Erratum: Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].

Engineering a versatile and retrievable cell macroencapsulation device for the delivery of therapeutic proteins.

Encapsulated cell therapy holds a great potential to deliver sustained levels of highly potent therapeutic proteins to patients and improve chronic disease management. A versatile encapsulation device that is biocompatible, scalable, and easy to administer, retrieve, or replace has yet to be validated for clinical applications. Here, we report on a cargo-agnostic, macroencapsulation device with optimized features for protein delivery. It is compatible with adherent and suspension cells, and can be administered and retrieved without burdensome surgical procedures. We characterized its biocompatibility and showed that different cell lines producing different therapeutic proteins can be combined in the device. We demonstrated the ability of cytokine-secreting cells encapsulated in our device and implanted in human skin to mobilize and activate antigen-presenting cells, which could potentially serve as an effective adjuvant strategy in cancer immunization therapies. We believe that our device may contribute to cell therapies for cancer, metabolic disorders, and protein-deficient diseases.

Targeting and eradicating latent CNS reservoirs of HIV-1: Original strategies and new models.

Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.

Corrigendum: Editorial: The relevance of molecular mechanisms in HIV-1 latency and reactivation from latency.

[This corrects the article DOI: 10.3389/fcimb.2023.1190867.].

Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics.

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT.

Novel role of UHRF1 in the epigenetic repression of the latent HIV-1.

BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.

Suicide gene therapy in cancer and HIV-1 infection: An alternative to conventional treatments.

Suicide Gene Therapy (SGT) aims to introduce a gene encoding either a toxin or an enzyme making the targeted cell more sensitive to chemotherapy. SGT represents an alternative approach to combat pathologies where conventional treatments fail such as pancreatic cancer or the high-grade glioblastoma which are still desperately lethal. We review the possibility to use SGT to treat these cancers which have shown promising results in vitro and in preclinical trials. However, SGT has so far failed in phase III clinical trials thus further improvements are awaited. We can now take advantages of the many advances made in SGT for treating cancer to combat other pathologies such as HIV-1 infection. In the review we also discuss the feasibility to add SGT to the therapeutic arsenal used to cure HIV-1-infected patients. Indeed, preliminary results suggest that both productive and latently infected cells are targeted by the SGT. In the last section, we address the limitations of this approach and how we might improve it.

Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy.

Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.

Resveratrol Inhibits HCoV-229E and SARS-CoV-2 Coronavirus Replication In Vitro.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19.

Inhibition of HIV-1 gene transcription by KAP1 in myeloid lineage.

HIV-1 latency generates reservoirs that prevent viral eradication by the current therapies. To find strategies toward an HIV cure, detailed understandings of the molecular mechanisms underlying establishment and persistence of the reservoirs are needed. The cellular transcription factor KAP1 is known as a potent repressor of gene transcription. Here we report that KAP1 represses HIV-1 gene expression in myeloid cells including microglial cells, the major reservoir of the central nervous system. Mechanistically, KAP1 interacts and colocalizes with the viral transactivator Tat to promote its degradation via the proteasome pathway and repress HIV-1 gene expression. In myeloid models of latent HIV-1 infection, the depletion of KAP1 increased viral gene elongation and reactivated HIV-1 expression. Bound to the latent HIV-1 promoter, KAP1 associates and cooperates with CTIP2, a key epigenetic silencer of HIV-1 expression in microglial cells. In addition, Tat and CTIP2 compete for KAP1 binding suggesting a dynamic modulation of the KAP1 cellular partners upon HIV-1 infection. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of HIV-1 latency in myeloid cells.

Evolution of a concept: From accessory protein to key virulence factor, the case of HIV-1 Vpr.

Back in 1989 some studies have shown that the viral protein Vpr was dispensable for HIV-1 replication in vitro. From then the concept of accessory or auxiliary protein for Vpr has emerged and it is still used to date. However, Vpr soon appeared to be very important for in vivo virus spread and pathogenesis. Vpr has been involved in many biological functions including regulation of reverse transcriptase activity, the nuclear import of the pre-integration complex (PIC), HIV-1 transcription, gene splicing, apoptosis and in cell cycle arrest. Thus, we might rather consider Vpr as a true virulence factor instead of just an accessory factor. At present, Vpr can be regarded as a potential and promising target in different strategies aiming to fight infected cells including latently infected cells.

Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex.

Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.

Toxoplasma gondii ROP16 kinase silences the cyclin B1 gene promoter by hijacking host cell UHRF1-dependent epigenetic pathways.

Toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii, is one of the most common infections in the world due to the lifelong persistence of this parasite in a latent stage. This parasite hijacks host signaling pathways through epigenetic mechanisms which converge on key nuclear proteins. Here, we report a new parasite persistence strategy involving T. gondii rhoptry protein ROP16 secreted early during invasion, which targets the transcription factor UHRF1 (ubiquitin-like containing PHD and RING fingers domain 1), and leads to host cell cycle arrest. This is mediated by DNMT activity and chromatin remodeling at the cyclin B1 gene promoter through recruitment of phosphorylated UHRF1 associated with a repressive multienzymatic protein complex. This leads to deacetylation and methylation of histone H3 surrounding the cyclin B1 promoter to epigenetically silence its transcriptional activity. Moreover, T. gondii infection causes DNA hypermethylation in its host cell, by upregulation of DNMTs. ROP16 is already known to activate and phosphorylate protective immunity transcription factors such as STAT 3/6/5 and modulate host signaling pathways in a strain-dependent manner. Like in the case of STAT6, the strain-dependent effects of ROP16 on UHRF1 are dependent on a single amino-acid polymorphism in ROP16. This study demonstrates that Toxoplasma hijacks a new epigenetic initiator, UHRF1, through an early event initiated by the ROP16 parasite kinase.

Microglial Cells: The Main HIV-1 Reservoir in the Brain.

Despite efficient combination of the antiretroviral therapy (cART), which significantly decreased mortality and morbidity of HIV-1 infection, a definitive HIV cure has not been achieved. Hidden HIV-1 in cellular and anatomic reservoirs is the major hurdle toward a functional cure. Microglial cells, the Central Nervous system (CNS) resident macrophages, are one of the major cellular reservoirs of latent HIV-1. These cells are believed to be involved in the emergence of drugs resistance and reseeding peripheral tissues. Moreover, these long-life reservoirs are also involved in the development of HIV-1-associated neurocognitive diseases (HAND). Clearing these infected cells from the brain is therefore crucial to achieve a cure. However, many characteristics of microglial cells and the CNS hinder the eradication of these brain reservoirs. Better understandings of the specific molecular mechanisms of HIV-1 latency in microglial cells should help to design new molecules and new strategies preventing HAND and achieving HIV cure. Moreover, new strategies are needed to circumvent the limitations associated to anatomical sanctuaries with barriers such as the blood brain barrier (BBB) that reduce the access of drugs.