Saved Queries

by Diogo Boreski, Valentine Fabienne Schmid, Priscila Longhin Bosquesi, Jean Leandro dos Santos, Cauê Benito Scarim, Viktor Reshetnikov and Chung Man Chin

Pharmaceuticals 2025, 18(2), 210; https://doi.org/10.3390/ph18020210 - 4 Feb 2025

Abstract

The development of new drugs is a lengthy and complex process regarding its conception and ideation, passing through in silico studies, synthesis, in vivo studies, clinical trials, approval, and commercialization, with an exceptionally low success rate. The lack of efficacy, safety, and suboptimal pharmacokinetic parameters are commonly identified as significant challenges in the discovery of new drugs. To help address these challenges, various approaches have been explored in medicinal chemistry, including the use of prodrug strategies. As a well-established approach, prodrug design remains the best option for improving physicochemical properties, reducing toxicity, and increasing selectivity, all while minimizing costs and saving on biological studies. This review article aims to analyze the current advances using the prodrug approach that has allowed the advance of drug candidates to clinical trials in the last 10 years. The approaches presented here aim to inspire further molecular optimization processes and highlight the potential of this strategy to facilitate the advancement of new compounds to clinical study phases. Full article

(This article belongs to the Special Issue Prodrugs: Design and Development)

18 pages, 287 KiB

Open AccessArticle

Characterization of Klebsiella pneumoniae Isolates Resistant to Cefiderocol from Hospitals and Outpatient Settings in Croatia

by Branka Bedenić, Josefa Luxner, Gernot Zarfel, Ana Benčić, Sanda Sardelić, Maja Anušić, Jasmina Vraneš, Verena Dobretzberger, Ivan Barišić and Andrea Grisold

Antibiotics 2025, 14(2), 154; https://doi.org/10.3390/antibiotics14020154 - 4 Feb 2025

Abstract

Background/Objectives: We conducted this study to evaluate the genotypic and phenotypic profiles of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates, exhibiting resistance to cefiderocol (FDC), focusing on antibiotic susceptibility, β-lactamase production, the genetic environment of bla_CARB and bla_ESBL genes and molecular epidemiology. FDC is now a last-line antibiotic for severe infections due to CRKP. Methods: Susceptibility to a wide range of antibiotics was determined by the disk diffusion and broth microdilution method. Carbapenemases were screened by a modified Hodge test while carbapenem hydrolysis was investigated using mCIM and eCIM tests. The screening for β-lactamase and fluoroquinolone cluster resistance genes was carried out by PCR. Plasmids were characterized by PCR-based replicon typing (PBRT). An inter-array genotyping CarbaResist test and whole genome sequencing (WGS) were applied on selected isolates. Results: All of the 31 isolates studied exhibited high-level resistance to amoxicillin–clavulanate, piperacillin–tazobactam, cefuroxime, expanded-spectrum cephalosporins (ESC), cefepime, ceftolozan–tazobactam and ciprofloxacin and the majority to gentamicin, and amikacin. Colistin and ceftazidime–avibactam preserved activity against 71% and 87% of the isolates, respectively. The combined disk method with clavulanic acid was positive in all but one isolate, indicating the production of an ESBL. Twenty-eight isolates carried one single carbapenemase-encoding gene, whereas three harbored double bla_CARB genes. Among the studied isolates, 61% carried bla_OXA-48, 29% bla_KPC and 12.9% bla_NDM genes. The inter-array genotyping CarbaResist test and WGS identified additional aminoglycoside-, sulphonamide- and trimethoprim-resistance genes. Conclusion: To our knowledge, this is the first study on FDC resistance in Croatia. The diffusion of FDC-resistant isolates was detected in both hospital and outpatient settings, emphasizing the need for a “One Health” approach. Full article

47 pages, 3036 KiB

Open AccessReview

Alternative Protein-Based Meat and Fish Analogs by Conventional and Novel Processing Technologies: A Systematic Review and Bibliometric Analysis

by Buse N. Gürbüz, Lorenzo M. Pastrana, Ricardo N. Pereira and Miguel A. Cerqueira

