Development of KoRV-pseudotyped lentiviral vectors for efficient gene transfer into freshly isolated immune cells.

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.

Optimized peptide nanofibrils as efficient transduction enhancers for in vitro and ex vivo gene transfer.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.

Correction: Click functionalized biocompatible gadolinium oxide core-shell nanocarriers for imaging of breast cancer cells.

[This corrects the article DOI: 10.1039/D2RA00347C.].

Click functionalized biocompatible gadolinium oxide core-shell nanocarriers for imaging of breast cancer cells.

Site-specific delivery using functionalized nanocarriers is in high demand in imaging applications of modern clinical research. To improve the imaging capabilities of conventionally used contrast agents and expand the targeting accuracy, functional gadolinium oxide based nanocarriers originated from homogeneous core shells structures (Gd2O3@SiO2@Fe3O4) were developed using a multilayer formation approach. The synthesis and chemical configuration for the covalent binding of macrocyclic chelating agents and estrogen targeting molecules on these nanocarriers were designed by a two-step chemical synthesis method. Initially, SiO2@Fe3O4 structures were prepared and encapsulated with a homogenous thin Gd2O3 overlayer. The exterior surface of the as-prepared carriers offered chemical binding with a breast cancer specific estrogen molecule, covalently grafted through a Click-Chemistry protocol. In the next step, to enhance the diagnostic imaging capabilities of these carriers, thiocyanate-linked chelator molecule, DOTA, was attached to the surface of estrogen bound Gd2O3@SiO2@Fe3O4 using basic reaction conditions. The active amino groups before and after conjugation of estrogen molecules on the surface were quantified using a fluorescamine based approach. Due to the covalent binding of the macrocyclic chelator to the Gd2O3@SiO2@Fe3O4 surface, core shell carriers showed potential radiolabeling efficiency using positron emitter radionuclide, gallium-68 (68Ga). Intracellular uptake of estrogen-conjugated carriers was evaluated with MCF7 breast cancer cell lines using confocal laser scanning microscopy and fluorescent flow cytometry. In addition, in vitro cytotoxicity studies of functional nanocarriers as compared to bare nanoparticles showed reduced toxicity to HEK-293 cells demonstrating the role of surface attached molecules in preventing direct exposure of the Gd2O3 surface to the cells. The as-developed gadolinium based nanocarriers presented excellent capabilities as biocompatible target-specific imaging probes which indicates great potential in the field of dual-mode contrast agents.

High efficiency capture of biomarker miRNA15a for noninvasive diagnosis of malignant kidney tumors.

To date, there are no preoperative and quantitative dynamics in clinical practice that can reliably differentiate between a benign and malignant renal cell carcinoma (RCC). For monitoring different analytes in body fluids, more than 40 different molecular biomarkers have been identified, however, they are associated with limited clinical sensitivity and/or non-optimal specificity due to their leaky nature. Previous work on RCC demonstrated the miRNA15a to be reliable and novel biomarker with 98.1% specificity and 100% sensitivity. Despite the high potential of miRNA15a biomarker, its clinical application is considerably hampered by the insensitive nature of the detection methods and low concentration of biomarker in samples that is aggravated by the high level of contamination due to other solutes present in body fluids. In this work, a non-invasive quantitative approach is demonstrated to overcome such diagnostics issues through biotin-streptavidin binding and fluorescence active magnetic nanocarriers that ensured prompt isolation, enrichment and purification of the biomarker miRNA15a from urine. The study demonstrates that detectable low levels of these miRNAs through miRNA capturing nanocarriers can potentially function as advanced diagnostic markers for the non-invasive investigation and early detection of renal cancer.

18F-Labeled magnetic nanovectors for bimodal cellular imaging.

Surface modification of nanocarriers enables selective attachment to specific molecular targets within a complex biological environment. Besides the enhanced uptake due to specific interactions, the surface ligands can be utilized for radiolabeling applications for bimodal imaging ensured by positron emission topography (PET) and magnetic resonance imaging (MRI) functions in one source. Herein, we describe the surface functionalization of magnetite (Fe3O4) with folic acid as a target vector. Additionally, the magnetic nanocarriers were conjugated with appropriate ligands for subsequent copper-catalyzed azide-alkyne cycloaddition or carbodiimide coupling reactions to successfully achieve radiolabeling with the PET-emitter 18F. The phase composition (XRD) and size analysis (TEM) confirmed the formation of Fe3O4 nanoparticles (6.82 nm ± 0.52 nm). The quantification of various surface functionalities was performed by Fourier-transform infrared spectroscopy (FT-IR) and ultraviolet-visible microscopy (UV-Vis). An innovative magnetic-HPLC method was developed in this work for the determination of the radiochemical yield of the 18F-labeled NPs. The as-prepared Fe3O4 particles demonstrated high radiochemical yields and showed high cellular uptake in a folate receptor overexpressing MCF-7 cell line, validating bimodal imaging chemical design and a magnetic HPLC system. This novel approach, combining folic acid-capped Fe3O4 nanocarriers as a targeting vector with 18F labeling, is promising to apply this probe for bimodal PET/MR-studies.

Receptor-Mediated In Vivo Targeting of Breast Cancer Cells with 17α-Ethynylestradiol-Conjugated Silica-Coated Gold Nanoparticles.

Efficient therapies for breast cancer remain elusive because of the lack of strategies for targeted transport and receptor-mediated uptake of synthetic drug molecules by cancer cells. Conjugation of nanoparticles (NPs) with active targeting ligands enabling selective molecular recognition of antigens expressed on the surface of cancer cells is promising for localization and treatment of malignant cells. In this study, covalent attachment of synthetic estrogen 17α-ethynylestradiol on the silica (SiO2) shell of silica-gold NPs (SiO2@Au) was undertaken to improve the cancer-targeting ability of the nano-biotags. Chemical and structural analysis of the bioconjugates examined in solution (UV-vis and ξ-potential) and solid state (Fourier transform infrared spectroscopy, X-ray diffractometry, and transmission electron microscopy) confirmed the identity of the carrier particles and surface-bound ligands. The mesoporous silica shell served as a reservoir for anticancer drugs (doxorubicin and quercetin) and to facilitate covalent attachment of receptor molecules by click chemistry protocols. The chemoselective recognition between the nanoconjugates and cell membranes was successfully demonstrated by the accumulation of nanoprobes in the tumor tissue of mice with subcutaneous breast cancer, whereas healthy cells were unaffected. The drug release studies showed sustained release kinetics over several weeks. These findings elaborate the exceptional selectivity and potential of estrogen-coated nano-biolabels in efficient diagnosis and detection of breast cancer cells.

Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties.

Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.