Search Results (154)

Over 35 years of history, the field of gene therapy has undergone much progress. The initial concept—the replacement of dysfunctional genes with correct ones—has advanced to the next stage and reached the level of precise genome editing. Dozens of gene therapy products based on viral and non-viral delivery platforms have been approved, marking the dawn of the gene therapy era. These viral vector strategies rely on adenoviruses, adeno-associated viruses, lentivirus-derived tools, and so on. From the middle of the gene therapy transition, despite the challenges and serious negative consequences, the lentiviral vector has emerged as a cornerstone and demonstrated benefits in fields ranging from basic science to gene therapy. Therefore, we outline the importance of lentiviral vectors in the gene therapy era by focusing on their roles in the clinical usage, derivation, and development of next-generation platforms, as well as their pseudotyping. Full article

Gaucher disease (GD) is an autosomal recessive disorder caused by the deficient activity of the lysosomal enzyme glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) remains the standard of care for non-neuropathic GD patients, its high cost significantly limits accessibility. To enhance production efficiency, we developed a lentiviral system encoding a codon-optimized GCase gene driven by the human elongation factor 1a (hEF1α) promoter for stable production in human cell lines. A functional lentiviral vector, LV_EF1α_GBA_Opt, was generated at a titer of 7.88 × 10⁸ LV particles/mL as determined by qPCR. Six transduction cycles were performed at a multiplicity of infection of 30–50. The transduced heterogeneous human cell population showed GCase-specific activity of 307.5 ± 53.49 nmol/mg protein/h, which represents a 3.21-fold increase compared to wild-type 293FT cells (95.58 ± 16.5 nmol/mg protein/h). Following single-cell cloning, two clones showed specific activity of 763.8 ± 135.1 and 752.0 ± 152.1 nmol/mg/h (clones 15 and 16, respectively). These results show that codon optimization, a lentiviral delivery system, and clonal selection together enable the establishment of stable human cell lines capable of producing high levels of biologically active, synthetic recombinant GCase in vitro. Further studies are warranted for the functional validation in GD patient-derived fibroblasts and animal models. Full article

Background and Purpose: Glioblastoma remains a therapeutic challenge with a poor prognosis despite multimodal treatments. Reporter-based magnetic resonance imaging (MRI) offers a promising approach for tumor visualization, but its efficacy depends on sufficient reporter gene expression. This study aimed to develop a chimeric element-regulated ferritin heavy chain 1 (FTH1) reporter system to enhance MRI-based glioma detection while enabling targeted therapy via transferrin receptor (TfR)-mediated drug delivery. Methods: Using gene cloning techniques, we constructed a chimeric FTH1 expression system comprising tumor-specific PEG3 promoter (transcriptional control), bFGF-2 5′UTR (translational enhancement), and WPRE (mRNA stabilization). Lentiviral vectors delivered constructs to U251 glioblastoma cells and xenografts. FTH1/TfR expression was validated by Western blot and immunofluorescence. Iron accumulation was assessed via Prussian blue staining and TEM. MRI evaluated T2 signal changes. Transferrin-modified doxorubicin liposomes (Tf-LPD) were characterized for size and drug loading and tested for cellular uptake and cytotoxicity in vitro. In vivo therapeutic efficacy was assessed in nude mouse models through tumor volume measurement, MR imaging, and histopathology. Results: The chimeric system increased FTH1 expression significantly over PEG3-only controls (p < 0.01), with an increase of nearly 1.5-fold compared to the negative and blank groups and approximately a two-fold increase relative to the single promoter group, with corresponding TfR upregulation. Enhanced iron accumulation reduced T2 relaxation times significantly (p < 0.01), improving MR contrast. Tf-LPD (115 nm, 70% encapsulation) showed TfR-dependent uptake, inducing obvious apoptosis in high-TfR cells compared with that in controls. In vivo, Tf-LPD reduced tumor growth markedly in chimeric-system xenografts versus controls, with concurrent MR signal attenuation. Conclusions: The chimeric regulatory strategy overcomes limitations of single-element systems, demonstrating significant potential for integrated glioma theranostics. Its modular design may be adaptable to other reporter genes and malignancies. Full article

