Transgene Expression Research Articles

In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors

Human adipose-derived stem cell (ASC) grafts have emerged as a powerful tool in regenerative medicine. However, ASC therapeutic potential is hindered by stressors throughout their use. Here we demonstrate the transgenic expression of the tardigrade-derived mitochondrial abundant heat soluble (MAHS) protein for improved ASC resistance to metabolic, mitochondrial, and injection shear stress. In vitro, MAHS-expressing ASCs demonstrate up to 61% increased cell survival following 72 h of incubation in phosphate buffered saline containing 20% media. Following up to 3.5% DMSO exposure for up to 72 h, a 14–49% increase in MAHS-expressing ASC survival was observed. Further, MAHS expression in ASCs is associated with up to 39% improved cell viability following injection through clinically relevant 27-, 32-, and 34-gauge needles. Our results reveal that MAHS expression in ASCs supports survival in response to a variety of common stressors associated with regenerative therapies, thereby motivating further investigation into MAHS as an agent for stem cell stress resistance. However, differentiation capacity in MAHS-expressing ASCs appears to be skewed in favor of osteogenesis over adipogenesis. Specifically, activity of the early bone formation marker alkaline phosphatase is increased by 74% in MAHS-expressing ASCs following 14 days in osteogenic media. Conversely, positive area of the neutral lipid droplet marker BODIPY is decreased by up to 10% in MAHS-transgenic ASCs following 14 days in adipogenic media. Interestingly, media supplementation with up to 40 mM glucose is sufficient to restore adipogenic differentiation within 14 days, prompting further analysis of mechanisms underlying interference between MAHS and differentiation processes.

A phytoplasma effector destabilizes chloroplastic glutamine synthetase inducing chlorotic leaves that attract leafhopper vectors

Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.

A stem cell-based assay platform demonstrates alpha-synuclein dependent synaptic dysfunction in patient-derived cortical neurons

Alpha-synuclein (αS)-rich Lewy bodies and neurites in the cerebral cortex correlate with the presence of dementia in Parkinson disease (PD) and Dementia with Lewy bodies (DLB), but whether αS influences synaptic vesicle dynamics in human cortical neurons is unknown. Using a new iPSC-based assay platform for measuring synaptic vesicle cycling, we found that in human cortical glutamatergic neurons, increased αS from either transgenic expression or triplication of the endogenous locus in patient-derived neurons reduced synaptic vesicle cycling under both stimulated and spontaneous conditions. Thus, using a robust, easily adopted assay platform, we show for the first time αS-induced synaptic dysfunction in human cortical neurons, a key cellular substrate for PD dementia and DLB.

SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans.

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

Maize Kernel-Associated Metagenomes Reveal Potential Microbe–Microbe Interactions that Underlie Fusarium Ear Rot Disease

As an alternative to host resistance, transgenic expression of entomocidal and antimicrobial proteins from Bacillus thuringiensis ( Bt) in maize can mitigate Fusarium ear rot (FER). This study evaluated FER in Bt and conventional maize and the role of microbes in the kernel-associated metagenome using quantitative reduced representation sequencing. Our results revealed significant differences in FER severity across environments and varieties and between inoculation treatments. The lower FER scores of conventional maize Spectrum 6416 relative to other varieties highlighted resistant alleles in maize germplasm, whereas strong correlations indicated FER-induced yield loss. Mitigation of FER by transgenes was validated by about threefold enrichment of Fusarium verticillioides ( Fv) postinoculation compared with nonsignificant Fv enrichment in conventional maize. Although the major causal pathogen of FER, Fv, was the most abundant species in the metagenomes ( r = 0.41 to 0.49), a similar degree of correlation was observed between FER and several Fusarium spp. ( r = 0.2 to 0.56). The potential FER-suppressing properties of Talaromyces stipitatus and Ustilago maydis were indicated by negative correlations with FER ( r = –0.22 to –0.42), Fv, and some Fusarium spp. The more FER-resistant varieties consistently enriched for potential FER-suppressing Burkholderia cenocepacia (negatively correlated with Fv). This suggests host genetic background-dependent recruitment of beneficial microbes that suppress pathogens and that microbe–microbe interactions play a role in FER severity. Quantitative and species/strain-level metagenomic profiles hold promise for identifying robust disease-suppressing microbes and microbes that act in synergy with pathogens and for developing holobiont-aware breeding strategies that account for host–microbiome coevolution and host genotype, microbiome/metagenome, and environment interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Comparative study on the expression characteristics of transgenes inserted into the Gt(ROSA)26Sor and H11 loci in mice.