Foods 2025, 14(3), 498; https://doi.org/10.3390/foods14030498 - 4 Feb 2025

Abstract

This study aimed to explore the extent of research on developing meat and fish analogs using alternative proteins. It examined the novel and conventional technologies employed to produce these analogs and identified the primary alternative proteins that were used in their production through a systematic literature review (SLR) using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and bibliometric analysis. The SLR resulted in 46 and 13 meat and fish analog records, respectively, according to defined selection and exclusion criteria. Meat analogs are mainly produced using extrusion, followed by the novel 3D printing and mixing technology. Additionally, fish analogs are mainly produced by mixing and 3D printing. Meat analogs are mainly produced from pulses, followed by cereal, fungi, microalgae, other sources, and insects. Similarly, pulse proteins were the most used alternative protein source for the fish analogs, followed by macro- and microalgae, plant, cereal, and fungal proteins. According to keyword analysis, rheological and textural properties are essential for meat and fish analogs. This review provides up-to-date information to clarify the critical role of alternative proteins and the utilization of novel technologies in the production of meat and fish analogs. It also gives essential insights into the expected increase in studies to determine sustainability and overcome challenges related to textural, sensorial, and nutritional properties. Full article

(This article belongs to the Section Food Engineering and Technology)

17 pages, 502 KiB

Open AccessReview

Towards a Rational Basis for the Selection of Probiotics to Improve Silkworm Health and Performance

by Siripuk Suraporn, Jisheng Liu, Feifei Ren, Luoluo Wang, Min Feng, Olle Terenius and Luc Swevers

Insects 2025, 16(2), 162; https://doi.org/10.3390/insects16020162 - 4 Feb 2025

Abstract

Pathogenic infections of silkworms constitute the greatest threat to sericulture. An attractive approach to the improvement in silkworm health and performance comprises the use of probiotics, i.e., microorganisms that confer beneficial properties such as an increased growth rate and resistance against pathogens. While this method has already resulted in promising results, generally, there is a lack of a rational basis for guidance on the selection of probiotics. This review attempts to organize useful information that needs to be considered for the successful application of probiotics: the constitution of the microbiota in silkworms and its origins; the interaction of the major silkworm pathogens with the microbiota; and the microorganisms that have been used so far as silkworm probiotics. Our analysis points to two major issues that seem of vital importance: (1) the absence of a “core microbiota” in silkworms which necessitates continuous supply of beneficial microorganisms according to environmental conditions and (2) the apparent negative impact that some other microorganisms can have on resistance against baculovirus infections. Recent findings have reported the beneficial effects of lactic acid bacteria (Lactobacillus sp.) when applied as probiotics in improving silkworm health and performance. Full article

(This article belongs to the Special Issue Genomics and Molecular Biology in Silkworm)

20 pages, 10762 KiB

Open AccessArticle

Biomimetic Chromatography/QSAR Investigations in Modeling Properties Influencing the Biological Efficacy of Phenoxyacetic Acid-Derived Congeners

by Małgorzata Janicka, Małgorzata Sztanke and Krzysztof Sztanke

Molecules 2025, 30(3), 688; https://doi.org/10.3390/molecules30030688 - 4 Feb 2025

Abstract

A hybrid method—combining liquid biomimetic chromatography techniques (immobilized artificial membrane chromatography and biopartitioning micellar chromatography) and Quantitative Structure–Activity Relationships—was used to derive helpful models for predicting selected biological properties such as penetration through the plant cuticle, the skin and the blood–brain barrier, and binding to human serum albumin of phenoxyacetic acid-derived congeners regarded as potential herbicides. Reliable, high-concept models were developed indicating the lipophilicity, polarizability, and sum of hydrogen bond donors and acceptors as properties that determine the biological efficacy of the title compounds. These models were validated by leave-one-out cross-validation. Modeling the toxicity of phenoxyacetic acid-derived congeners to red blood cells allowed the identification of the most toxic substances as well as those molecular descriptors that determine their hemolytic properties. Full article

(This article belongs to the Special Issue In Silico Methods Applied in Drug and Pesticide Discovery, 2nd Edition)

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11 pages, 2497 KiB

Open AccessArticle

A Bioassay Analysis of Uranium and Lead in Urine Samples from a High Natural Background Radiation Area in Indonesia

by Very Susanto, Radhia Pradana, Eka Djatnika Nugraha, Prihadi Sumintadireja, Oumar Bobbo Modibo, Ilsa Rosianna, Nastiti Rahajeng, Haeranah Ahmad, Rusbani Kurniawan, Leons Rixson, Atika Yuningsih, Yusraini Dian Inayati Siregar, Asep Saepuloh, Wahyudi Wahyudi, Hirofumi Tazoe, Naofumi Akata and Shinji Tokonami

