Adeno-associated Virus Vectors Research Articles

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.

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.

PGD 2 /DP1 axis promotes liver regeneration by secreting Wnt2 in KCs in mice.

The liver possesses a remarkable regenerative capacity in response to injuries or viral infections. Various growth factors and cytokines are involved in regulating liver regeneration. Prostaglandin D 2 , a pro-resolution lipid mediator, is the most abundant hepatic prostanoid. However, the role of prostaglandin D 2 in the injury-induced liver regeneration remains unclear. Two-thirds partial hepatectomy (70% PH), massive hepatectomy (85% resection), and carbon tetrachloride-induced chronic injury were performed in mice to study the mechanisms of live regeneration. Hepatic prostaglandin D 2 production was elevated in mice after PH. Global deletion of D prostanoid receptor (DP) 1, but not DP2, slowed PH-induced liver regeneration in mice, as evidenced by lower liver weight to body weight ratio, less Ki67 + hepatocyte proliferation, and G2/M phase hepatocytes. In addition, DP1 deficiency, specifically in resident KCs, and not in endothelial cells or HSCs, retarded liver regeneration in mice after PH. Conversely, the overexpression of exogenous DP1 in KCs accelerated liver regeneration in mice. Mechanistically, DP1 activation promoted Wnt2 transcription in a PKA/CREB-dependent manner in resident KCs and mediated hepatocyte proliferation through Frizzled8/β-catenin signaling. Adeno-associated virus vector serotype 8-mediated Frizzled8 knockdown in hepatocytes attenuated accelerated liver regeneration in KC-DP1 transgenic mice after PH. Treatment with the DP1 receptor agonist BW245C promotes PH-induced liver regeneration in mice. DP1 activation mediates crosstalk between KCs and hepatocytes through Wnt2 and facilitates liver regeneration. Hence, DP1 may serve as a novel therapeutic target in acute and chronic liver diseases.

Delivery of CDNF by AAV-mediated gene transfer protects dopamine neurons and regulates ER stress and inflammation in an acute MPTP mouse model of Parkinson’s disease

Cerebral dopamine neurotrophic factor (CDNF) and its close structural relative, mesencephalic astrocyte-derived neurotrophic factor (MANF), are proteins with neurotrophic properties. CDNF protects and restores the function of dopamine (DA) neurons in rodent and non-human primate (NHP) toxin models of Parkinson’s disease (PD) and therefore shows promise as a drug candidate for disease-modifying treatment of PD. Moreover, CDNF was found to be safe and to have some therapeutic effects on PD patients in phase 1/2 clinical trials. However, the mechanism underlying the neurotrophic activity of CDNF is unknown. In this study, we delivered human CDNF (hCDNF) to the brain using an adeno-associated viral (AAV) vector and demonstrated the neurotrophic effect of AAV-hCDNF in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. AAV-hCDNF resulted in the expression of hCDNF in the striatum (STR) and substantia nigra (SN), and no toxic effects on the nigrostriatal pathway were observed. Intrastriatal injection of AAV-hCDNF reduced motor impairment and partially alleviated gait dysfunction in the acute MPTP mouse model. In addition, gene therapy with AAV-hCDNF had significant neuroprotective effects on the nigrostriatal pathway and decreased the levels of interleukin 1beta (IL-1β) and complement 3 (C3) in glial cells in the acute MPTP mouse model. Moreover, AAV-hCDNF reduced C/EBP homologous protein (CHOP) and glucose regulatory protein 78 (GRP78) expression in astroglia. These results suggest that the neuroprotective effects of CDNF may be mediated at least in part through the regulation of neuroinflammation and the UPR pathway in a mouse MPTP model of PD in vivo.

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes thatexceed the AAV capacity.

When size matters: A novel compact Cas12a variant for in vivo genome editing.

A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.

In vivo neural regeneration via AAV-NeuroD1 gene delivery to astrocytes in neonatal hypoxic-ischemic brain injury

BackgroundNeonatal hypoxic-ischemic brain injury (HIBI) is a significant contributor to neonatal mortality and long-term neurodevelopmental disability, characterized by massive neuronal loss and reactive astrogliosis. Current therapeutic approaches for neonatal HIBI have been limited to general supportive therapy because of the lack of methods to compensate for irreversible neuronal loss. This study aimed to establish a feasible regenerative therapy for neonatal HIBI utilizing in vivo direct neuronal reprogramming technology.MethodsNeonatal HIBI was induced in ICR mice at postnatal day 7 by permanent right common carotid artery occlusion and exposure to hypoxia with 8% oxygen and 92% nitrogen for 90 min. Three days after the injury, NeuroD1 was delivered to reactive astrocytes of the injury site using the astrocyte-tropic adeno-associated viral (AAV) vector AAVShH19. AAVShH19 was engineered with the Cre-FLEX system for long-term tracking of infected cells.ResultsAAVShH19-mediated ectopic NeuroD1 expression effectively converted astrocytes into GABAergic neurons, and the converted cells exhibited electrophysiological properties and synaptic transmitters. Additionally, we found that NeuroD1-mediated in vivo direct neuronal reprogramming protected injured host neurons and altered the host environment, i.e., decreased the numbers of activated microglia, reactive astrocytes, and toxic A1-type astrocytes, and decreased the expression of pro-inflammatory factors. Furthermore, NeuroD1-treated mice exhibited significantly improved motor functions.ConclusionsThis study demonstrates that NeuroD1-mediated in vivo direct neuronal reprogramming technology through AAV gene delivery can be a novel regenerative therapy for neonatal HIBI.