Investigation into the difference in mitochondrial-cytosolic calcium coupling between adult cardiomyocyte and hiPSC-CM using a novel multifunctional genetic probe.

Ca2+ cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca2+ handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca2+ dynamics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically encoded Ca2+ probe capable of simultaneously measuring cytosolic and mitochondrial Ca2+ in real time. Using this novel probe, we determined and compared mitochondrial Ca2+ activity and the coupling with cytosolic Ca2+ dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca2+ in sync with cytosolic Ca2+, hiPSC-CMs showed high cell-wide variability in mitochondrial Ca2+ activity that is poorly coordinated with cytosolic Ca2+. We then revealed that mitochondrial-sarcoplasmic reticulum (SR) tethering, as well as the inter-mitochondrial network connection, is underdeveloped in hiPSC-CM compared to ACM, which may underlie the observed spatiotemporal decoupling between cytosolic and mitochondrial Ca2+ dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca2+ coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca2+ coupling in hiPSC-CMs. Physiological relevance: The present study will advance our understanding of the role of mitochondria in Ca2+ handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.

Correction: Chen et al. Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma. Biomedicines 2022, 10, 130.

In the original publication [...].

Process improvement of adeno-associated virus (AAV) production.

Adeno-associated viruses (AAVs) have been well characterized and used to deliver therapeutic genes for diseases treatment in clinics and basic research. This study used the triple transient transfection of AAV-DJ/8 as a model expression system to develop and optimize the laboratory production of AAV for research and pre-clinical applications. Specifically, various production parameters, including host cell, transfection reagent, cell density, ratio of plasmid DNA and cells, gene size, and production mode, were tested to determine the optimal process. Our results showed that the adherent production using HEK 293AAV with calcium transfection generated the highest volumetric productivity of 7.86x109 gc/mL. The optimal suspensive production using HEK 293F had best AAV productivity of 5.78x109 gc/mL in serum-free medium under transfection conditions of transfection density of 0.4x106 cells/mL, plasmid DNA:cells ratio of 1.6 µg:106 cells and synthesized cationic liposomes as transfection reagent. The similar AAV productivity was confirmed at scales of 30 mL - 450 mL in shaker and/or spinner flasks. The in vitro transfection and in vivo infection efficiency of the harvested AAV-DJ/8 carrying luciferase reporter gene was confirmed using cell line and xenograft mouse model, respectively. The minimal or low purification recovery rate of AAV-DJ/8 in ion-exchange chromatography column and affinity column was observed in this study. In summary, we developed and optimized a scalable suspensive production of AAV to support the large-scale preclinical animal studies in research laboratories.

Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma.

Glioblastomas, accounting for approximately 50% of gliomas, comprise the most aggressive, highly heterogeneous, and malignant brain tumors. The objective of this study was to develop and evaluate a new targeted therapy, i.e., highly potent natural compound verrucarin A (Ver-A), delivered with monoclonal antibody-directed extracellular vesicle (mAb-EV). First, the high surface expression of epidermal growth factor receptor (EGFR) in glioblastoma patient tissue and cell lines was confirmed using immunohistochemistry staining, flow cytometry, and Western blotting. mAb-EV-Ver-A was constructed by packing Ver-A and tagging anti-EGFR mAb to EV generated from HEK293F culture. Confocal microscopy and the In Vivo Imaging System demonstrated that mAb-EV could penetrate the blood-brain barrier, target intracranial glioblastoma xenografts, and deliver drug intracellularly. The in vitro cytotoxicity study showed IC50 values of 2-12 nM of Ver-A. The hematoxylin and eosin staining of major organs in the tolerated dose study indicated minimal systemic toxicity of mAb-EV-Ver-A. Finally, the in vivo anti-tumor efficacy study in intracranial xenograft models demonstrated that EGFR mAb-EV-Ver-A effectively inhibited glioblastoma growth, but the combination with VEGF mAb did not improve the therapeutic efficacy. This study suggested that mAb-EV is an effective drug delivery vehicle and natural Ver-A has great potential to treat glioblastoma.

Anti-EGFR antibody-drug conjugate for triple-negative breast cancer therapy.

Triple-negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody-drug conjugate (ADC) to target EGFR+ TNBC and deliver high-potency drug. First, we constructed an ADC by conjugating anti-EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo-SMCC. Second, we confirmed the TNBC-targeting specificity of anti-EGFR ADC by evaluating its surface binding and internalization in MDA-MB-468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live-cell and live-animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC-targeting. Finally, both in vitro toxicity assay and in vivo anti-cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti-EGFR ADC has a great potential to against TNBC.

