Adeno-associated Virus Vectors Research Articles

Abstract Tu046: Mechanisms of Epicardium-Directed Cardiac Repair

Background: In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability. Methods: We used the Wt1 (Wilms Tumor 1) CreERT2 ; R26 tdTomato mouse line for tamoxifen-inducible epicardial-specific lineage tracing and Pdgfra nGFP to label cardiac fibroblasts ( Wt1 CreERT2 ; R26 tdT ; Pdgfra nGFP ). Tagged epicardial-derived cells (tdTomato + ) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed in vitro studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI. Results: We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with Wnt signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in Wt1 -expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition. Conclusions: Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.

Retinal Penetrating Adeno-Associated Virus.

The most common method of delivery of genes to the outer retina uses recombinant adeno-associated virus (AAV) injected into the subretinal space using a surgical procedure. In contrast, most drugs are delivered to the retina using an intravitreal approach in an office setting. The objective of the current study was to develop AAV vectors that can reach the outer retina via intravitreal injection. Recently, we described a molecular chaperone (Nuc1) that enhanced the penetration of small and large molecules, including AAV, into the retina. The Nuc1 amino acid sequence or a truncated version of Nuc1 (IKV) was genetically incorporated into an exposed loop of AAV2/9 VP1 protein. These novel recombinant AAV vectors expressing green fluorescent protein (GFP) or nuclear factor erythroid 2 p45-related factor 2 (Nrf2) were injected into the vitreous of C57Bl/6J or Nrf2 knockout mice, respectively. The amount of GFP expression or oxidative stress as measured by 8-Hydroxy-2'-deoxyguanosine staining in C57Bl/6J or Nrf2 knockout mice, respectively, was quantified. Incorporation of Nuc1 into AAV2/9 did not lead to significant expression of GFP in the murine retina. However, incorporation of IKV into AAV2/9 led to robust expression of GFP in photoreceptors and retinal pigment epithelium (RPE) via the intravitreal and subretinal routes of delivery. Furthermore, expression of Nrf2 using an IKV vector led to a reduction in oxidative stress in the retina of C57Bl/6J and Nrf2 knockout mice. We have developed a novel AAV vector that enables delivery of transgenes to the outer retina of mice, including photoreceptors and RPE following intravitreal injection.

Brain-Derived Neurotrophic Factor (BDNF) in the Frontal Cortex Enhances Social Interest in the BTBR Mouse Model of Autism.

A large body of evidence implies the involvement of brain-derived neurotrophic factor (BDNF) in the pathogenesis of autism spectrum disorders (ASDs). A deficiency of BDNF in the hippocampus and frontal cortex of BTBR mice (a model of autism) has been noted in a number of studies. Earlier, we showed that induction of BDNF overexpression in the hippocampus of BTBR mice reduced anxiety and severity of stereotyped behavior, but did not affect social interest. Here, we induced BDNF overexpression in the frontal cortex neurons of BTBR mice using an adeno-associated viral vector, which resulted in a significant increase in the social interest in the three-chamber social test. At the same time, the stereotypy, exploratory behavior, anxiety-like behavior, and novel object recognition were not affected. Therefore, we have shown for the first time that the presence of BDNF in the frontal cortex is critical for the expression of social interest in BTBR mice, since compensation for its deficiency in this structure eliminated the autism-like deficiencies in the social behavior characteristic for these animals.

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

Innovative fixed bed bioreactor platform: Enabling linearly scalable adherent cell biomanufacturing with real-time biomass prediction from nutrient consumption.

Scalable single-use adherent cell-based biomanufacturing platforms are essential for unlocking the full potential of cell and gene therapies. The primary objective of this study is to design and develop a novel fixed bed bioreactor platform tailored specifically for scaling up adherent cell culture. The bioreactor comprises a packed bed of vertically stacked woven polyethylene terephthalate mesh discs, sandwiched between two-fluid guide plates. Leveraging computational fluid dynamics modeling, we optimized bioreactor design to achieve uniform flow with minimal shear stress. Residence time distribution measurements demonstrated excellent flow uniformity with plug flow characteristics. Periodic media sampling coupled with offline analysis revealed minimal gradients of crucial metabolites (glucose, glutamine, lactate, and ammonia) across the bioreactor during cell growth. Furthermore, the bioreactor platform demonstrated high performance in automated cell harvesting, with ≈96% efficiency and ≈98% viability. It also exhibited linear scalability in both operational parameters and performance for cell culture and adeno-associated virus vector production. We developed mathematical models based on oxygen uptake rates to accurately predict cell growth curves and estimate biomass in real-time. This study demonstrates the effectiveness of the developed fixed-bed bioreactor platform in enabling scalable adherent cell-based biomanufacturing with high productivity and process control.