Current osteoarthritis (OA) therapies fail to yield long-term clinical benefits, due in part to the lack of a mechanism for the targeted and confined delivery of therapeutics to OA joints. This study evaluates if M2 macrophages are effective cell vehicles for the targeted and confined delivery of therapeutic genes to OA joints. CT bioluminescence in vivo cell tracing and fluorescent microscopy reveal that intraarticularly injected M2 macrophages were recruited to and retained at inflamed synovia. The feasibility of an M2 macrophage-based adoptive gene transfer strategy for OA was assessed using IL-1Ra as the therapeutic gene in a mouse tibial plateau injury model. Mouse M2 macrophages were transduced with lentiviral vectors expressing IL-1Ra or GFP. The transduced macrophages were intraarticularly injected into injured joints at 7 days post-injury and OA progression was monitored with plasma COMP and histology at 4 weeks. The IL-1Ra-expressing M2 macrophage treatment reduced plasma COMP, increased the area and width of the articular cartilage layer, decreased synovium thickness, and reduced the OARSI OA score without affecting the osteophyte maturity and meniscus scores when compared to the GFP-expressing M2 macrophage-treated or PBS-treated controls. When the treatment was given at 5 weeks post-injury, at which time OA should have developed, the IL-1Ra-M2 macrophage treatment also reduced plasma COMP, had a greater articular cartilage area and width, decreased synovial thickness, and reduced the OARSI OA score without an effect on the meniscus and osteophyte maturity scores at 8 weeks post-injury. In conclusion, the IL-1Ra-M2 macrophage treatment, given before or after OA was developed, delayed OA progression, indicating that the M2 macrophage-based adoptive gene transfer strategy for OA is tenable. Full article

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder causing systemic skeletal dysplasia due to a deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) enzyme activity, leading to the impaired degradation and accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate. While treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available, they have significant limitations regarding efficacy in skeletal tissues and long-term safety, highlighting the need for more effective therapies. We evaluated a novel gene therapy approach using a dual Integrase-deficient lentiviral vector (IDLV) to deliver an expression cassette that includes human GALNS cDNA and Cas9 sgRNA, targeting the upstream region of the mouse Galns initial codon. This approach leverages the endogenous promoter to drive transgene expression. We assessed in vitro transduction, editing, and functional correction in NIH3T3 and MPS IVA mouse fibroblasts. In vivo efficacy was successfully evaluated via the facial vein injection in MPS IVA newborn mice. In vitro, this IDLV platform demonstrated supraphysiological GALNS activity in cell lysate, resulting in the normalization of KS levels. In vivo direct IDLV platform in newborn MPS IVA mice led to sustained plasma GALNS activity, reduced plasma KS, and favorable biodistribution. Partial correction of heart and bone pathology was observed, with no vector toxicity and minimal antibody responses. This dual IDLV-CRISPR/Cas9 approach effectively mediated targeted GALNS knock-in, yielding sustained enzyme activity, reduced KS storage, and partial pathological amelioration in MPS IVA mice. In conclusion, IDLVs represent an efficient, safe platform for delivering the CRISPR/Cas9 gene editing system for MPS IVA. Full article

