Biology subjects

Daniele, M.

Publications and source records attributed to Daniele, M..

6 recordsLinked to original sources

bioRxiv2025

Adaptive Machine Learning Framework enables Unprecedented Yield and Purity of Adeno-Associated Viral Vectors for Gene Therapy

Adeno-associated viral (AAV) vectors for gene therapy are becoming integral to modern medicine, providing therapeutic options for diseases once deemed incurable. Currently, optimizing viral vector purification is a critical bottleneck in the gene therapy industry, impacting product efficacy and safety as well as accessibility and cost to patients. Traditional optimization methods are resource-intensive and often fail to adjust the purification process parameters to maximize the resulting product yield and quality. To address this challenge, we developed a machine learning framework that leverages Bayesian optimization to systematically refine affinity chromatography parameters (sample load, flow rate, and the formulation of chromatographic media) to improve AAV purification. The efficiency of this closed-loop workflow in iteratively optimizing the vectors yield, purity, and transduction efficiency was demonstrated by purifying clinically-relevant serotypes AAV2, AAV5, and AAV9 from HEK293 cell lysates using the affinity adsorbent AAVidity. We show that three cycles of Bayesian optimization elevated yields from a baseline of 70% to 99%, while reducing host-cell impurities by 230-to-400-fold across all serotypes. The optimized parameters consistently produced vectors with high purity and preserved high transduction activity, essential for therapeutic efficacy and safety, demonstrating serotype versatility - a key challenge in AAV manufacturing. By streamlining parameter optimization and enhancing productivity, our adaptive machine learning framework accelerates process development and reduces costs, advancing the accessibility and clinical translation of AAV-based gene therapies.

molecular biology↗

bioRxiv2025

Integrating affinity chromatography in the platform process for Adenovirus purification

Adenoviral vectors (AdVs) are gaining prominence in cancer therapy and vaccine development, posing the need for a modern AdV manufacturing platform. Current AdV purification by ion-exchange chromatography indeed struggles to achieve the products yield and purity of processes that employ affinity technologies. Addressing these challenges, this study presents the first affinity-based process that delivers high product yield and clearance of host cell proteins and DNA (HCPs and hcDNA) in two chromatography steps. The affinity capture utilizes resins functionalized with peptide ligands that target AdV hexon proteins (AEFFIWNA and TNDGPDYSSPLTGSG), and provide high capacity (>5{middle dot}1010 vp per mL of resin) and yield under mild elution conditions ([~]50% at pH 8.0). Peptide-functionalized adsorbents prepared using different matrices (polymethylmethacrylate vs. agarose) were initially tested to compare the purification performance. AEFFIWNA-SulfoLink resin was selected for its yield of cell- transducing AdVs ([~]50%) and removal of HCPs and hcDNA (144-fold and 56-fold). Similarly, TNDGPDYSSPLTGSG-Toyopearl(R) resin afforded [~]50% yield and >50-fold reduction of con- taminants. Additional gains in product purity were achieved by optimizing the washing step, which removed free hexon proteins and additional HCPs. All peptide-functionalized resins maintained their purification performance for ten cycles upon regeneration at pH [~]2.0. The purification process was assembled to include clarification, affinity capture in bind-and-elute mode using AEFFIWNA-SulfoLink resin, and polishing in flow-through mode using mixed- mode resins. The optimized process provided a yield [~]50% of cell-infecting units (IFU) and a product titer [~]107 IFU/mL, along with residual HCP and hcDNA levels ([~]10 ng/mL and 44 ng per dose, respectively) that meet clinical requirements.

bioengineering↗

bioRxiv2024

Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed in E. coli

Process Analytical Technologies (PAT) are critical for efficient and automated bioprocessing; moreover, sensor and assay-driven automation will be necessary for the realization of Industry 4.0. Herein, we demonstrate methods for analyzing product yield from a pilot-scale bioreactor (300L) and conduct cross-method comparisons. We used a system of recombinant green fluorescent protein (GFPuv) expressed in Escherichia coli (E. coli), which is a simple, cost-effective model for evaluation at both laboratory and pilot scales. By comparing inline bioreactor measurements, plate reader assays, and a novel image analysis pipeline, we identify optimal harvest timelines and demonstrate the strengths and limitations of each technique. Results indicate peak cell density and GFPuv expression between 12.5h and 18h post-inoculation, with declining viability thereafter. Comparisons across techniques suggest that imaging methods may be more effective in capturing adverse outcomes, such as increased membrane permeability or cell death, which typically occur at later stages of operation. This integrated analysis offers actionable insights for optimizing biomanufacturing workflows and advances the development of scalable PAT applications, bridging the gap between laboratory research and industrial implementation.

biochemistry↗

bioRxiv2023

Simple Design for Membrane-Free Microphysiological Systems to Model the Blood-Tissue Barriers

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ{middle dot}cm-2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations. HIGHLIGHTSO_LIBarrier-type organs-on-a-chip are popular due to their mimicry of a variety of tissue constructs and interfaces. C_LIO_LITypical barrier-type organs-on-a-chip rely upon microperforated membranes and complex assembly, which limits both ease of fabrication the desired barrier performance. C_LIO_LIA membrane-free barrier-type organ-on-a-chip is designed, which uses simple Y-channel microfluidics and photopolymerization to form a precise "blood-tissue interface." C_LIO_LIFabrication of the membrane-free design can be easily parallelized and scaled-up. C_LI

bioengineering↗

bioRxiv2023

A new microphysiological system shows hypoxia primes human ISCs for interleukin-dependent rescue of stem cell activity

Background & AimsHypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces inflammatory hypoxia, a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. MethodshISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1 response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. ResultsThe novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1 at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins ConclusionsHypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

cell biology↗

bioRxiv2020

Ivermectin repurposing for COVID-19 therapy: Safety and pharmacokinetic assessment of a novel nasal spray formulation in a pig model

High ivermectin (IVM) concentrations suppress in vitro SARS-CoV-2 replication. Nasal IVM spray (N-IVM-spray) administration may contribute to attaining high drug concentrations in nasopharyngeal (NP) tissue, a primary site of virus entrance/replication. The safety and pharmacokinetic performance of a new N-IVM spray formulation in a piglet model were assessed. Crossbred piglets (10-12 kg) were treated with either one or two (12 h apart) doses of N-IVM-spray (2 mg, 1 puff/nostril) or orally (0.2 mg/kg). The overall safety of N-IVM-spray was assessed (clinical, haematological, serum biochemical determinations), and histopathology evaluation of the application site tissues performed. The IVM concentration profiles measured in plasma and respiratory tract tissues (nasopharynx and lungs) after the nasal spray treatment (one and two applications) were compared with those achieved after the oral administration. Animals tolerated well the novel N-IVM-spray formulation. No local/systemic adverse events were observed. After nasal administration, the highest IVM concentrations were measured in NP and lung tissues. Significant increases in IVM concentration profiles in both NP-tissue and lungs were observed after the 2-dose nasal administrations. The nasal/oral IVM concentration ratios in NP and lung tissues (at 6 h post-dose) markedely increased by repeating the spray application. The fast attainment of high and persistent IVM concentrations in NP tissue is the main advantage of the nasal over the oral route. These original results are encouraging to support the undertaking of further clinical trials to evaluate the safety/efficacy of the nasal IVM spray application in the treatment and/or prevention of COVID-19.

pharmacology and toxicology↗