Enhancer AAVs for targeting spinal motor neurons and descending motor pathways in rodents and macaque.

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, which function to integrate information from the brain and control movement through direct innervation of effector muscles, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. The most promising candidate enhancers were then extensively characterized using imaging and molecular techniques and further tested in rat and macaque to show conservation of LMN labeling. Additionally, we combined enhancer elements into a single vector to achieve simultaneous labeling of upper motor neurons (UMNs) and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.

Sub-genomic flaviviral RNA elements increase the stability and abundance of recombinant AAV vector transcripts.

Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.

An efficient AAV vector system of Rec2 serotype for intravenous injection to study metabolism in brown adipocytes in vivo

ObjectiveRecombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. MethodsTo achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. ResultsWe demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. ConclusionTaken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes.

Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs.

Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in egg-derived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA. In vitro, the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges. This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro, i.e., it significantly increases the fibrogenic factors Col1(α1), Col3(α1), and α-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1(α1), Col3(α1), and α-SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo, modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica. The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis.

Preclinical evaluation of tissue-selective gene therapies for congenital generalised lipodystrophy

Lipodystrophy is a rare disorder which can be life-threatening. Here individuals fail to develop or maintain appropriate adipose tissue stores. This typically causes severe metabolic complications, including hepatic steatosis and lipoatrophic diabetes. There is no cure for lipodystrophy, and treatment options remain very limited. Here we evaluate whether tissue-selective adeno-associated virus (AAV) vectors can provide a targeted form of gene therapy for lipodystrophy, using a preclinical lipodystrophic mouse model of Bscl2 deficiency. We designed AAV vectors containing the mini/aP2 or thyroxine-binding globulin promoter to selectively target adipose or liver respectively. The AAV-aP2 vectors also contained the liver-specific microRNA-122 target sequence, restricting hepatic transgene expression. Systemic delivery of AAV-aP2 vectors overexpressing human BSCL2 restored adipose tissue development and metabolic health in lipodystrophic mice without detectable expression in the liver. High doses (1 × 1012 GCs) of liver-selective vectors led to off target expression and adipose tissue development, whilst low doses (1 × 1010 GCs) expressed selectively and robustly in the liver but did not improve metabolic health. This reveals that adipose tissue-selective, but not liver directed, AAV-mediated gene therapy is sufficient to substantially recover metabolic health in generalised lipodystrophy. This provides an exciting potential new avenue for an effective, targeted, and thereby safer therapeutic intervention.

Adeno-associated viral vector gene therapy: Challenges for the paediatric hepatologist.

Hepatoxicity associated with recombinant adeno-associated virus gene therapy is being increasingly encountered by hepatologists in tertiary and quaternary referral units due to the recent increase of these therapies for neuromuscular and haematological disorders. The challenges in managing the condition stem from a lack of good-quality evidence on the appropriate protocols for immunosuppressants due to lack of representative animal models. There is a need for protocols for diagnosing and treating hepatotoxicity and this possible with further research to understand the problem and its management. The review also highlights the importance of a multidisciplinary team in managing hepatotoxicity and recommends further research to better identify at-risk individuals, define the extent of the problem and assess the long-term effects of liver injury and immunosuppressants.

Optimization of an adeno-associated viral vector for epidermal keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

Unraveling the Prefrontal Cortex-Basolateral Amygdala Pathway's Role on Schizophrenia's Cognitive Impairments: A Multimodal Study in Patients and Mouse Models.