Environments 2025, 12(2), 51; https://doi.org/10.3390/environments12020051 - 4 Feb 2025

Abstract

Heavy metal pollution is a major environmental concern due to the high toxicity of heavy metals in humans. High natural background radiation areas (HNBRAs) contain high concentrations of the radioactive element ²³⁸U, which decays into ²⁰⁶Pb, in their soil, crops, and water. Concentrations of the heavy metals lead (Pb) and uranium (U) are, thus, correlated with HNBRAs. Mamuju in Indonesia is a recently studied HNBRA where high concentrations of Pb and U in the soil have been reported. The present study analyzes Mamuju residents’ exposure to Pb and U. Two zones in the study area were selected for comprehensive assessment. North Botteng was chosen to represent the HNBRA, and Topoyo was selected as the control zone, with 22 urine samples collected from each zone. The samples were analyzed using a quadrupole inductively coupled plasma mass spectrometer (ICP-MS). The average concentrations of Pb measured in the urine samples were 1.31 mg L⁻¹ and 0.77 mg L⁻¹ in North Botteng and Topoyo, respectively. These values are higher than the urine Pb reference value of 5 µg L⁻¹. The urine Pb concentrations in both studied zones were alarmingly high, which may have serious health effects on the population and should warrant action to reduce Pb exposure in this area. The committed effective dose from the ingestion of ²³⁸U in North Botteng was higher than in Topoyo, measuring 36.0 mSv and 8.9 mSv, respectively. The area most affected by the ingestion of ²³⁸U was the red bone marrow, followed by the bone surface. Full article

(This article belongs to the Special Issue Environmental Pollutant Exposure and Human Health)

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16 pages, 1151 KiB

Open AccessArticle

Study on the Cutter–Granite Interaction Mechanism in High-Temperature Geothermal Wells

by Yan Yang, Dongdong Song, Kuilin Huang, Haitao Ren, Yingxin Yang, Shunzuo Qiu and Zequan Huang

Energies 2025, 18(3), 719; https://doi.org/10.3390/en18030719 - 4 Feb 2025

Abstract

In high-temperature geothermal wells, the formation usually has extremely high abrasiveness, hardness, and temperature, which pose severe challenges to drilling tools. Among them, the interaction between the cutter of the drill bit and the rock is the key factor determining the rock-breaking efficiency of PDC (Polycrystalline Diamond Composite) drill bits. To further explore the rock-breaking mechanism of cutters on granite, this study adopts a combination of experimental and simulation methods to conduct systematic research. The results indicate that the specific crushing work increases and then decreases with rising temperature, reaching a minimum of 0.388 J/mm³ at 200 °C. In the temperature range of 300 °C to 500 °C, the specific crushing work is 15% lower than at room temperature. The specific crushing work during instant cooling is 12–25% lower than that during self-cooling, with instant cooling showing higher rock-breaking efficiency. As the rake angle increases, the specific crushing work initially decreases and then increases. The smallest specific crushing work, 0.383 J/mm³, occurs at a rake angle of 10°, where the number of debris and particle size are maximized. With deeper cutting depths, the specific crushing work gradually decreases, resulting in more debris, larger particle sizes, and higher cutter surface temperatures. These findings clarify the variation laws of rock load, cutting tooth distribution, and rock fragmentation state when the PDC bit breaks rocks. A rake angle of 10° can be used as the selection of cutting tooth inclination angle for PDC bit design, providing a theoretical basis for the design and application of PDC bits in high-temperature geothermal drilling and holding significant guiding importance. Considering that increasing the depth of penetration can cause uneven wear of the cutter, the drilling parameters can be controlled under certain conditions to achieve a penetration depth of 2 mm, thereby improving the rock-breaking efficiency and working life of the PDC bit. Full article

(This article belongs to the Section J: Thermal Management)

24 pages, 2017 KiB

Open AccessArticle

Earthquake-Resilient Housing Setback Distances and Open Road Networks for Sustainable Urbanization: A Case Study in Elbistan (Türkiye)

by Burak Korkmazyürek, Sümeyye Kahraman and Erkan Polat

Sustainability 2025, 17(3), 1254; https://doi.org/10.3390/su17031254 - 4 Feb 2025

