PHLDA1-PRDM1 mediates the effect of lentiviral vectors on fate-determination of human retinal progenitor cells.

Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but their long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also up-regulated by lentiviral vectors exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 axis.

Preclinical safety assessment of modified gamma globin lentiviral vector-mediated autologous hematopoietic stem cell gene therapy for hemoglobinopathies.

Previously, we reported the development of a human Aγ-globin gene lentivirus (LV), GbG, which expresses high levels of HbF to correct the sickle cell anemia (SCA) phenotype in the Berkeley SCA mouse model, and then modified the γ-globin gene by substituting glycine at codon 16 with aspartic acid in the Aγ-globin gene to generate GbGM LV. In the present study, we evaluated the long-term safety of human Aγ-globin gene carrying GbGM LV in wild-type mice after primary and secondary transplants of GbGM-modified hematopoietic stem cells (HSC) over 18 months. The safety of the GbGM bone marrow transplant was assessed by monitoring the effects on body weight, hematology, histopathology, malignancy formation, and survival. Mice transplanted with Mock-transduced and spleen focus forming virus (SFFV) γ-retroviral vector (RV)-transduced HSC served as negative and positive controls, respectively. The mean donor-cell engraftment was comparable across Mock, GbGM LV, and SFFV RV groups. There were no significant differences in body weight, clinical signs, immunophenotype, or histopathology in the GbGM-treated mice compared to controls. Four SFFV RV-treated mice, but none of the GbGM-treated mice, developed donor-derived, vector-positive lymphomas as demonstrated by flow cytometry analysis and in situ hybridization. These results highlight the safety of the administration of GbGM LV-modified HSC with long-term follow-up after primary and secondary transplants in mice. This data supported the initiation of phase 1/2 first-in-human SCA clinical trial in the United States.

Novel Vpx virus-like particles to improve cytarabine treatment response against acute myeloid leukemia.

Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.

Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.

BACKGROUND: Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. METHODS: As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. RESULTS: In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. CONCLUSIONS: These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

CCDC59 alleviates bleomycin-induced inflammation and pulmonary fibrosis by increasing SP-B and SP-C expression in mice.

BACKGROUND: Pulmonary fibrosis is a progressive disease with high incidence and poor prognosis. It is urgent to explore new therapeutic methods for pulmonary fibrosis. As a new treatment method, gene therapy has attracted more and more attention. CCDC59 is a transcriptional coactivator of SP-B and SP-C. Our study mainly aims to explore the effect of overexpression of CCDC59 gene in pulmonary fibrosis of mice. METHODS: CCDC59 overexpressing lentivirus was constructed and then concentrated. RT-qPCR, Western blotting, and immunofluorescence assays were used to detect the expression of CCDC59, SP-B and SP-C protein in cell line and lung tissues after infected with lentivirus. Immunohistochemical staining and hematoxylin-eosin staining assays were used to assess the degree of fibrosis and ELISA assay was used to detect the concentrations of inflammatory factors, SP-B, and SP-C in bronchoalveolar lavage fluid of mice. Dynamic changes of mice lung function at various time points were assessed by lung function test assay. HIPPO pathway and proliferation capacity of alveolar type II epithelial cells were evaluated by immunofluorescence staining and Western blotting. RESULTS: Results showed that endotracheal instillation of CCDC59 overexpressed lentivirus significantly alleviated bleomycin-induced inflammation and pulmonary fibrosis in mice. Overexpression of CCDC59 protein in type II alveolar epithelial cells can enhance the expression of SP-B and SP-C. Overexpression of CCDC59 protein significantly protected against pulmonary inflammatory response and improved lung function of mice. Overexpression of CCDC59 protein significantly alleviated the hyperactivation of HIPPO pathway and increased the proliferative capacity of type II alveolar epithelial cells in lung. CONCLUSION: CCDC59 can alleviate inflammation and pulmonary fibrosis in mice by upregulating the expression of SP-B and SP-C in type II alveolar epithelial cells and alleviating the hyperactivation of HIPPO pathway. Our study offers a new potential treatment for pulmonary fibrosis.

[Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity].

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by ß1 (PSMB6), ß2 (PSMB7), and ß5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of ß1, ß2, and ß5 with their respective immuno-subunits ß1i (PSMB9), ß2i (PSMB10), and ß5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9-eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

Heart immunoengineering by lentiviral vector-mediated genetic modification during normothermic ex vivo perfusion.

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shß2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.

Towards a Cure for Diamond-Blackfan Anemia: Views on Gene Therapy.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.

Transcription Factor-Mediated Generation of Dopaminergic Neurons from Human iPSCs-A Comparison of Methods.

Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.

Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection.

