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Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4 + T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand ( CD40LG ) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile., Competing Interests: L.N., C.A., M.R., V.V., A.V., S.F., S.P., D.C., and A.J. are inventors of patent applications owned by Ospedale San Raffaele S.r.l. and Fondazione Telethon ETS, including one patent application on CD40LG gene editing. L.N. is founder, quota holder, and consultant of GeneSpire S.r.l., (© 2023 The Authors.)

Decoration of nanoparticles with specific molecules such as antibodies, peptides, and proteins that preserve their biological properties is essential for the recognition and internalization of their specific target cells. Inefficient preparation of such decorated nanoparticles leads to nonspecific interactions diverting them from their desired target. We report a simple two-step procedure for the preparation of biohybrid nanoparticles containing a core of hydrophobic quantum dots coated with a multilayer of human serum albumin. These nanoparticles were prepared by ultra-sonication, crosslinked using glutaraldehyde, and decorated with proteins such as human serum albumin or human transferrin in their native conformations. These nanoparticles were homogeneous in size (20-30 nm), retained the fluorescent properties of quantum dots, and did not show a "corona effect" in the presence of serum. The uptake of transferrin-decorated quantum dot nanoparticles was observed in A549 lung cancer and SH-SY5Y neuroblastoma cells but not in non-cancerous 16HB14o- or retinoic acid dopaminergic neurons differentiated SH-SY5Y cells. Furthermore, digitoxin-loaded transferrin-decorated nanoparticles decreased the number of A549 cells without effect on 16HB14o-. Finally, we analyzed the in vivo uptake of these biohybrids by murine retinal cells, demonstrating their capacity to selectively target and deliver into specific cell types with excellent traceability.

The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~10 13 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cardiac explant-derived cells (cEDC), also referred as cardiac progenitors cells (CPC) (Barile et al., Cardiovasc Res 103(4):530-541, 2014; Barile et al., Cardiovasc Res 114(7):992-1005, 2018), represent promising candidates for the development of cell-based therapies, a novel and interesting treatment for cardioprotective strategy in heart failure (Kreke et al., Expert Rev Cardiovasc Ther 10(9):1185-1194, 2012). CPC have been tested in a preclinical setting for direct cell transplantation and tissue engineering or as a source for production of extracellular vesicles (EV) (Oh et al., J Cardiol 68(5):361-367, 2016; Barile et al., Eur Heart J 38(18):1372-1379, 2017; Rosen et al., J Am Coll Cardiol 64(9):922-937, 2014). CPC cultured as cardiospheres derived cells went through favorable Phase 1 and 2 studies demonstrating safety and possible efficacy (Makkar et al., Lancet 379(9819):895-904, 2012; Ishigami et al., Circ Res 120(7):1162-1173, 2017; Ishigami et al., Circ Res 116 (4):653-664, 2015; Tarui et al., J Thorac Cardiovasc Surg 150(5):1198-1207, 1208 e1191-1192, 2015). In this context and in view of clinical applications, cells have to be prepared and released according to Good Manufacturing Practices (GMP) (EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part I-basic requirements for medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ; EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part IV-guidelines on good manufacturing practices specific to advanced therapy medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ). This chapter describes GMP-grade methods for production and testing of a CPC Master Cell Bank (MCB), consisting of frozen aliquots of cells that may be used either as a therapeutic product or as source for the manufacturing of Exo for clinical trials.The MCB production method has been designed to isolate and expand CPC from human cardiac tissue in xeno-free conditions (Andriolo et al., Front Physiol 9:1169, 2018). The quality control (QC) methods have been implemented to assess the safety (sterility, endotoxin, mycoplasma, cell senescence, tumorigenicity) and identity/potency/purity (cell count and viability, RT-PCR, immunophenotype) of the cells (Andriolo et al., Front Physiol 9:1169, 2018).

Author(s): C. Bonini (corresponding author) [1]; M. Grez [2]; C. Traversari [3]; F. Ciceri [1]; S. Marktel [1, 4]; G. Ferrari [5]; M. Dinauer [6]; M. Sadat [6]; A. Aiuti [...]

Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 10 7  TU mL -1  in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 10 7  TU mL -1  in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 10 7  TU mL -1  day -1  for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34 + cells, reducing the LV quantities required for gene and cell therapy applications., (© 2024 CSL Innovation GmbH and The Authors. Biotechnology Journal published by Wiley‐VCH GmbH.)

Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro , and prevent the early decline in cardiac function after myocardial infarction in vivo in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>10 13 particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency in vitro , showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals., Competing Interests: The authors have declared that no competing interests exist.