Chronic myeloid leukemia (CML) is a leading example of a malignancy where a molecular targeted therapy revolutionized treatment but has rarely led to cures. Overcoming tyrosine kinase inhibitor (TKI) drug resistance remains a challenge in the treatment of CML. We have recently identified miR-185 as a predictive biomarker where reduced expression in CD34⁺ treatment-naïve CML cells was associated with TKI resistance. We have also identified PAK6 as a target gene of miR-185 that was upregulated in CD34⁺ TKI-nonresponder cells. However, its role in regulating TKI resistance remains largely unknown. In this study, we specifically targeted PAK6 in imatinib (IM)-resistant cells and CD34⁺ stem/progenitor cells from IM-nonresponders using a lentiviral-mediated PAK6 knockdown strategy. Interestingly, the genetic and pharmacological suppression of PAK6 significantly reduced proliferation and increased apoptosis in TKI-resistant cells. Cell survivability was further diminished when IM was combined with PAK6 knockdown. Importantly, PAK6 inhibition in TKI-resistant cells induced cell cycle arrest in the G2-M phase and cellular senescence, accompanied by increased levels of DNA damage-associated senescence markers. Mechanically, we identified a PAK6-mediated MDM2-p21 axis that regulates cell cycle arrest and senescence. Thus, PAK6 plays a critical role in determining alternative cell fates in leukemic cells, and targeting PAK6 may offer a therapeutic strategy to selectively eradicate TKI-resistant cells. Full article

Background/Objectives: Radiolabeled biomolecules specifically targeting overexpressed structures on tumor cells hold great potential for prostate cancer (PCa) imaging and therapy. Due to heterogeneous target expression, single radiopharmaceuticals may not detect or treat all lesions, while simultaneously applying two or more radiotracers potentially improves staging, stratification, and therapy of cancer patients. This study explores a dual-tracer SPECT approach using [¹¹¹In]In-RM2 (targeting the gastrin-releasing peptide receptor, GRPR) and [^99mTc]Tc-PSMA-I&S (targeting the prostate-specific membrane antigen, PSMA) as a proof of concept. To mimic heterogeneous tumor lesions in the same individual, we aimed to establish a dual xenograft mouse model for preclinical evaluation. Methods: CHO-K1 cells underwent lentiviral transduction for human GRPR or human PSMA overexpression. Six-to-eight-week-old female immunodeficient mice (NOD SCID) were subsequently inoculated with transduced CHO-K1 cells in both flanks, enabling a dual xenograft with similar target density and growth of both xenografts. Respective dual-isotope imaging and γ-counting protocols were established. Target expression was analyzed ex vivo by Western blotting. Results: In vitro studies showed similar target-specific binding and internalization of [¹¹¹In]In-RM2 and [^99mTc]Tc-PSMA-I&S in transduced CHO-K1 cells compared to reference lines PC-3 and LNCaP. However, in vivo imaging showed negligible tumor uptake in xenografts of the transduced cell lines. Ex vivo analysis indicated a loss of the respective biomarkers in the xenografts. Conclusions: Although the technical feasibility of a dual-tracer SPECT imaging approach using ¹¹¹In and ^99mTc has been demonstrated, the potential of [^99mTc]Tc-PSMA-I&S and [¹¹¹In]In-RM2 in a dual-tracer cocktail to improve PCa diagnosis could not be verified. The animal model, and in particular the transduced cell lines developed exclusively for this project, proved to be unsuitable for this purpose. The in/ex vivo experiments indicated that results from an in vitro model may not necessarily be successfully transferred to an in vivo setting. To assess the potential of this dual-tracer concept to improve PCa diagnosis, optimized in vivo models are needed. Nevertheless, our strategies address key challenges in dual-tracer applications, aiming to optimize future SPECT imaging approaches. Full article

Lentiviral vectors (LVs) have become a fundamental tool in gene therapy due to their unique ability to transduce both dividing and non-dividing cells, transfer large genes of up to 10 kb, and facilitate stable, long-term expression of therapeutic genes into target cells. A key application of LVs is the ex vivo genetic modification of patient-derived cells, such as the production of CAR-T cells by transducing isolated T cells with LVs to express the CAR gene, enabling them to target and destroy cancer cells once infused back into the patient. However, these ex vivo gene therapy drugs are often dismally unaffordable due to the complex procedures involved, including cell isolation, genetic modification, and expansion, along with the significant risks associated with immune conditioning to ensure successful engraftment. To overcome these barriers, direct in vivo transgene delivery to physiologically relevant cells has been explored, bypassing the need for ex vivo manipulations and reducing costs. Yet, a major challenge in this approach is engineering LV cell tropism to ensure the precise targeting of specific cells while avoiding off-target effects. Recent advances in modifying LV surface proteins have shown promise, including the successful in vivo generation of CAR T cells and ensuing clinical trials. This review is aimed at providing an up-to-date account of the progress in engineering LV tropism, covering the utility of different heterologous viral envelopes and their engineering to achieve cell-type-specific delivery and host immune evasion, and highlighting the potential of in vivo gene therapy to improve the affordability and accessibility of life-saving treatments. Full article

