Cultivation, Differentiation, and Lentiviral Transduction of Human-Induced Pluripotent Stem Cell (hiPSC)-Derived Glutamatergic Neurons for Studying Human Tau.

Tau pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these disorders,  such as Alzheimer's disease, the neuronal axonal microtubule-binding Tau protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of Tau is accompanied by an abnormally high phosphorylation state of the Tau protein, and several downstream pathological consequences (e.g., loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of Tau sorting, missorting, and associated pathologies have been addressed in rodent models, few studies have addressed human Tau in physiological disease-relevant human neurons. Thus, suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene-carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, that is, synapse formation, Tau isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we describe a simple protocol for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation.

Capsid-dependent lentiviral restrictions.

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5, have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

An empowered, clinically viable hematopoietic stem cell gene therapy for the treatment of multisystemic mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.

Lentiviral gene therapy with IGF2-tagged GAA normalizes the skeletal muscle proteome in murine Pompe disease.

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor. Lentiviral gene therapy with LV-IGF2.GAAco showed superior efficacy in heart, skeletal muscle, and brain of Gaa -/- mice compared to gene therapy with untagged LV-GAAco. Here, we used quantitative mass spectrometry using TMT labeling to analyze the muscle proteome and the response to gene therapy in Gaa -/- mice. We found that muscle of Gaa -/- mice displayed altered levels of proteins including those with functions in the CLEAR signaling pathway, autophagy, cytoplasmic glycogen metabolism, calcium homeostasis, redox signaling, mitochondrial function, fatty acid transport, muscle contraction, cytoskeletal organization, phagosome maturation, and inflammation. Gene therapy with LV-GAAco resulted in partial correction of the muscle proteome, while gene therapy with LV-IGF2.GAAco resulted in a near-complete restoration to wild type levels without inducing extra proteomic changes, supporting clinical development of lentiviral gene therapy for Pompe disease. SIGNIFICANCE: Lysosomal glycogen accumulation is the primary cause of Pompe disease, and leads to a cascade of pathological events in cardiac and skeletal muscle and in the central nervous system. In this study, we identified the proteomic changes that are caused by Pompe disease in skeletal muscle of a mouse model. We showed that lentiviral gene therapy with LV-IGF2.GAAco nearly completely corrects disease-associated proteomic changes. This study supports the future clinical development of lentiviral gene therapy with LV-IGF2.GAAco as a new treatment option for Pompe disease.

Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

Chimeric TßRII-SE/Fc overexpression by a lentiviral vector exerts strong antitumoral activity on colorectal cancer-derived cell lines in vitro and on xenografts.

The TGF signaling pathway is a key regulator of cancer progression. In this work, we report for the first time the antitumor activity of TßRII-SE/Fc, a novel peptibody whose targeting domain is comprised of the soluble endogenous isoform of the human TGF-ß type II receptor (TßRII-SE). Overexpression of TßRIISE/Fc reduces in vitro cell proliferation and migration while inducing cell cycle arrest and apoptosis in human colorectal cancer-derived cell lines. Moreover, TßRII-SE/Fc overexpression reduces tumorigenicity in BALB/c nude athymic mice. Our results revealed that TRII-SE/Fc-expressing tumors were significantly reduced in size or were even incapable of developing. We also demonstrated that the novel peptibody has the ability to inhibit the canonical TGF-ß and BMP signaling pathways while identifying SMAD-dependent and independent proteins involved in tumor progression that are modulated by TßRII-SE/Fc. These findings provide insights into the underlying mechanism responsible for the antitumor activity of TßRII-SE/Fc. Although more studies are required to demonstrate the effectiveness and safety of the novel peptibody as a new therapeutic for the treatment of cancer, our initial in vitro and in vivo results in human colorectal tumor-derived cell lines are highly encouraging. Our results may serve as the foundation for further research and development of a novel biopharmaceutical for oncology.

Third-generation lentiviral gene therapy rescues function in a mouse model of Usher 1B.

Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6 months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear.

Biodistribution of lentiviral transduced adipose-derived stem cells for "ex-vivo" regional gene therapy for bone repair.

Ex-vivo gene therapy has been shown to be an effective method for treating bone defects in pre-clinical models. As gene therapy is explored as a potential treatment option in humans, an assessment of the safety profile becomes an important next step. The purpose of this study was to evaluate the biodistribution of viral particles at the defect site and various internal organs in a rat femoral defect model after implantation of human ASCs transduced with lentivirus (LV) with two-step transcriptional activation (TSTA) of bone morphogenetic protein-2 (LV-TSTA-BMP-2). Animals were sacrificed at 4-, 14-, 56-, and 84-days post implantation. The defects were treated with either a standard dose (SD) of 5 million cells or a high dose (HD) of 15 million cells to simulate a supratherapeutic dose. Treatment groups included (1) SD LV-TSTA-BMP-2 (2) HD LV-TSTA-BMP-2, (3) SD LV-TSTA-GFP (4) HD LV-TSTA-GFP and (5) SD nontransduced cells. The viral load at the defect site and ten organs was assessed at each timepoint. Histology of all organs, ipsilateral tibia, and femur were evaluated at each timepoint. There were nearly undetectable levels of LV-TSTA-BMP-2 transduced cells at the defect site at 84-days and no pathologic changes in any organ at all timepoints. In conclusion, human ASCs transduced with a lentiviral vector were both safe and effective in treating critical size bone defects in a pre-clinical model. These results suggest that regional gene therapy using lentiviral vector to treat bone defects has the potential to be a safe and effective treatment in humans.

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages.

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.

A glucose-sensing mechanism with glucose transporter 1 and pyruvate kinase in the area postrema regulates hepatic glucose production in rats.