The Gt(ROSA)26Sor ( ROSA26) and H11 loci are commonly used as safe harbors for the construction of targeted transgenic mice. However, it remains unclear whether these two loci have distinct effects on transgene expression. In this study, we insert three differently colored fluorescent protein expression cassettes (EGFP, tdTomato and mTagBFP2) driven by the CAG promoter into the ROSA26 and H11 loci. We generate five single-transgenic mouse models and a triple-transgenic mouse model expressing three distinct fluorescent proteins simultaneously. Our results reveal that the efficiency of transgene expression is greater at the ROSA26 locus with a reverse orientation relative to the transcription of the ROSA26 gene. In most tissues examined, the efficiency of transgene expression at the ROSA26 locus exceeds that at the H11 locus. Moreover, the expression profiles of identical transgenes display discrepancies across various tissues, and notably, substantial heterogeneity in transgene expression is discernible within cells of the same tissue. Our findings offer a valuable reference for the selection of safe harbors and strategies for the construction of transgenic mouse models.

Misregulation of bromotyrosine compromises fertility in male Drosophila

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

The infectivity of AAV9 is influenced by the specific location and extent of chemically modified capsid residues

BackgroundSeveral treatments for genetic diseases utilizing recombinant adeno-associated viruses (AAVs) have recently gained approval. However, the development of a greater number of therapeutic AAVs is constrained by certain limitations. While extensive efforts have concentrated on screening AAV genetic libraries, an alternative strategy involves modifying the AAV capsid by attaching various moieties. The capsid of AAV plays a pivotal role in transducing target cells and evading immune responses, making modifications a key avenue for engineering improved variants.ResultsIn our study, we replaced specific AAV9 capsid residues with an unnatural amino acid bearing a bioorthogonal group, identifying four positions with no adverse impact on production. Utilizing click chemistry, we attached varying proportions of Cy5.5 to these positions, allowing us to assess the impact of these modifications on AAV9 infectivity in cultured cells. Our findings reveal that both the position and degree of capsid modification significantly affect AAV transduction. While higher amounts of attached molecules lead to an increased number of AAV genomes within cells, this does not positively impact transgene expression. Conversely, a negative impact on transgene expression is observed when the AAV capsid is highly modified, with the degree of this effect associated with the modified residue.ConclusionCareful control of both the degree and specific position of capsid modifications is crucial for optimizing transduction efficiency and minimizing undesired effects on transgene expression. These results underscore the importance of precision in AAV capsid modification to achieve optimal transduction efficiency while mitigating potential drawbacks on transgene expression.

Modified lentiviral globin gene therapy for pediatric β0/β0 transfusion-dependent β-thalassemia: A single-center, single-arm pilot trial.

β0/β0 thalassemia is the most severe type of transfusion-dependent β-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a β-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in β0/β0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22months. The treatment extended the lifespan of red blood cells by over 42days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all β-thalassemia.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2+ lymphoid malignancies irrespective of BIM status

AbstractVenetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which calls for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient–derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL1L11/BIM status.

Persistent GDNF Expression 45 Months after Putaminal Infusion of AAV2-GDNF in a Patient with Parkinson's Disease.

Gene therapy by convection-enhanced delivery of type 2 adeno-associated virus-glial cell derived neurotrophic factor (AAV2-GDNF) to the bilateral putamina seeks to increase GDNF gene expression and treat Parkinson's disease (PD). A 63-year-old man with advanced PD received AAV2-GDNF in a clinical trial. He died from pneumonia after anterior cervical discectomy and fusion 45 months later. An autopsy included brain examination for GDNF transgene expression. Putaminal catecholamine concentrations were compared to in vivo 18F-Fluorodopa (18F-FDOPA) positron emission tomography (PET) scanning results before and 18 months after AAV2-GDNF infusion. Parkinsonian progression stabilized clinically. Postmortem neuropathology confirmed PD. Bilateral putaminal regions previously infused with AAV2-GDNF expressed the GDNF gene. Total putaminal dopamine was 1% of control, confirming the striatal dopaminergic deficiency suggested by baseline 18F-DOPA-PET scanning. Putaminal regions responded as expected to AAV2-GDNF. After AAV2-GDNF infusion, infused putaminal regions showed increased GDNF gene expression, tyrosine hydroxylase immunoreactive sprouting, catechol levels, and 18F-FDOPA-PET signal, suggesting the regenerative potential of AAV2-GDNF in PD. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Transgenic Expression of Rubisco Accumulation Factor2 and Rubisco Subunits Increases Photosynthesis and Growth in Maize.

