Adeno-associated Virus Vectors Research Articles

Novel mechanisms of action of emerging therapies of hereditary thrombotic thrombocytopenic purpura.

Hereditary thrombotic thrombocytopenic purpura (hTTP) is caused by deficiency of plasma ADAMTS13 activity, resulting from ADAMTS13 mutations. ADAMTS13 cleaves ultra large von Willebrand factor (VWF), thus reducing its multimer sizes. Hereditary deficiency of plasma ADAMTS13 activity leads to the formation of excessive platelet-VWF aggregates in small arterioles and capillaries, resulting in hTTP. PubMed search from 1956-2024 using thrombotic thrombocytopenic purpura and therapy identified 3,675 articles. Only the articles relevant to the topic were selected for discussion, which focuses on pathophysiology, clinical presentations, and mechanisms of action of emerging therapeutics for hTTP. Current therapies include infusion of plasma, or coagulation factor VIII, or recombinant ADAMTS13. Emerging therapies include anti-VWF A1 aptamers or nanobody and gene therapies with adeno-associated viral vector or self-inactivated lentiviral vector or a Sleeping Beauty transposon system for a long-term expression of a functional ADAMTS13 enzyme. Frequent plasma infusion remains to be the standard of care in most parts of the world, while recombinant ADAMTS13 has become the treatment of choice for hTTP in some of the Western countries. The success of gene therapies in preclinical models may hold a promise for future development of these novel approaches for a cure of hTTP.

How safe is gene therapy? : Second death after Duchenne therapy

Duchenne muscular dystrophy (DMD) is asevere monogenic hereditary disease with early manifestation and aprogressive course. Treatment options have so far been limited. Gene therapy opens up new options for DMD patients. Against the background of afurther death following DMD gene therapy, the side effects and risks of the gene therapeutics already approved or undergoing clinical trials will be evaluated and alternative gene therapeutics will be described. Based thereon, the future of DMD gene therapy will be discussed. For the first time, in June 2023, delandistrogene moxeparvovec (SRP-9001), agene replacement therapy based on an adeno-associated virus (AAV) vector, was approved in the USA for children aged 4-5years with DMD. Other promising gene therapies are in preclinical development or clinical trials, including CRISPR/Cas9-mediated strategies to restore dystrophin expression. Two deaths following DMD gene therapy with high-dose AAV vectors were attributed to AAV-mediated immune responses. The pre-existing disease underlying the therapy is most likely involved in the fatal AAV toxicity. Although gene therapy applications of AAV vectors are generally considered safe, the systemic administration of high vector doses can lead to severe side effects with apotentially fatal outcome in individual patients, especially after activation of the immune system. In the future, new methods for immunosuppression, reduction of AAV dose and alternative vectors will therefore increasingly come to the fore.

Selective Enhancement of REM Sleep in Male Rats through Activation of MT1 Receptors Located in the Locus Coeruleus Norepinephrine neurons.

Sleep disorders affect millions of people around the world and have a high comorbidity with psychiatric disorders. While current hypnotics mostly increase non-rapid eye movement sleep (NREMS), drugs acting selectively on enhancing rapid eye movement sleep (REMS) are lacking. This polysomnographic study in male rats showed that the first-in-class selective melatonin MT1 receptor partial agonist UCM871 increases the duration of REMs without affecting that of NREMS. The REMS-promoting effects of UCM871 occurred by inhibiting, in a dose-response manner, the firing activity of the locus coeruleus (LC) norepinephrine (NE) neurons, which express MT1 receptors. The increase of REMS duration and the inhibition of LC-NE neuronal activity by UCM871 were abolished by MT1 pharmacological antagonism and by an adeno-associated viral (AAV) vector which selectively knocked down MT1 receptors in the LC-NE neurons. In conclusion, MT1 receptor agonism inhibits LC-NE neurons and triggers REMS, thus representing a novel mechanism and target for REMS disorders and/or psychiatric disorders associated with REMS impairments.Significance Statement Rapid eye movement sleep (REMS) is involved in the processes of memory consolidation and emotional regulation, but drugs selectively enhancing REMS are scant. Herein, we show that the first-in-class selective melatonin MT1 receptor agonist UCM871, by inhibiting the activity of norepinephrine neurons in the locus coeruleus, an important nucleus regulating the sleep/wake cycle, selectively increases the duration of REMS. These findings enhance our current understanding of the neurobiology and pharmacology of REMS and provide a possible novel mechanism and target for disorders associated with REMS dysfunctions.