Inborn errors of metabolism (IEMs) are a group of disorders resulting from defects in enzymes in metabolic pathways. These disorders impact the processing of metabolites, leading to a wide array of effects on each organ system. Advances in genetic screening have allowed for the early identification and intervention of IEMs, traditionally in the form of enzyme replacement or vitamin supplementation. However, many IEMs disrupt essential metabolic pathways where simple supplementation proves ineffective, resulting in substantial disease burden. In the case of renal IEMs, metabolic pathway disruption leads to the onset of chronic kidney disease (CKD). For these diseases, genetic therapy provides hope. Over the past few decades, the technology for genetic therapy has emerged as a promising solution to these disorders. These therapies aim to correct the source of the defect in the genetic code so that patients may live full, unencumbered lives. In this review, we searched a large database to identify IEMs that affect the kidney and investigated the current landscape and progression of gene therapy technology. Multiple promising genetic therapies were identified for IEMs affecting the kidney, including primary hyperoxaluria, argininemia, glycogen storage diseases Ia and Ib, and Fabry disease. Emerging gene therapy approaches using adeno-associated virus (AAV) vectors, lentiviral vectors, and CRISPR/Cas9 techniques hold promising potential to provide curative treatments for additional single-mutation disorders. Full article

Chimeric Antigen Receptor T-cell (CAR-T) therapy has emerged as a transformative approach for cancer treatment, demonstrating remarkable success in patients with relapsed and refractory hematological malignancies. However, challenges persist in optimizing CAR-T cell production and improving therapeutic outcomes. One of the major hurdles is the efficiency of retroviral or lentiviral transduction during CAR-T cell manufacturing. Additionally, the heterogeneity of T-cell populations isolated from patients can impact CAR-T cell effectiveness and persistence in vivo. This article explores a novel strategy to address these challenges by focusing on serum-free medium and additive optimization. We propose a unique approach that incorporates the culturing of T cells in Nutri-T medium, along with 24 h of exposure to combined low concentrations of BX795 and rosuvastatin, to enhance the transduction efficacy and functionality of CAR-T cells. The results presented here provide promising insights into the potential of this strategy to produce more effective CAR-T cells for immunotherapy, ultimately advancing the field and benefiting cancer patients worldwide. Full article

Hereditary Tyrosinemia Type-1 (HT1), an inherited error of metabolism caused by a mutation in the fumarylacetoacetate hydrolase gene, is associated with liver disease, severe morbidity, and early mortality. The use of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) has almost eradicated the acute HT1 symptoms and childhood mortality. However, patient outcomes remain unsatisfactory due to the neurocognitive effects of NTBC and the requirement for a strict low-protein diet. Gene therapy (GT) offers a potential single-dose cure for HT1, and there is now abundant preclinical data showing how a range of vector-nucleotide payload combinations could be used with curative intent, rather than continued reliance on amelioration. Unfortunately, there have been no HT1-directed clinical trials reported, and so it is unclear which promising pre-clinical approach has the greatest chance of successful translation. Here, to fill this knowledge gap, available HT1 preclinical data and available clinical trial data pertaining to liver-directed GT for other diseases are reviewed. The aim is to establish which vector-payload combination has the most potential as a one-dose HT1 cure. Analysis provides a strong case for progressing lentiviral-based approaches into clinical trials. However, other vector-payload combinations may be more scientifically and commercially viable, but these options require additional investigation. Full article