This study investigated the role of the medial prefrontal cortex (mPFC)-basolateral amygdala (BLA) pathway in schizophrenia (SCZ)-related cognitive impairments using various techniques. This study utilized clinical scales, magnetic resonance imaging, single-cell RNA sequencing, and optogenetics to investigate the mPFC-BLA pathway in SCZ patients. In the mouse model, 6-week-old methylazoxymethanol acetate-induced mice demonstrated significant cognitive deficits, which were addressed through stereotaxic injections of an adeno-associated viral vector to unveil the neural connection between the mPFC and BLA. Significant disparities in brain volume and neural activity, particularly in the dorsolateral prefrontal cortex (DLPFC) and BLA regions, were found between SCZ patients and healthy controls. Additionally, we observed correlations indicating that reduced volumes of the DLPFC and BLA were associated with lower cognitive function scores. Activation of the mPFC-BLA pathway notably improved cognitive performance in the SCZ model mice, with the targeting of excitatory or inhibitory neurons alone failing to replicate this effect. Single-cell transcriptomic profiling revealed gene expression differences in excitatory and inhibitory neurons in the BLA of SCZ model mice. Notably, genes differentially expressed in the BLA of these model mice were also found in the blood exosomes of SCZ patients. Our research provides a comprehensive understanding of the role of the PFC-BLA pathway in SCZ, underscoring its significance in cognitive impairment and offering novel diagnostic and therapeutic avenues. Additionally, our research highlights the potential of blood exosomal mRNAs as noninvasive biomarkers for SCZ diagnosis, underscoring the clinical feasibility and utility of this method.

Restoration of peripheral neuropathy in Fabry mice via intrathecal administration of an adeno-associated virus vector encoding mGLA cDNA

Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a pathological variant of the α-galactosidase A (GLA) gene that results in deficient GLA activity. GLA deficiency leads to the accumulation of globotriaosylceramide (Gb3) and lyso-Gb3 in many tissues. A certain number of FD patients have burning pain or acroparesthesia in the feet and hands since childhood. Enzyme replacement therapy (ERT) is available for FD patients. However, ERT does not dramatically improve these FD-related peripheral neuropathic pain. We generated an adeno-associated virus serotype PHP.eB (AAV-PHP.eB) vector encoding mouse GLA cDNA, which was administered to FD mice intrathecally (it) or intravenously (iv). In the it-administered AAV (it-AAV) FD mice, the GLA enzyme activity in the lumbar dorsal root ganglion (DRG) was significantly greater than that in the untreated (NT) FD mice, and the level of activity was similar to that in wild-type (WT) B6 mice. However, in iv-administered AAV (iv-AAV) FD mice, GLA activity in the DRG did not increase compared to that in NT FD mice. Gb3 storage in the DRG of it-AAV FD mice was reduced compared to that in the DRG of NT FD mice. However, compared with NT FD mice, iv-AAV FD mice did not exhibit a significant reduction in the expression of the Gb3 substrate. Compared with WT mice, FD mice were thermally hyposensitive at 52 °C according to the hot plate test. The it-AAV FD mice showed significant recovery from thermal hyposensitivity. However, the iv-AAV FD mice did not exhibit significant improvement in thermal hyposensitivity. These results suggest that the intrathecal delivery of AAV-PHP.eB-mGLA may be a valuable tool for the treatment of FD-related peripheral neuropathic pain.

Characterization of residual microRNAs in AAV vector batches produced in HEK293 mammalian cells and Sf9 insect cells

With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA invivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

Foxq1 activates CB2R with oleamide to alleviate POCD.