Abstract

Urban roads must remain open after an earthquake, requiring the urban transportation network and surrounding buildings to meet earthquake-resilient standards. The 6 February 2023 Kahramanmaraş (Türkiye) earthquakes showed that Türkiye’s cities fail to meet these standards, making debris-blocked roads a significant challenge. Currently, there are no global normative standards for earthquake-resilient transportation and buildings. In countries like Türkiye, the main issues are zoning standards for earthquake-resilient urbanization and ensuring that roads stay open post-earthquake. This study investigates the earthquake suitability of residential setback distances using a quantitative methodology based on field data. This study recommends standards for earthquake-resilient setback distances to maintain open roads after an earthquake. Elbistan (Kahramanmaraş), impacted by the 2023 earthquakes, was selected as the research area. The goal is for this research to serve as a reference in the global literature and guide the development of earthquake-resilient zoning regulations for Türkiye’s cities. Research findings show that residential setback distances in Türkiye are inadequate for earthquake resilience. In the context of sustainable urbanization, the study emphasizes the need to integrate resilience into city planning. Earthquake-resilient zoning standards and setback distances can help to ensure that cities are better prepared for natural disasters and more sustainable communities for the future. Full article

(This article belongs to the Section Hazards and Sustainability)

23 pages, 1163 KiB

Open AccessReview

Identifying Biomarkers for Remyelination and Recovery in Multiple Sclerosis: A Measure of Progress

by Vito A. G. Ricigliano, Silvia Marenna, Serena Borrelli, Valentina Camera, Edgar Carnero Contentti, Natalia Szejko, Christos Bakirtzis, Sanja Gluscevic, Sara Samadzadeh, Hans-Peter Hartung, Krzysztof Selmaj, Bruno Stankoff, Giancarlo Comi and ECF Young Investigators/Fellows Initiative

Biomedicines 2025, 13(2), 357; https://doi.org/10.3390/biomedicines13020357 - 4 Feb 2025

Abstract

Background: Multiple sclerosis (MS) pathology is characterized by acute and chronic inflammation, demyelination, axonal injury, and neurodegeneration. After decades of research into MS-related degeneration, recent efforts have shifted toward recovery and the prevention of further damage. A key area of focus is the remyelination process, where researchers are studying the effects of pharmacotherapy on myelin repair mechanisms. Multiple compounds are being tested for their potential to foster remyelination in different clinical settings through the application of less or more complex techniques to assess their efficacy. Objective: To review current methods and biomarkers to track myelin regeneration and recovery over time in people with MS (PwMS), with potential implications for promyelinating drug testing. Methods: Narrative review, based on a selection of PubMed articles discussing techniques to measure in vivo myelin repair and functional recovery in PwMS. Results: Non-invasive tools, such as structural Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET), are being implemented to track myelin repair, while other techniques like evoked potentials, functional MRI, and digital markers allow the assessment of functional recovery. These methods, alone or in combination, have been employed to obtain precise biomarkers of remyelination and recovery in various clinical trials on MS. Conclusions: Combining different techniques to identify myelin restoration in MS could yield novel biomarkers, enhancing the accuracy of clinical trial outcomes for remyelinating therapies in PwMS. Full article

(This article belongs to the Special Issue Advanced Research in Neuroprotection)

21 pages, 1131 KiB

Open AccessFeature PaperArticle

Body Awareness Does Not Need a Pedigree: Mixed-Breed Dogs Rely More on Self-Representation Than Social Learning in a Spatial Task

by Petra Dobos and Péter Pongrácz

Animals 2025, 15(3), 432; https://doi.org/10.3390/ani15030432 - 4 Feb 2025

Abstract

Body awareness allows the individual to negotiate spatial tasks by referencing their own body. Here, we tested whether biologically meaningful factors, such as an alternative solution (detour around an obstacle) and learning from a human demonstrator, would affect dogs’ reliance on their body size in an aperture test. We hypothesized that the dogs would choose the socially reinforced solution over a shortcut, and they would choose the shortcut when the opening was comfortably large. We tested N = 45 adult, mixed-breed dogs, by using a 3 m long, transparent fence. The dogs had three trials with closed doors and then three trials with either a small (but passable) or a large open door. In the demonstration group the experimenter performed a detour before the first three trials, then we opened the large door. The dogs preferred the shortcut, where they relied on body awareness, as they either hesitated or opted for a detour when the small door was open. The dogs who watched the demonstration for longer in trial 3 chose the door less often in trial 4, which indicates social learning. By testing mixed-breed dogs, our results are minimally influenced by functional breed selection and serve as valuable baseline for testing complex socio-cognitive traits in companion dogs. Full article

