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Gene delivery to fertilized eggs is often the first step in creation of transgenic animals, CRISPR knock-out, or early developmental studies. The zona pellucida, a hardened glycoprotein matrix surrounding the mammalian fertilized eggs, often complicates gene delivery by forming a barrier against transfection reagents and viruses. High efficiency techniques to perforate or penetrate the zona allow for access and gene delivery to fertilized eggs. However, these techniques often rely on highly skilled technologists, are costly, and require specialized equipment for micromanipulation, laser perforation, or electroporation. Here, we report that adenoassociated viruses (AAVs) with serotypes 1 or DJ can efficiently diffuse across the zona to deliver genes without any manipulations to fertilized eggs. We observe lowered rates of embryo development after treatment of embryos with all AAV serotypes. However, we were able to reduce adverse effects on embryo development by exposing embryos to AAVs at later stages of in vitro development. AAVs have low immune response and do not incorporate into their host chromosomes to cause insertional mutations. Hence, AAVs can serve as a highly effective tool for transient delivery of genes to fertilized mammalian eggs.

Adeno-associated virus (AAV)-based vectors have transformed into powerful elements of genetic medicine with proven therapeutic efficacy and a good safety profile. Over the years, efforts to transduce hematopoietic stem cells (HSCs) with AAV2 vectors have, however, been challenging. While there was evidence that AAV2 delivered vector genomes to primitive, quiescent, multipotential, self-renewing, in vivo engrafting HSCs, transgene expression was elusive. In this study, we review the evolution of AAV transduction of HSC, starting with AAV2 vectors leading to the isolation of a family of naturally occurring AAVs from human CD34(+) HSC, the AAVHSC. The stem cell-derived AAVHSCs have turned out to have remarkable potentials for genetic therapies well beyond the hematopoietic system. AAVHSCs have tropism for a wide variety of peripheral tissues, including the liver, muscle, and the retina. They cross the blood–brain barrier and transduce cells of the central nervous system. Preclinical gene therapy studies underway using AAVHSC vectors are discussed. We review the notable ability of AAVHSCs to mediate efficient, seamless homologous recombination in the absence of exogenous nuclease activity and discuss the therapeutic implications. We also discuss early results from an AAVHSC-based clinical gene therapy trial that is underway for the treatment of phenylketonuria. Thus, the stem cell-derived AAVHSC, offer a multifaceted platform for in vivo gene therapy and genome editing for the treatment of inherited diseases.

Background: Adeno-associated Virus (AAV) vectors are the most promising vehicles for therapeutic gene delivery to the retina. To develop a practical gene delivery tool, achieving high AAV transduction efficiency in specific cell types is often required. AAV-mediated targeted expression in retinal bipolar cells is needed in certain applications such as optogenetic therapy, however, the transduction efficiency driven by endogenous cell-specific promoters is usually low. Methods that can improve AAV transduction efficiency in bipolar cells need to be developed. Objective: The study aimed to examine the effect of proteasome inhibitors on AAV-mediated transduction efficiency in retinal bipolar cells. Methods: Quantitative analysis of fluorescent reporter protein expression was performed to assess the effect of two proteasome inhibitors, doxorubicin and MG132, on AAV-mediated transduction efficiency in retinal bipolar cells in mice. Results: Our results showed that doxorubicin can increase the AAV transduction efficiency in retinal bipolar cells in a dose-dependent manner. We also observed doxorubicin-mediated cytotoxicity in retinal neurons, but the cytotoxicity could be mitigated by the coapplication of dexrazoxane. Three months after the coapplication of doxorubicin (300 μM) and dexrazoxane, the AAV transduction efficiency in retinal bipolar cells increased by 33.8% and no cytotoxicity was observed in all the layers of the retina. Conclusion: Doxorubicin could enhance the AAV transduction efficiency in retinal bipolar cells in vivo. The potential long-term cytotoxicity caused by doxorubicin to retinal neurons could be partially mitigated by dexrazoxane. The coapplication of doxorubicin and dexrazoxane may serve as a potential adjuvant regimen for improving AAV transduction efficiency in retinal bipolar cells.

Inflammation plays an essential role in the development of lumbar disc degeneration (LDD), although the exact effects of macrophage subtypes on LDD remain unclear. Based on previous studies, we hypothesized that M2-polarization of local macrophages and simultaneous suppression of their production of fibrotic transforming growth factor beta 1 (TGFβ1) could inhibit progression of LDD. Thus, we applied an orthotopic injection of adeno-associated virus (AAV) carrying shRNA for DNA Methyltransferase 1 (DNMT1) and/or shRNA for TGFβ1 under a macrophage-specific CD68 promoter to specifically target local macrophages in a mouse model for LDD. We found that shDNMT1 significantly reduced levels of the pro-inflammatory cytokines TNFα, IL-1β and IL-6, significantly increased levels of the anti-inflammatory cytokines IL-4 and IL-10, significantly increased M2 macrophage polarization, significantly reduced cell apoptosis in the disc degeneration zone and significantly reduced LDD-associated pain. The anti-apoptotic and anti-pain effects were further strengthened by co-application of shTGFβ1. Together, these data suggest that M2 polarization of macrophages induced by both epigenetic modulation and suppressed production and release of TGFβ1 from polarized M2 macrophages, may have a demonstrable therapeutic effect on LDD.