Postoperative cognitive dysfunction (POCD) is a major concern, particularly among older adults. This study used social isolation (ISO) and multiomics analyses in aged mice to investigate potential mechanisms underlying POCD development. Aged mice were divided into two groups: ISO and paired housing (PH). Oleamide and the cannabinoid receptor type 2 (CB2R) antagonist AM630 were administered intraperitoneally, while Foxq1 adeno-associated viral (AAV) vector was injected directly into the hippocampus. Intramedullary tibial surgeries were subsequently performed to establish the POCD models. Behavioral tests comprising the Y-maze, open field test, and novel object recognition were conducted 2 days after surgery. Hippocampal and serum inflammatory cytokines were assessed. Following surgery, ISO mice demonstrated intensified cognitive impairments and escalated inflammatory markers. Integrative transcriptomic and metabolomic analysis revealed elevated oleamide concentrations in the hippocampus and serum of PH mice, with associative investigations indicating a close relationship between the Foxq1 gene and oleamide levels. While oleamide administration and Foxq1 gene overexpression substantially ameliorated postoperative cognitive performance and systemic inflammation in mice, CB2R antagonist AM630 impeded these enhancements. The Foxq1 gene and oleamide may be crucial in alleviating POCD. While potentially acting through CB2R-mediated pathways, these factors may modulate neuroinflammation and attenuate proinflammatory cytokine levels within the hippocampus, substantially improving cognitive performance postsurgery. This study lays the groundwork for future research into therapeutic approaches targeting the Foxq1-oleamide-CB2R axis, with the ultimate goal of preventing or mitigating POCD.

AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS.MethodsWe used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice.ResultsSOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice.ConclusionsAAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.

Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles

Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics.

Hippocampus-specific knockdown of Shati/Nat8l impairs cognitive function and electrophysiological response in mice

Shati/Nat8l was identified as an upregulated molecule in the nucleus accumbens (NAc) of mice following repeated methamphetamine administration. Region-specific roles of this molecule are associated with psychiatric disorders. In the present study, we examined the importance of Shati/Nat8l in the hippocampus because of its high expression in this region. Mice with a hippocampus-specific knockdown of Shati/Nat8l (hippocampal Shati-cKD) were prepared by the microinjection of adeno-associated virus (AAV) vectors carrying Cre into the hippocampus of Shati/Nat8lflox/flox mice, and their phenotypes were investigated. Drastic reduction in the expression and function of Shati/Nat8l in the hippocampus was observed in Shati-cKD mice. These mice exhibited cognitive dysfunction in behavioral experiments and impaired the electrophysiological response to the stimuli, which elicits long-term potentiation. Shati/Nat8l in the hippocampus is suggested to possibly play an important role in synaptic plasticity to maintain cognitive function. This molecule could be a therapeutic target for hippocampus-related disorders such as dementia.

MiRNA-103-3p promotes neural cell autophagy by activating Wnt/β-catenin signaling via targeting rab10 in a rat model of depression

To explore the neuroprotective role of Rab10 gene in depression and the mechanism mediating its effect. Forty-eight male SD rats were randomized into a control group and 3 chronic unpredictable mild stress (CUMS) groups (n=12). The rats in the latter 3 groups were subjected to injections of normal saline, an adeno-associated viral (AAV) vector, or a Rab10-overexpressing AAV vector in the lateral ventricle after CUMS modeling. The depressive behavioral changes of the rats were assessed using behavioral tests. The TargetScan database was used to predict the miRNA interacting with Rab10 and the binding sites. The interaction between miRNA-103-3p and Rab10 was investigated using dual-luciferase and radioimmunoprecipitation (RIP) assay. The effect of corticosterone treatment on PC12 cell viability was assessed with CCK-8 assay. In corticosterone-stimulated PC12 cells, the changes in BDNF, CREB, p62, Beclin-1, Wnt3a, Gsk3β, phosphorylated (p)-Gsk3β, and β-catenin protein expressions following transfection with the Rab10-overexpressing AAV vector and a miRNA-103-3p inhibitor, alone or in combination, were analyzed using qRT-PCR and Western blotting. Injection of Rab10-overexpressing AVV vector into the lateral ventricle significantly improved depressive behaviors of CUMS rats. The mRNA and proteins expression of Rab10 were significantly down-regulated in the hippocampus of CUMS rats and in corticosteronestimulated PC12 cells. Bioinformatics analysis and the results of double luciferase and RIP experiments confirmed the targeting relationship between miRNA-103-3p and Rab10. In PC12 cells, overexpression of Rab10 or silencing miRNA-103-3p activated the Wnt/β-catenin signaling pathway, up-regulated the expressions of BDNF, CREB and Beclin-1, and down-regulated the expression of p62 protein; silencing Rab10 obviously blocked the effect of miRNA-103-3p inhibitor. In mouse models of depression, miRNA-103-3p activates Wnt/β-catenin signaling via targeting rab10 to improve neural plasticity and promotes neural cell autophagy.