The area postrema (AP) of the brain is exposed to circulating metabolites and hormones. However, whether AP detects glucose changes to exert biological responses remains unknown. Its neighboring nuclei, the nucleus tractus solitarius (NTS), responds to acute glucose infusion by inhibiting hepatic glucose production, but the mechanism also remains elusive. Herein, we characterized AP and NTS glucose-sensing mechanisms. Infusion of glucose into the AP, like the NTS, of chow rats suppressed glucose production during the pancreatic (basal insulin)-euglycemic clamps. Glucose transporter 1 or pyruvate kinase lentiviral-mediated knockdown in the AP negated AP glucose infusion to lower glucose production, while the glucoregulatory effect of NTS glucose infusion was also negated by knocking down glucose transporter 1 or pyruvate kinase in the NTS. Furthermore, we determined that high-fat (HF) feeding disrupts glucose infusion to lower glucose production in association with a modest reduction in the expression of glucose transporter 1, but not pyruvate kinase, in the AP and NTS. However, pyruvate dehydrogenase activator dichloroacetate infusion into the AP or NTS that enhanced downstream pyruvate metabolism and recapitulated the glucoregulatory effect of glucose in chow rats still failed to lower glucose production in HF rats. We discovered that a glucose transporter 1- and pyruvate kinase-dependent glucose-sensing mechanism in the AP (as well as the NTS) lowers glucose production in chow rats and that HF disrupts the glucose-sensing mechanism that is downstream of pyruvate metabolism in the AP and NTS. These findings highlight the role of AP and NTS in mediating glucose to regulate hepatic glucose production.

Increased lentivirus titer using ultra expression vectors.

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers.

Production of CRISPRi-engineered primary human mammary epithelial cells with baboon envelope pseudotyped lentiviral vectors.

Primary human mammary epithelial cells (pHMECs) are known to be remarkably difficult to engineer genetically. Here, we present a protocol for efficient transduction of pHMECs using a baboon retroviral envelope glycoprotein for pseudotyping of lentiviral vectors (BaEV-LVs). We describe the preparation of the BaEV-LVs, the isolation of pHMECs from breast samples, and the subsequent transduction of pHMECs. We also detail the use of CRISPRi technology to efficiently silence gene expression in pHMECs, which can then be used for functional assays. For complete details on the use and execution of this protocol, please refer to Richart et al. (2022).1.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Lentivirus-shRNA Mediated Prolyl Hydroxylase 2 Knockdown Increases HIF-1α and Inhibits Nucleus Pulposus Cells Degeneration.

Hypoxia-inducible factor (HIF) plays a crucial role in regulating the hypoxia-inducible state of nucleus pulposus cells in the intervertebral disc. In addition, the oxygen-dependent conversion of HIF-1α in nucleus pulposus cells is controlled by the protein proline 4-hydroxylase domain (PHD) family. To explore whether HIF-1α can be regulated by modulating PHD homologs to inhibit nucleus pulposus degeneration, PHD2-shRNAs were designed and a PHD2 interference vector was constructed. The expression of HIF-1α and PHD2 genes in the nucleus pulposus cells in the experimental group was detected by RT-PCR, and the expression of HIF-1α, MMP-2, Aggrecan, and Col II proteins in the P0-P3 cells in the experimental group and the control group was detected by Western blotting. The apoptosis of P0-P3 nucleus pulposus cells was detected by flow cytometry. After lentivirus infection, the interference efficiency of the PHD2 gene decreased with cell passage. The apoptosis of P1-P3 cells in the experimental group was significantly lower than that in the control group or degeneration group. Compared to the control group, the expression of HIF-1α, Aggrecan, and Col II proteins increased significantly, and the expression of MMP-2 protein decreased significantly. In conclusion, interference with PHD2 can upregulate the expression of HIF-1α, accelerate anabolism, reduce catabolism, inhibit apoptosis of nucleus pulposus cells, and then these can inhibit degeneration of nucleus pulposus cells. Our results can provide an effective therapeutic target in intervertebral discs during intervertebral disc degeneration, and this may have important clinical significance.

Neutralizing peripheral circulating IL1ß slows the progression of ALS in a lentivirus-infected OPTNE478G mouse model.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is irreversible and fatal within 3-5 years, with limited options for treatment. It is imperative to develop a symptom-based treatment that may increase the survival of ALS patients and improve their quality of life. Inflammation status, especially elevated interleukin 1ß (IL1ß), has been reported to play a critical role in ALS progression. Our study determined that neutralizing circulating IL1ß slows down the progression of ALS in an ALS mouse model. METHODS: The ALS mouse model was developed by microinjection of lentivirus-carrying OPTNE478G (optineurin, a mutation from ALS patients) into the intra-motor cortex of mice. Peripheral circulating IL1ß was neutralized by injecting anti-IL1ß antibody into the tail vein. Enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) were carried out to determine the protein and gene expression levels of IL1ß. TUNEL assay was used to assess the neural cell death. Immunofluorescent staining of MAP2 and CASP3 was accomplished to evaluate neuronal cell apoptosis. Glial fibrillary acidic protein staining was performed to analyze the number of astrocytes. Rotarod test, grip strength test, balance beam test, and footprint test were conducted to assess the locomotive function after anti-IL1ß treatment. RESULTS: The model revealed that neuroinflammation contributes to ALS progression. ALS mice exhibited elevated neuroinflammation and IL1ß secretion. After anti-IL1ß treatment, ALS mice revealed decreased neural cell death and astrogliosis and gained improved muscle strength and motor ability. CONCLUSIONS: Blocking IL1ß is a promising strategy to slow down the progression of ALS.

Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML.

We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real­time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 109 and 2.61 × 109 IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.

Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications.

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34+ cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34+ cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34+ cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.

Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells.

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.