Protective role of RIPK1 scaffolding against HDV-induced hepatocyte cell death and the significance of cytokines in mice.

Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNAR KO and WT mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.

Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Methodological validation of sedimentation velocity analytical ultracentrifugation method for Adeno-Associated Virus and collaborative calibration of system suitability substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and High Sedimentation Coefficient Substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF( high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the RSD (relative standard deviation) could be within 5%. Even for empty or partial less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4×1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Due to the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control)laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Effects of unilateral hippocampal surgical procedures needed for calcium imaging on mouse behavior and adult hippocampal neurogenesis

Hippocampus is essential for episodic memory formation, lesion studies demonstrating its role especially in processing spatial and temporal information. Further, adult hippocampal neurogenesis (AHN) in the dentate gyrus (DG) has also been linked to learning. To study hippocampal neuronal activity during events like learning, in vivo calcium imaging has become increasingly popular. It relies on the use of adeno-associated viral (AAV) vectors, which seem to lead to a decrease in AHN when applied on the DG. More notably, imaging requires the implantation of a relatively large lens into the tissue. Here, we examined how injection of an AAV vector and implantation of a 1-mm-diameter lens into the dorsal DG routinely used to image calcium activity impact the behavior of adult male C57BL/6 mice. To this aim, we conducted open-field, object-recognition and object-location tasks at baseline, after AAV vector injection, and after lens implantation. Finally, we determined AHN from hippocampal slices using a doublecortin-antibody. According to our results, the operations needed for in vivo imaging of the dorsal DG did not have adverse effects on behavior, although we noticed a decrease in AHN ipsilaterally to the operations. Thus, our results suggest that in vivo imaging can be safely used to, for example, correlate patterns of calcium activity with learned behavior. One should still keep in mind that the defects on the operated side might be functionally compensated by the (hippocampus in the) contralateral hemisphere.

IGF2BP2 regulates the inflammation of fibroblast-like synoviocytes via GSTM5 in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with an unknown etiology. RA cannot be fully cured and requires lengthy treatment, imposing a significant burden on both individuals and society. Due to the lack of specific drugs available for treating RA, exploring a key new therapeutic target for RA is currently an important task. Activated fibroblast-like synoviocytes (FLSs) play a crucial role in the progression of RA, which release interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α resulting in abnormal inflammatory reaction in the synovium. A previous study has highlighted the correlation of m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) with inflammation-related diseases in human. However, the role of IGF2BP2 in the inflammatory reaction of FLSs during RA progression has not been assessed. In this study, IGF2BP2 expression was decreased in the synovial tissues of RA patients and collagen-induced arthritis (CIA) rats. Intra-articular injection of an adeno-associated virus (AAV) vector overexpressing IGF2BP2 relieved paw swelling, synovial hyperplasia and cartilage destruction in CIA rats. IGF2BP2 overexpression also inhibited lipopolysaccharide (LPS)-mediated RA fibroblast-like synoviocytes (RA-FLSs) migration and invasion accompanied by a decreased level of inflammatory factors in vitro. Conversely, IGF2BP2 suppression promoted RA-FLSs migration and invasion with an elevated level of inflammatory factors in vitro. The sequencing result showed that glutathione S-transferase Mu 5 (GSTM5), a key antioxidant gene, was the target mRNA of IGF2BP2. Further experiments demonstrated that IGF2BP2 strengthened the stability of GSTM5 mRNA, leading to weakened inflammatory reaction and reduced expression of matrix metalloproteinase 9 and 13 (MMP9, MMP13). Therefore, IGF2BP2-GSTM5 axis may represent a potential therapeutic target for RA treatment.