Antibody-Drug Conjugate to Treat Meningiomas.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody-drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of <5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of <10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

Anti-CD47 Monoclonal Antibody-Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) are frequently recurrent due to the development of drug resistance post chemotherapy. Both the existing literature and our study found that surface receptor CD47 (cluster of differentiation 47) was upregulated in chemotherapy-treated TNBC cells. The goal of this study was to develop a monoclonal antibody (mAb)-based targeting strategy to treat TNBC after standard treatment. Specifically, a new mAb that targets the extracellular domain of receptor CD47 was developed using hybridoma technology and produced in fed-batch culture. Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the anti-CD47 mAb effectively targeted human and mouse TNBC cells and xenograft models with high specificity. The antibody-drug conjugate (ADC) carrying mertansine was constructed and demonstrated higher potency with reduced IC50 in TNBC cells than did the free drug and significantly inhibited tumor growth post gemcitabine treatment in MDA-MB-231 xenograft NSG model. Finally, whole blood analysis indicated that the anti-CD47 mAb had no general immune toxicity, flow cytometry analysis of lymph nodes revealed an increase of CD69+ NK, CD11c+ DC, and CD4+ T cells, and IHC staining showed tumoral infiltration of macrophage in the 4T1 xenograft BALB/cJ model. This study demonstrated that targeting CD47 with ADC has great potential to treat TNBCs as a targeted therapy.

Targeted Liposomal Chemotherapies to Treat Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) are highly aggressive and recurrent. Standard cytotoxic chemotherapies are currently the main treatment options, but their clinical efficacies are limited and patients usually suffer from severe side effects. The goal of this study was to develop and evaluate targeted liposomes-delivered combined chemotherapies to treat TNBCs. Specifically, the IC50 values of the microtubule polymerization inhibitor mertansine (DM1), mitotic spindle assembly defecting taxane (paclitaxel, PTX), DNA synthesis inhibitor gemcitabine (GC), and DNA damage inducer doxorubicin (AC) were tested in both TNBC MDA-MB-231 and MDA-MB-468 cells. Then we constructed the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) tagged liposomes and confirmed its TNBC cell surface binding using flow cytometry, internalization with confocal laser scanning microscopy, and TNBC xenograft targeting in NSG female mice using In Vivo Imaging System. The safe dosage of anti-EGFR liposomal chemotherapies, i.e., <20% body weight change, was identified. Finally, the in vivo anti-tumor efficacy studies in TNBC cell line-derived xenograft and patient-derived xenograft models revealed that the targeted delivery of chemotherapies (mertansine and gemcitabine) can effectively inhibit tumor growth. This study demonstrated that the targeted liposomes enable the new formulations of combined therapies that improve anti-TNBC efficacy.

Dual-Targeted Extracellular Vesicles to Facilitate Combined Therapies for Neuroendocrine Cancer Treatment.

Neuroendocrine (NE) cancers arise from cells within the neuroendocrine system. Chemotherapies and endoradiotherapy have been developed, but their clinical efficacy is limited. The objective of this study was to develop a dual-targeted extracellular vesicles (EV)-delivered combined therapies to treat NE cancer. Specifically, we produced EV in stirred-tank bioreactors and surface tagged both anti-somatostatin receptor 2 (SSTR 2) monoclonal antibody (mAb) and anti-C-X-C motif chemokine receptor 4 (CXCR4) mAb to generate mAbs-EV. Both live-cell confocal microscopy imaging and In Vivo Imaging System (IVIS) imaging confirmed that mAbs-EV specifically targeted and accumulated in NE cancer cells and NE tumor xenografts. Then the highly potent natural cytotoxic marine compound verrucarin A (Ver-A) with IC50 of 2.2-2.8 nM and microtubule polymerization inhibitor mertansine (DM1) with IC50 of 3.1-4.2 nM were packed into mAbs-EV. The in vivo maximum tolerated dose study performed in non-tumor-bearing mice indicated minimal systemic toxicity of mAbs-EV-Ver-A/DM1. Finally, the in vivo anticancer efficacy study demonstrated that the SSTR2/CXCR4 dual-targeted EV-Ver-A/DM1 is more effective to inhibit NE tumor growth than the single targeting and single drug. The results from this study could expand the application of EV to targeting deliver the combined potent chemotherapies for cancer treatment.