Lentiviral vectors derived from human immunodeficiency virus type I are widely used to deliver functional gene copies to mammalian cells for research and gene therapies. Post-transcriptional splicing of lentiviral vector transgene in transduced host and transfected producer cells presents barriers to widespread application of lentiviral vector-based therapies. The present study examined effects of indole derivative compound IDC16 on splicing of lentiviral vector transcripts in producer cells and corresponding yield of infectious lentiviral vectors. Indole IDC16 was shown previously to modify alternative splicing in human immunodeficiency virus type I. Human embryonic kidney 293T cells were transiently transfected by 3rd generation backbone and packaging plasmids using polyethyleneimine. Reverse transcription-quantitative polymerase chain reaction of the fraction of unspliced genomes in human embryonic kidney 293T cells increased up to 31% upon the indole's treatment at 2.5 uM. Corresponding yield of infectious lentiviral vectors decreased up to 4.5-fold in a cell transduction assay. Adjusting timing and duration of IDC16 treatment indicated that the indole's disruption of early stages of transfection and cell cycle had a greater effect on exponential time course of lentiviral vector production than its reduction of post-transcriptional splicing. Decrease in transfected human embryonic kidney 293T proliferation by IDC16 became significant at 10 uM. These findings indicated contributions by early-stage transfection, cell proliferation, and post-transcriptional splicing in transient transfection of human embryonic kidney 293T cells for lentiviral vector production.

Prospects and limitations of cumate-inducible lentivirus as a tool for investigating VEGF-A-mediated pathology in diabetic retinopathy.

Diabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR. In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability. Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry. Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis. In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mechanistic studies in cellular drug discovery, its use is not a feasible approach to model DR in in vivo studies due to cumate-induced retinal toxicity.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

Liposome-lentivirus for miRNA therapy with molecular mechanism study.

BACKGROUND: Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability. METHODS: We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs). RESULTS: MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3ß at ser 9 (p-GSK-3ß S9) to inactivate GSK3ß, and facilitate translocation of ß-catenin into the nucleus to activate the Wnt/ß-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3ß/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs. CONCLUSIONS: These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery.

Stable Expression by Lentiviral Transduction of Cells.

Lentiviral gene transfer represents a versatile and powerful method for genetic transduction of many cell lines and primary cells including "hard-to-transfect" cells. As a consequence of the integration of the recombinant lentiviral vector into the cellular genome, the transgene is stably maintained, and long-term producing cells are established. Here, we describe the current state of the art and give details for lab-scale production of lentiviral vectors as well as for infection and titration of the viral vectors.

Development of a novel three-dimensional injection guide for trigeminal ganglia.

BACKGROUND: Trigeminal ganglion (TG) plays an important role in the process of orthodontic pain. It's necessary to design an accurate, precise and minimally invasive trigeminal ganglion injection guide plate to study TG. METHODS: Micro-CT was used to obtain the Dicom format data, and three-dimensional (3D) software (mimics and magics23.03) was used to reconstruct 3D head models. Design and modifications of the TG injection guide plate were performed in Magic 23.03 software, and the guide plate was produced by a 3D stereolithography printer. X-ray, micro-CT, Evans blue, and virus transduction were used to demonstrate the accuracy of the guide-assisted injection. Pain levels were evaluated after using the injection guide by a bite force test and Von Frey test. RESULTS: X-ray and micro-CT tests confirmed that the injection needle reached the bilateral trigeminal ganglia fossa. The Evans blue test and virus transduction proved that the injected drug could be accurately injected into the bilateral trigeminal ganglion and the lentivirus could be successfully transfected. The percentage of accurate injection was 10/10 (bilateral trigeminal ganglia). Orofacial pain induced by the trigeminal ganglion injection was mild and returned to baseline within seven days. CONCLUSION: The injection guide described in this study is viable and reliable for the delivery of drugs and virus transduction into the trigeminal ganglia.

Non-clinical safety assessment of novel drug modalities: Genome safety perspectives on viral-, nuclease- and nucleotide-based gene therapies.

Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.

Surface-modified injectable poly(ethylene-glycol) diacrylate-based cryogels for localized gene delivery.

Lentiviral transduction is widely used in research, has shown promise in clinical trials involving gene therapy and has been approved for CAR-T cell immunotherapy. However, most modifications are doneex vivoand rely on systemic administration of large numbers of transduced cells for clinical applications. A novel approach utilizingin situbiomaterial-based gene delivery can reduce off-target side effects while enhancing effectiveness of the manipulation process. In this study, poly(ethylene glycol) diacrylate (PEGDA)-based scaffolds were developed to enablein situlentivirus-mediated transduction. Compared to other widely popular biomaterials, PEGDA stands out due to its robustness and cost-effectiveness. These scaffolds, prepared via cryogelation, are capable of flowing through surgical needles in bothin vitroandin vivoconditions, and promptly regain their original shape. Modification with poly(L-lysine) (PLL) enables lentivirus immobilization while interconnected macroporous structure allows cell infiltration into these matrices, thereby facilitating cell-virus interaction over a large surface area for efficient transduction. Notably, these preformed injectable scaffolds demonstrate hemocompatibility, cell viability and minimally inflammatory response as shown by ourin vitroandin vivostudies involving histology and immunophenotyping of infiltrating cells. This study marks the first instance of using preformed injectable scaffolds for delivery of lentivectors, which offers a non-invasive and localized approach for delivery of factors enablingin situlentiviral transduction suitable for both tissue engineering and immunotherapeutic applications.

Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry.

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

Challenges in the analysis of pharmaceutical lentiviral vector products by orthogonal and complementary physical (nano)particle characterization techniques.

Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to âˆ¼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.