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco assembly factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco Accumulation Factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing RAF2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least six weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy, or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown.

SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model

Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 x 1013 viral genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile-affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.

Targeted knock-in of a NCF1 coding sequence into the endogenous NCF2 locus leads to myeloid phenotypic correction of p47phox-deficient chronic granulomatous disease

The p47phox-deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 cDNA knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67phox, an NADPH oxidase subunit that closely interacts with p47phox and is predominantly expressed in myeloid cells. This approach restored p47phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47phox-deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47phox and p67phox, ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

OCT Intensity of the Region between Outer Retina Band 2 and Band 3 as a Biomarker for Retinal Degeneration and Therapy.

Optical coherence tomography (OCT) is widely used to probe retinal structure and function. This study investigated the outer retina band (ORB) pattern and reflective intensity for the region between bands 2 and 3 (Dip) in three mouse models of inherited retinal degeneration (Rs1KO, TTLL5KO, RPE65KO) and in human AMD patients from the A2A database. OCT images were manually graded, and reflectivity signals were used to calculate the Dip ratio. Qualitative analyses demonstrated the progressive merging band 2 and band 3 in all three mouse models, leading to a reduction in the Dip ratio compared to wildtype (WT) controls. Gene replacement therapy in Rs1KO mice reverted the ORB pattern to one resembling WT and increased the Dip ratio. The degree of anatomical rescue in these mice was highly correlated with level of transgenic RS1 expression and with the restoration of ERG b-wave amplitudes. While the inner retinal cavity was significantly enlarged in dark-adapted Rs1KO mice, the Dip ratio was not altered. A reduction of the Dip ratio was also detected in AMD patients compared with healthy controls and was also positively correlated with AMD severity on the AMD score. We propose that the ORB and Dip ratio can be used as non-invasive early biomarkers for retina health, which can be used to probe therapeutic gene expression and to evaluate the effectiveness of therapy.

Aeluropus littoralis stress-associated protein promotes water deficit resilience in engineered durum wheat

Global climate change-related water deficit negatively affect the growth, development and yield performance of multiple cereal crops, including durum wheat. Therefore, the improvement of water-deficit stress tolerance in durum wheat varieties in arid and semiarid areas has become imperative for food security. Herein, we evaluated the water deficiency resilience potential of two marker-free transgenic durum wheat lines (AlSAP-lines: K9.3 and K21.3) under well-watered and water-deficit stress conditions at both physiological and agronomic levels. These two lines overexpressed the AlSAP gene, isolated from the halophyte grass Aeluropus littoralis, encoding a stress-associated zinc finger protein containing the A20/AN1 domains. Under well-watered conditions, the wild-type (WT) and both AlSAP-lines displayed comparable performance concerning all the evaluated parameters. Ectopic transgene expression exerted no adverse effects on growth and yield performance of the durum wheat plants. Under water-deficit conditions, no significant differences in the plant height, leaf number, spike length, and spikelet number were observed between AlSAP-lines and WT plants. However, compared to WT, the AlSAP-lines exhibited greater dry matter production, greater flag leaf area, improved net photosynthetic rate, stomatal conductance, and water use efficiency. Notably, the AlSAP-lines displayed 25 % higher grain yield (GY) than the WT plants under water-deficit conditions. The RT-qPCR-based selected stress-related gene (TdDREB1, TdLEA, TdAPX1, and TdBlt101-2) expression analyses indicated stress-related genes enhancement in AlSAP-durum wheat plants under both well-watered and water-deficit conditions, potentially related to the water-deficit resilience. Collectively, our findings support that the ectopic AlSAP expression in durum wheat lines enhances water-deficit resilience ability, thereby potentially compensate for the GY loss in arid and semi-arid regions.

Development of an Improved Adenovirus Vector and Its Application to the Treatment of Lifestyle-Related Diseases.