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Engineered IgM and IgG cleaving enzymes for mitigating antibody neutralization and complement activation in AAV gene transfer

Systemic dosing of adeno-associated viral (AAV) vectors poses potential risk of adverse side-effects including complement activation triggered by anti-capsid immunity. Due to the multifactorial nature of toxicities observed in this setting, a wide spectrum of immune modulatory regimens (IMR) are being investigated in the clinic. Here, we discover an IgM cleaving enzyme (IceM) that degrades human IgM, a key trigger in the anti-AAV immune cascade. We then engineer a fusion enzyme (IceMG) with dual proteolytic activity against human IgM and IgG. IceMG cleaves B cell surface antigen receptors (BCR) and inactivates phospholipase gamma signaling in vitro. Importantly, IceMG is more effective at inhibiting complement activation compared to an IgG cleaving enzyme alone. Upon IV dosing, IceMG rapidly and reversibly clears circulating IgM and IgG in macaques. Antisera from these animals treated with IceMG shows decreased ability to neutralize AAV and activate complement. Consistently, pre-conditioning with IceMG restores AAV transduction in mice passively immunized with human antisera. Thus, IgM cleaving enzymes show promise in simultaneously addressing multiple aspects of anti-AAV immunity mediated by B cells, circulating antibodies and complement. These studies have implications for improving safety of AAV gene therapies and possibly broader applications including organ transplantation and autoimmune diseases.

Nurr1 overexpression in the primary motor cortex alleviates motor dysfunction induced by intracerebral hemorrhage in the striatum in mice

Hemorrhage-induced injury of the corticospinal tract (CST) in the internal capsule (IC) causes severe neurological dysfunction in both human patients and rodent models of intracerebral hemorrhage (ICH). A nuclear receptor Nurr1 (NR4A2) is known to exert anti-inflammatory and neuroprotective effects in several neurological disorders. Previously we showed that Nurr1 ligands prevented CST injury and alleviated neurological deficits after ICH in mice. To prove direct effect of Nurr1 on CST integrity, we examined the effect of Nurr1 overexpression in neurons of the primary motor cortex on pathological consequences of ICH in mice. ICH was induced by intrastriatal injection of collagenase type VII, where hematoma invaded into IC. Neuron-specific overexpression of Nurr1 was induced by microinjection of synapsin I promoter-driven adeno-associated virus (AAV) vector into the primary motor cortex. Nurr1 overexpression significantly alleviated motor dysfunction but showed only modest effect on sensorimotor dysfunction after ICH. Nurr1 overexpression also preserved axonal structures in IC, while having no effect on hematoma-associated inflammatory events, oxidative stress, and neuronal death in the striatum after ICH. Immunostaining revealed that Nurr1 overexpression increased the expression of Ret tyrosine kinase and phosphorylation of Akt and ERK1/2 in neurons in the motor cortex. Moreover, administration of Nurr1 ligands 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane or amodiaquine increased phosphorylation levels of Akt and ERK1/2 as well as expression of glial cell line-derived neurotrophic factor and Ret genes in the cerebral cortex. These results suggest that the therapeutic effect of Nurr1 on striatal ICH is attributable to the preservation of CST by acting on cortical neurons.

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.

Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells.

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.

Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Enhancing prime editor flexibility with coiled-coil heterodimers

BackgroundPrime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.ResultsHerein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.ConclusionsOur innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

Membrane-Targeted palGFP Predominantly Localizes to the Plasma Membrane but not to Neurosecretory Vesicle Membranes in Rat Oxytocin Neurons.