(This article belongs to the Section Human-Animal Interactions, Animal Behaviour and Emotion)

23 pages, 16066 KiB

Open AccessArticle

Forecasting the River Ice Break-Up Date in the Upper Reaches of the Heilongjiang River Based on Machine Learning

by Zhi Liu, Hongwei Han, Yu Li, Enliang Wang and Xingchao Liu

Water 2025, 17(3), 434; https://doi.org/10.3390/w17030434 - 4 Feb 2025

Abstract

Ice-jam floods (IJFs) are a significant hydrological phenomenon in the upper reaches of the Heilongjiang River, posing substantial threats to public safety and property. This study employed various feature selection techniques, including the Pearson correlation coefficient (PCC), Grey Relational Analysis (GRA), mutual information (MI), and stepwise regression (SR), to identify key predictors of river ice break-up dates. Based on this, we constructed various machine learning models, including Extreme Gradient Boosting (XGBoost), Backpropagation Neural Network (BPNN), Random Forest (RF), and Support Vector Regression (SVR). The results indicate that the ice reserves in the Oupu to Heihe section have the most significant impact on the ice break-up date in the Heihe section. Additionally, the accumulated temperature during the break-up period and average temperature before river ice break-up are identified as features closely related to the river’s opening in all four feature selection methods. The choice of feature selection method notably impacts the performance of the machine learning models in predicting the river ice break-up dates. Among the models tested, XGBoost with PCC-based feature selection achieved the highest accuracy (RMSE = 2.074, MAE = 1.571, R² = 0.784, NSE = 0.756, TSS = 0.950). This study provides a more accurate and effective method for predicting river ice break-up dates, offering a scientific basis for preventing and managing IJF disasters. Full article

(This article belongs to the Special Issue Mathematical, Physical, Chemical, and Biological Methods for Ice and Water Problems)

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21 pages, 2480 KiB

Open AccessReview

Recent Advances in ZIF Membrane: Fabrication, Separation Ability and Its Application

by Jingyuan Zhang, Jiatong Han, Xin Chen, Dan Xu, Xiaobin Wen, Yiming Zhao, Yanyan Huang, Xin Ding, Ge Chen, Donghui Xu, Xiaomin Xu and Guangyang Liu

Nanomaterials 2025, 15(3), 239; https://doi.org/10.3390/nano15030239 - 4 Feb 2025

Abstract

With the growth of the population and the development of industry and agriculture, water resources are experiencing contamination by numerous pollutants, posing a threat to the aquatic environment and human health. Zeolitic imidazolate framework (ZIF) membranes, as a solution for water pollutant treatment, not only have the advantages of high efficiency adsorption, good selectivity, stability, and easy recyclability, but they also can be modified or derivatized through surface functionalization, compositing, or structural tuning, which can further endow the membranes with other functions, such as catalysis and degradation. In order to improve the performance of ZIF membranes, it is crucial to select suitable preparation methods to optimize the microstructure of the membranes and to improve the separation performance and stability of the membranes. This review systematically summarizes the current major preparation methods of ZIF membranes and their respective advantages and disadvantages, providing an overview of the applications of ZIF membranes in the treatment of water pollutants, such as dyes, antibiotics, and heavy metal ions. Future development prospects are also discussed, with the expectation that future research will optimize the synthesis methods to enhance the mechanical strength of the membranes and improve their selectivity, permeability, and anti-fouling properties through modifications or functionalization. This article is expected to provide theoretical support for the application of ZIF membranes in water pollution treatment. Full article

(This article belongs to the Special Issue Advances and Recent Trends in Functional Membranes and Nanostructured Materials for Environmental Applications)

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25 pages, 1235 KiB

Open AccessArticle

Artifical Intelligence-Based Smart Security System Using Internet of Things for Smart Home Applications

by Hakilo Sabit

Electronics 2025, 14(3), 608; https://doi.org/10.3390/electronics14030608 - 4 Feb 2025