The number of patients with lifestyle-related diseases such as type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has continued to increase worldwide. Therefore, development of innovative therapeutic methods targeting lifestyle-related diseases is required. Gene therapy has attracted considerable attention as an advanced medical treatment. Safe and high-performance vectors are essential for the practical application of gene therapy. Replication-incompetent adenovirus (Ad) vectors are widely used in clinical gene therapy and basic research. Here, we developed a novel Ad vector, named Ad-E4-122aT, exhibiting higher and longer-term transgene expression and lower hepatotoxicity than conventional Ad vectors. We also elucidated the mechanisms underlying Ad vector-induced hepatotoxicity during the early phase using Ad-E4-122aT. Next, we examined the therapeutic effects of the genes of interest, namely zinc finger AN1-type domain 3 (ZFAND3), lipoprotein lipase (LPL), and lysophospholipid acyltransferase 10 (LPLAT10), on lifestyle-related diseases using Ad-E4-122aT. We showed that the overexpression of ZFAND3 in the liver improved glucose tolerance and insulin resistance. Liver-specific LPL overexpression suppressed hepatic lipid accumulation and improved glucose metabolism. LPLAT10 overexpression in the liver suppressed postprandial hyperglycemia by increasing glucose-stimulated insulin secretion. Furthermore, we also focused on foods to advance research on the pathophysiology and treatment of lifestyle-related diseases. Cranberry and calamondin, which are promising functional foods, attenuated the progression of MASLD/NAFLD. Our findings will aid the development of new therapeutic methods, including gene therapy, for lifestyle-related diseases such as T2DM and MASLD/NAFLD.

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa (Medicago sativa. L.) is a globally significant autotetraploid legume forage crop. However, despite its importance, establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge. In this study, we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa, a variety widely cultivated in Northeast China. Subsequently, we created a single transcript CRISPR/Cas9 (CRISPR_2.0) toolkit, incorporating multiplex gRNAs, designed for gene editing in Gongnong 1. Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter, a widely employed polymerase II constitutive promoter known for strong transgene expression in dicots. To assess the toolkit’s efficiency, we targeted PALM1, a gene associated with a recognizable multifoliate phenotype. Utilizing the CRISPR_2.0 toolkit, we directed PALM1 editing at two sites in the wild-type Gongnong 1. Results indicated a 35.1% occurrence of editing events all in target 2 alleles, while no mutations were detected at target 1 in the transgenic-positive lines. To explore more efficient sgRNAs, we developed a rapid, reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation, incorporating the visible reporter MtLAP1. This screening system demonstrated that most purple visible hairy roots underwent gene editing. Notably, sgRNA3, with an 83.0% editing efficiency, was selected using the visible hairy root system. As anticipated, tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype. These palm1 lines demonstrated an average crude protein yield increase of 21.5% compared to trifoliolate alfalfa. Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Miniaturization of hiPSC-derived 3D neural cultures in stirred-tank bioreactors for parallelized preclinical assessment of rAAV.

Introduction: Engineered 3D models employing human induced pluripotent stem cell (hiPSC) derivatives have the potential to recapitulate the cell diversity and structure found in the human central nervous system (CNS). Therefore, these complex cellular systems offer promising human models to address the safety and potency of advanced therapy medicinal products (ATMPs), such as gene therapies. Specifically, recombinant adeno-associated viruses (rAAVs) are currently considered highly attractive for CNS gene therapy due to their broad tropism, low toxicity, and moderate immunogenicity. To accelerate the clinical translation of rAAVs, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. The integration of hiPSC-derived CNS models in rAAV development will require, amongst other factors, robust, small-scale, high-throughput culture platforms that can feed the preclinical trials. Methods: Herein, we pioneer the miniaturization and parallelization of a 200mL stirred-tank bioreactor-based 3D brain cell culture derived from hiPSCs. We demonstrate the applicability of the automated miniaturized Ambr® 15 Cell Culture system for the maintenance of hiPSC-derived neurospheroids (iNSpheroids), composed of neuronal and glial cells. Critical process parameters were optimized, namely, cell density and agitation mode. Results: Under optimized conditions, stable iNSpheroid cultures were attained in the microbioreactors for at least 15days, with high cell viability and astrocytic and neuronal phenotype maintenance. This culture setup allowed the parallelization of different rAAVs, in different multiplicity of infections (MOIs), to address rAAV-host interactions at a preclinical scale. The iNSpheroids were exposed to rAAV2- and rAAV9-eGFP in the microbioreactors. Transgene expression was detected 14days post-transduction, revealing different astrocyte/neuron tropism of the two serotypes. Discussion: We advocate that the iNSpheroid cultures in miniaturized bioreactors are reliable and reproducible screening tools for addressing rAAV transduction and tropism, compatible with preclinical demands.