effect of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcome to that of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activities, nor augmented Hcn2-mediated pacing. In conclusion, at functional non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development. ### Competing Interest Statement J.W., O.F.K., H.L.T, G.J.J.B., A.R.B., K.N., and O.F.K. are employees of PacingCure BV. O.F.K., H.L.T,. and G.J.J.B. report ownership interest in PacingCure BV. S.S. and C.T. were employees of Revvity Gene Delivery GmbH, a wholly-owned subsidiary of PerkinElmer Inc. C.T. receives shares from the company. H.Y.L, K.H.P., and L.C.P. are employees of Naason Science Inc. L.C.P reports ownership interest in Naason Science Inc. J.W., R.V., V.M.C., and G.J.J.B. filed patent applications concerning application of uORF technologies. Other authors declare no conflicts of interest. European Research Council, https://ror.org/0472cxd90 Health Holland, https://ror.org/056cwr036 Horizon 2020 Dutch Research Council European Innovation Council

In most clinical trials, oncolytic viruses (OAds) based on human adenovirus type 5 (HAdV-C5) were explored, and only rarely a complete switch to another adenovirus type was evaluated. This review highlights the broader diversity of human adenoviruses and discusses advances in engineering non-HAdV-C5 OAds. We will discuss ongoing research to refine adenoviral constructs derived from alternative adenovirus species, including chimeric viruses, to optimize viral delivery and enhance antitumor immune responses. We summarize translational and clinical studies using these alternative OAds, emphasizing their therapeutic promise despite remaining challenges. Unlocking the full potential of diverse OAds could significantly expand cancer treatment options.

The adenoviral vector-based AstraZeneca and Janssen COVID-19 vaccines have been associated with rare cases of thrombosis, believed to be triggered, among other factors, by vector binding to the blood protein platelet factor 4 (PF4). To identify vectors with lower thrombosis risk, we screened 50 natural and hexon-modified adenoviruses (Ads). Unlike the applied COVID-19 vaccines and most tested vectors, Ad34 and Ad80, as well as Ad5 vectors with deleted or chemically shielded hexon hyper-variable region 1 (HVR1), did not bind to PF4. Furthermore, interactions with PF4 substantially modified Ad5 infectivity in various immortalized and primary cells, suggesting that PF4 may influence existing vector tropism. Finally, HVR1-deleted Ad5 and Ad34 vectors expressing SARS-CoV-2 spike S1 domain were tested as vaccine candidates in mice and induced robust cellular immune responses. Therefore, the identified PF4 non-binding vectors may represent safe and efficient candidates for clinical applications.

BackgroundThe development of mucosal adenovirus (Ad) vaccine vectors is considered one of the next frontiers to protect vulnerable patients from respiratory and gastrointestinal pathogens. An efficient delivery to or through the oral cavity necessitates a thorough understanding of Ad interactions with saliva for oral, buccal or sublingual vaccine delivery, which could additionally prove instrumental in the containment of natural Ad infections but remains unexplored. Therefore, we investigated the influence of saliva on Ad infectivity, emphasizing its intrinsic antiviral role against particular Ad types in various epithelial cell cultures.MethodsA saliva pool was created from healthy donors (n=16) and incubated with ChAdOx1 or human Ads from 20 different types prior to infection of human immortalized epithelial cells. All human Ads used were replication-competent and expressed a GLN cassette containing a green-fluorescent protein, nano-luciferase, and neomycin resistance. Loss-of-function experiments were conducted by immunoprecipitation or enzymatic digestion of specific saliva components to decipher related mechanisms.ResultsTemporal and inter-individual variability in saliva samples were observed, validating the use of a saliva pool to represent the population. Saliva strongly influenced Ad infectivity, in general through inhibiting species B types and enhancing species D and E Ads, that include the vaccine vector platforms Ad26 and ChAdOx1. Interestingly, Ad20 presented the highest infectivity enhancement, as well as superior to average salivary mucus crossing rates. Furthermore, saliva immunoglobulins and human neutrophil peptides marginally influenced the Ad infectivity, while sialic acid inhibited all tested Ad types.ConclusionSaliva may have a protective role against infection by certain types of Ads. This discovery highlights a potential limitation in the efficacy of next-generation oral Ad vaccine vectors. Consequently, our study underscores the importance of identifying and utilizing saliva-resistant Ad vectors to optimize Ad-based vaccination strategies.