Lentiviral vector-transduced T cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the effects of host genetic variation on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge gap, we characterized hepatic gene delivery by lentiviral vectors across the Collaborative Cross (CC) mouse genetic reference population. For 24 weeks, we periodically measured hepatic luciferase expression from lentiviral vectors in 41 CC mouse strains. Hepatic and splenic vector copy numbers were determined. We report that the CC mouse strains showed highly diverse outcomes following lentiviral gene delivery. For the first time, a moderate correlation between mouse-strain-specific sleeping patterns and transduction efficiency was observed. We associated two quantitative trait loci (QTLs) with intrastrain variations in transduction phenotypes, which mechanistically relates to the phenomenon of metastable epialleles. An additional QTL was associated with the kinetics of hepatic transgene expression. Genes found in the above QTLs are potential targets for personalized gene therapy protocols. Importantly, we identified two mouse strains that open new directions for characterizing continuous viral vector silencing and HIV latency. Our findings suggest that wide-range patient-specific outcomes of viral vector-based gene therapy should be expected. Thus, novel clinical protocols should be considered for non-fatal diseases. Full article

Background/Objectives: Over the last decades, our projects have been dedicated to clarifying immunopathological and virological events associated with Human Immunodeficiency Virus (HIV) infection. Methods: By using non-human primate models of pathogenic and non-pathogenic lentiviral infections, we aimed at identifying the cells and tissues in which the virus persists, despite antiretroviral therapy (ART). Indeed, the eradication of viral reservoirs is a major challenge for HIV cure. Results: We present a series of results performed in rhesus macaques of Chinese origin deciphering the virological and immunological events associated with ART that can be of interest for people living with HIV. Conclusions: This model could be of interest for understanding in whole body the clinical alteration that persist despite ART. Full article

Background: Chimeric antigen receptor (CAR) T cell therapy has shown significant promise in treating hematological malignancies, yet its application in solid tumors, particularly those expressing the epidermal growth factor receptor (EGFR), remains limited. This study investigates the potential of CAR-engineered peripheral blood mononuclear cells (PBMCs) as a novel adoptive cell therapy against EGFR-positive cancers. Methods: Lentiviral transduction at an MOI of 50 was performed to generate specific anti-EGFR second generation CAR-effector cells. The transduced PBMCs were stimulated with cytokines and CD3/CD28 beads to enhance their proliferation and activation. Flow cytometric and real-time cell analysis were performed at various effector-to-target ratios to explore the cytotoxic potential of CAR-PBMCs. Results: CAR-PBMCs exhibited improved targeting and cytotoxicity against EGFR-positive cancer cell lines MDA-MB-468 and SK-BR-3, compared to untransduced controls, with unsignificant effects on allogeneic PBMCs. Conclusion: CAR-PBMCs hold considerable potential as a therapeutic strategy for EGFR-positive solid tumors, warranting further clinical investigation. Full article

Achieving the precise targeting of lentiviral vectors (LVs) to specific cell populations is crucial for effective gene therapy, particularly in cancer treatment where the modulation of the tumor microenvironment can enhance anti-tumor immunity. Programmed cell death protein 1 (PD-1) is overexpressed on activated tumor-infiltrating T lymphocytes, including regulatory T cells that suppress immune responses via FOXP3 expression. We developed PD1-targeted LVs by incorporating the anti-PD1 nanobody nb102c3 into receptor-blinded measles virus H and VSV-G_mut glycoproteins. We assessed the retargeting potential of nb102c3 and evaluated transduction efficiency in activated T lymphocytes. FOXP3 expression was suppressed using shRNA delivered by these LVs. Our results demonstrate that PD1-targeted LVs exerted pronounced tropism towards PD1⁺ cells, enabling the selective transduction of activated T lymphocytes while sparing naive T cells. The suppression of FOXP3 in Tregs reduced their suppressive activity. PD1-targeted glycoprotein H provided greater specificity, whereas the VSV-G_mut, together with the anti-PD1 pseudoreceptor, achieved higher viral titers but was less selective. Our study demonstrates that PD1-targeted LVs may offer a novel strategy to modulate immune responses within the tumor microenvironment with the potential for developing new therapeutic strategies aimed at enhancing anti-tumor immunity. Full article