Abstract

This study presents the design and development of an AI-based Smart Security System leveraging IoT technology for smart home applications. This research focuses on exploring and evaluating various artificial intelligence (AI) and Internet of Things (IoT) options, particularly in video processing and smart home security. The system is structured around key components: IoT technology elements, software management of IoT interactions, AI-driven video processing, and user information delivery methods. Each component’s selection is based on a comparative analysis of alternative approaches, emphasizing the advantages of the chosen solutions. This study provides an in-depth discussion of the theoretical framework and implementation strategies used to integrate these technologies into the security system. Results from the system’s deployment and testing are analyzed, highlighting the system’s performance and the challenges faced during integration. This study also addresses how these challenges were mitigated through specific adaptations. Finally, potential future enhancements are suggested to further improve the system, including recommendations on how these upgrades could advance the functionality and effectiveness of AI-based Smart Security Systems in smart home applications. Full article

(This article belongs to the Special Issue Advances in AI, IoT and Smart Sensors for Digital Healthcare Applications)

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15 pages, 484 KiB

Open AccessArticle

Exploring the Utility of ChatGPT in Cleft Lip Repair Education

by Monali Mahedia, Rachel N. Rohrich, Kaiser O’Sahil Sadiq, Lauren Bailey, Lucas M. Harrison and Rami R. Hallac

J. Clin. Med. 2025, 14(3), 993; https://doi.org/10.3390/jcm14030993 - 4 Feb 2025

Abstract

Background/Objectives: The evolving capabilities of large language models, such as generative pre-trained transformers (ChatGPT), offer new avenues for disseminating health information online. These models, trained on extensive datasets, are designed to deliver customized responses to user queries. However, as these outputs are unsupervised, understanding their quality and accuracy is essential to gauge their reliability for potential applications in healthcare. This study evaluates responses generated by ChatGPT addressing common patient concerns and questions about cleft lip repair. Methods: Ten commonly asked questions about cleft lip repair procedures were selected from the American Society of Plastic Surgeons’ patient information resources. These questions were input as ChatGPT prompts and five board-certified plastic surgeons assessed the generated responses on quality of content, clarity, relevance, and trustworthiness, using a 4-point Likert scale. Readability was evaluated using the Flesch reading ease score (FRES) and the Flesch–Kincaid grade level (FKGL). Results: ChatGPT responses scored an aggregated mean rating of 2.9 out of 4 across all evaluation criteria. Clarity and content quality received the highest ratings (3.1 ± 0.6), while trustworthiness had the lowest rating (2.7 ± 0.6). Readability metrics revealed a mean FRES of 44.35 and a FKGL of 10.87, corresponding to approximately a 10th-grade literacy standard. None of the responses contained grossly inaccurate or potentially harmful medical information but lacked citations. Conclusions: ChatGPT demonstrates potential as a supplementary tool for patient education in cleft lip management by delivering generally accurate, relevant, and understandable information. Despite the value that AI-powered tools can provide to clinicians and patients, the lack of human oversight underscores the importance of user awareness regarding its limitations. Full article

(This article belongs to the Special Issue Plastic Surgery: Innovations and Future Directions)

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21 pages, 3112 KiB

Open AccessArticle

Improved Recombinant Adeno-Associated Viral Vector Production via Molecular Evolution of the Viral Rep Protein

by Thomas Steininger, Veronika Öttl, Linda E. Franken, Cornelius Frank, Philip Ohland, Miriam Lopez Ferreiro, Stefan Klostermann, Johannes Fritsch, Evelyn Hirschauer, Anna Sandmeir, Luisa D. Hilgenfeld, Florian Semmelmann, Marie-Sofie Dürr, Fabian Konkel, Gregor Pechmann, Sabine Linder, Markus Haindl, Mustafa N. Yazicioglu, Philippe Ringler, Matthias E. Lauer, Denis Phichith, Stefan Seeber and Julia Fakhiri Show full author list Hide full author list

Int. J. Mol. Sci. 2025, 26(3), 1319; https://doi.org/10.3390/ijms26031319 - 4 Feb 2025

Abstract

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency. Full article

(This article belongs to the Special Issue Virus Engineering and Applications)

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