Recent advances in viral vector technology, specifically using adeno-associated virus (AAV) vectors, have significantly expanded possibilities in neuronal tracing. We have utilized the Cre/loxP system in combination with AAV techniques in rats to explore the subcellular localization of palmitoylation signal-tagged GFP (palGFP) in oxytocin-producing neurosecretory neurons. A distinctive branching pattern of single axons was observed at the level of the terminals in the posterior pituitary. Despite challenges in detecting palGFP signals by fluorescent microscopy, immunoelectron microscopy demonstrated predominant localization on the plasma membrane, with a minor presence on the neurosecretory vesicle membrane. These findings suggest that membrane-anchored palGFP may undergo exocytosis, translocating from the plasma membrane to the neurosecretory vesicle membrane. In this study, we observed characteristic axon terminal structures in the posterior pituitary of oxytocin neurons. This study indicates the importance of understanding the plasma membrane-specific sorting system in neuronal membrane migration and encourages future studies on the underlying mechanisms.

Engineering miniature CRISPR-Cas Un1Cas12f1 for efficient base editing

Adeno-associated virus (AAV) is a relatively safe and efficient vector for gene therapy. However, due to its 4.7-kilobase limit of cargo, SpCas9-mediated base editors can't be packaged into a single AAV vector, which hinders its clinical application. The development of efficient miniature base editors becomes an urgent need. Un1Cas12f1 is a class II V-F type CRISPR-Cas protein with only 529 amino acids. Although Un1Cas12f1 has been engineered to be a base editor in mammalian cells, the base editing efficiency is less than 10%, which limits its therapeutic applications. Here, we developed hypercompact and high-efficiency base editors by engineering Un1Cas12f1, fusing non-specific DNA binding protein Sso7d and truncating sgRNA, termed as STUminiBEs. We demonstrated the robust A-to-G conversion (54% on average) by STUminiABE or C-to-T conversion (45% on average) by STUminiCBE. We packaged STUminiCBE into AAVs and successfully introduced a premature stop codon on the PCSK9 gene in mammalian cells. In sum, STUminiBEs are efficient miniature base editors and could readily be packaged into AAVs for biological research or biomedical applications.

Phosphorylation of AS160-Serine 704 is Not Essential for Exercise-increase in Insulin-stimulated Glucose Uptake by Skeletal Muscles from Female or Male Rats.

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle from rodents and humans of both sexes. We recently found that concurrent mutation of three key sites to prevent their phosphorylation (Ser588, Thr642, and Ser704) on Akt substrate of 160 kDa (AS160; also known as TBC1D4) reduced the magnitude of the enhancement of postexercise ISGU (PEX-ISGU) by muscle from male, but not female rats. However, we did not test the role of individual phosphorylation sites on PEX-ISGU. Accordingly, our current aim was to test if AS160 Ser704 phosphorylation (pSer704) is required for elevated PEX-ISGU by muscle. AS160-knockout (AS160-KO) rats (female and male) were studied when either sedentary or 3 hours after acute exercise. Adeno-associated virus (AAV) vectors were used to enable muscle expression of wildtype-AS160 (AAV-WT-AS160) or AS160 mutated Ser704 to alanine to prevent phosphorylation (AAV-1P-AS160). Paired epitrochlearis muscles from each rat were injected with AAV-WT-AS160 or AAV-1P-AS160. We discovered that regardless of sex: 1) AS160 abundance in AS160-KO rats was similar in paired muscles expressing WT-AS160 versus 1P-AS160; 2) muscles from exercised versus sedentary rats had greater ISGU, and PEX-ISGU was slightly greater for muscles expressing 1P-AS160 versus contralateral muscles expressing WT-AS160; 3) pAS160 Thr642 was lower in muscles expressing 1P-AS160 versus paired muscles expressing WT-AS160. These results indicate that pAS160 Ser704 was not essential for elevated PEX-ISGU by skeletal muscle from rats of either sex. Furthermore, elimination of the postexercise increase in pAS160 Thr642 did not lessen the postexercise effect on ISGU.

Transduction and Genome Editing of the Heart with Adeno-Associated Viral Vectors Loaded onto Electrospun Polydioxanone Nonwoven Fabrics.

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.