Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment

Advancing manufacture of hiPSC-derived hepatocytes with improved functionality: A nature-inspired protocol

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CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1(-/-) mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites

Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT gene.

Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive metabolic disorder characterized by defects in enzymes involved in glyoxylate metabolism. PH1 is a life-threatening disease caused by the absence, deficiency or mistargeting of the hepatic alanine-glyoxylate aminotransferase (AGT) enzyme. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a PH1 patient being compound heterozygous for the most common mutation c.508G>A (G170R), a mistargeting mutation, and c.364C>T (R122*), a previously reported nonsense mutation in AGTX. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development

Dengue Virus Inhibits the Production of Type I Interferon in Primary Human Dendritic Cells▿ †

Dengue virus (DENV) infects human immune cells in vitro and likely infects dendritic cells (DCs) in vivo. DENV-2 productive infection induces activation and release of high levels of chemokines and proinflammatory cytokines in monocyte-derived DCs (moDCs), with the notable exception of alpha/beta interferon (IFN-α/β). Interestingly, DENV-2-infected moDCs fail to prime T cells, most likely due to the lack of IFN-α/β released by moDCs, since this effect was reversed by addition of exogenous IFN-β. Together, our data show that inhibition of IFN-α/β production by DENV in primary human moDCs is a novel mechanism of immune evasion