ABSTRACTDirected evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus vector (AdV) development until now, mainly due to insufficient complexities of randomized libraries.Clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that the technology of randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for vector retargeting. Here we developed Adenovirus Directed EVOlution (ADEVO) protocols based on fiber knob peptide display.As a proof-of-concept, HAdV-C5-derived libraries were constructed following three distinct protocols and selected on A549-DCAR cells that lack the HAdV-C5 primary receptor, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6x10^5 unique variants, an approximate 100-fold improvement compared to previously published AdV libraries. After selection, the most enriched variants did not display enhanced infectivity but rather more efficient replication and cell lysis. This warrants investigations into potential unsuspected involvement of the fiber protein in adenovirus replication.GRAPHICAL ABSTRACT

ABSTRACT While recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated in vivo terminal resolution (CTR) aiding high efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based rAd-libraries employing the efficiency of CTR. We utilized frequent, pre-existing genomic sequences to allow insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In a second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement (HFR) allows introduction of >10 6 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5×10 4 modified rAd per cm 2 of transfected cell culture. GRAPHICAL ABSTRACT

Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.

Objectives Investigating the expression and prognostic significance of adenovirus receptors DSG-2, CXADR and CD46 in head and neck cancer. Methods 104 patients with HNSCC (77 OPSCC, 27 LSCC) were retrospectively included in the study. Immunohistochemical staining was performed on all selected slides to detect the expression of DSG-2, CXADR, CD46 and the immunoreactive score (IRS) was determined from the number of positively stained tumor cells and their staining intensity. Furthermore, the respective HPV status was determined by immunohistochemical staining against p16 and HPV-PCR. Results 81.7% of the tumors showed DSG-2, 34.6% of the tumors showed CXADR and 57.7% of the tumors showed CD46 expression. A high DSG-2 IRS correlated significantly with an advanced tumor size (p= 0.003), increased grading (p=0.012) and positive HPV status (p=0.024) in OPSCC. A high CXADR IRS was significantly associated with a positive lymph node status (p= 0.041) in LSCC and an advanced AJCC stage (p= 0.012) and a positive HPV status (p= 0.009) in OPSCC. No significant correlation could be shown regarding CD46 expression and clinical tumor data. There was no effect of DSG-2, CXADR, and CD46 expression on 5-year overall and on 5-year disease-free survival. Conclusion No prognostic significance of the expression of DSG-2, CXADR or CD46 in HNSCC was seen. DSG-2, CXADR and CD46 are expressed in HNSCC, so that optimization of oncotherapy with adenoviral vectors appears promising. Due to the significantly increased expression of DSG-2 and CXADR in advanced OPSCC tumors, there is potential for optimizing oncotherapy here in particular.

Adenoviruses typically cause mild illnesses, but severe diseases may occur primarily in immunodeficient individuals, particularly children. Recently, adenoviruses have garnered significant interest as a versatile tool in gene therapy, tumor treatment, and vaccine vector development. Over the past two decades, the advent of recombineering, a method based on homologous recombination, has notably enhanced the utility of adenoviral vectors in therapeutic applications. This review summarizes recent advancements in the use of human adenoviral vectors in medicine and discusses the pivotal role of recombineering in the development of these vectors. Additionally, it highlights the current achievements and potential future impact of therapeutic adenoviral vectors.

Adenoviruses are important human pathogens that are widespread and mainly associated with respiratory and gastrointestinal infections. In a previous study on human adenovirus (HAdV) seroprevalence, we observed reduced binding antibody levels against a range of HAdV types in sera collected from students in 2021 compared to sera collected before the SARS-CoV-2 pandemic. In this follow-up study, we wanted to verify this observation in a cohort of regular blood donors for whom serial samples were available. Therefore, HAdV-specific binding antibody levels were analyzed in sera collected over a 5-year period from 2018 to 2022 in a cohort of 60 regular donors to the blood bank of the University Hospital in Greifswald, Germany. Using ELISA-based assays, we quantified the binding antibody responses against 39 HAdV types. On the cohort level, we found largely stable antibody levels over the analyzed time period, with the highest antibody responses against HAdV-C1, -D25, -D26, -E4, -D10, -D27, -C5, -D75, -C2, and -C6. Only minor but significant reductions in comparison to the first serum samples from 2018 were detected for antibody levels in 2021 and 2022 against the low-prevalent types HAdV-A31, -D8, -D20, -D37, -D65, and -D69. On the other hand, we detected fluctuations in antibody levels on the individual level, with strong increases in antibody levels indicative of novel antigen contact. Interestingly, we frequently found simultaneous changes in antibody responses against multiple HAdV types, resulting in strong correlations of antibody responses against distinct clusters of HAdVs suggesting extensive cross-reactivity of HAdV-specific antibodies. To our knowledge, this is the first study of antibodies against a broad range of HAdV types in serum samples collected from a cohort of individuals over a prolonged period, and our data provide important insight into the long-term stability of HAdV-specific antibody levels. In this cohort of regular blood donors, we did not observe any major impact of the SARS-CoV-2 pandemic on HAdV immunity. Correlations of changes in antibody levels against different types indicate cross-reactivity of HAdV-specific antibodies that are important to consider for HAdV vector development. Our data also reveal possible candidates for future development of HAdV-based vectors.