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughputin vivoscreening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENTHere, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCsin vivo. To our knowledge, this is the first report demonstratingin vivoneuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF–enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 (DNM1) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1-targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product., Graphical Abstract, Toxic mutations in DNM1, a gene involved in the proper functioning of brain cells, cause developmental and epileptic encephalopathy—a severe class of epilepsy with no cure. Aimiuwu and colleagues show that a single gene therapy treatment via RNA interference eliminated the toxic allele sufficiently to curb severe lethal seizures, eliminate motor and developmental deficits, and extend survival of treated mice.

The messenger RNA (mRNA) 3′ untranslated regions (3′ UTRs), as cis-regulated elements bound by microRNAs (miRNAs), affect their gene translation. However, the role of the trans-regulation of 3′ UTRs during heart dysfunction remains elusive. Compared with administration of angiogenic factor with G-patch and forkhead-associate domains 1 (Aggf1), ectopic expression of Aggf1 with its 3′ UTR significantly suppressed cardiac dysfunction in angiotensin II-infused mice, with upregulated expression of both Aggf1 and myeloid cell leukemia 1 (Mcl1). Along their 3′ UTRs, Mcl1 and Aggf1 mRNAs share binding sites for the same miRNAs, including miR-105, miR-101, and miR-93. We demonstrated that the protein-coding Mcl1 and Aggf1 mRNAs communicate and co-regulate each other’s expression through competition for these three miRNAs that target both transcripts via their 3′ UTRs. Our results indicate that Aggf1 3′ UTR, as a trans-regulatory element, accelerates the cardioprotective role of Aggf1 in response to hypertensive conditions by elevating Mcl1 expression. Our work broadens the scope of gene therapy targets and provides a new insight into gene therapy strategies involving 3′ UTRs.

Nerve growth factor (NGF) gene therapy rescues and stimulates cholinergic neurons, which degenerate in Alzheimer's disease (AD). In a recent clinical trial for AD, intraparenchymal adeno-associated virus serotype 2 (AAV2)-NGF delivery was safe but did not improve cognition. Before concluding that NGF gene therapy is ineffective, it must be shown that AAV2-NGF successfully engaged the target cholinergic neurons of the basal forebrain. In this study, patients with clinically diagnosed early- to middle-stage AD received a total dose of 2 × 10(11) vector genomes of AAV2-NGF by stereotactic injection of the nucleus basalis of Meynert. After a mean survival of 4.0 years, AAV2-NGF targeting, spread, and expression were assessed by immunolabeling of NGF and the low-affinity NGF receptor p75 at 15 delivery sites in 3 autopsied patients. NGF gene expression persisted for at least 7 years at sites of AAV2-NGF injection. However, the mean distance of AAV2-NGF spread was only 0.96 ± 0.34 mm. NGF did not directly reach cholinergic neurons at any of the 15 injection sites due to limited spread and inaccurate stereotactic targeting. Because AAV2-NGF did not directly engage the target cholinergic neurons, we cannot conclude that growth factor gene therapy is ineffective for AD. Upcoming clinical trials for AD will utilize real-time magnetic resonance imaging guidance and convection-enhanced delivery to improve the targeting and spread of growth factor gene delivery.

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.

Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design., Adeno-associated viruses (AAVs) can be targeted in a tissue-specific manner, but their tissue accumulation cannot be assessed in a non-invasive manner. Here the authors conjugate a multivalent chelator labelled with Cu-64 to the surface of AAVs and image the brain accumulation of the PHB.eB capsid by PET.

Regulation of gene expression by viral vectors is an effective method for researchers to explore the function of gene products in a target tissue. The choroid plexus (CP) is an important target for gene therapy of neuropsychiatric diseases such as Alzheimer's disease and major depressive disorder. However, viral tropism in CP has not been well studied as a result of limited viral vector applications. To identify CP-specific viral vectors, we intracerebroventricularly administered six different serotypes of adeno-associated virus (AAV) vectors (AAV2/1, AAV2/5, AAV2/8, AAV2/9, AAV2-BR1, and AAV2-PHP.eB) and lentivirus in adult mice. Tropism in CP was compared among these viruses. We found that AAV2/5 and AAV2/8 displayed remarkable infections in CP, while AAV2/1 infected both ependymal cells and cells in the CP. Except for the low infection intensity of AAV2/9 and lentivirus in the CP, no infection intensity was found for CP tissues injected with AAV2-BR1 or AAV2-PHP.eB. Green fluorescence protein expression in the CP after AAV2/5 infection was confirmed by Western blotting. AAV2/5-mediated tropism in epithelial cells of the CP was verified by immunostaining with transthyretin. In this study, we identified for the first time that serotype-specific AAVs 5 and 8 may be robust research tools for intracerebroventricular gene delivery.

Allograft vasculopathy (AV) is the primary limiting factor for long-term graft survival. An increased activity of matrix metalloproteinases (MMPs) contributes to neointima formation in AV and represents a potential therapeutic target. Adeno-associated virus (AAV)-mediated gene therapy comprises a potentially benign vector model for the long-term expression of MMP antagonists.Aortic allografts from DBA/2 mice were incubated with control buffer, AAV-enhanced green fluorescence protein (EGFP), or tissue inhibitor of metalloproteinases 1 (TIMP-1)-loaded AAV (AAV-TIMP-1) and transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight) was administered daily. Explantation as well as histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography.Intima-to-media area ratio and neointima formation were significantly reduced in the AAV-TIMP-1 treatment group compared with those in the control group (by 40%; p0.001) and the AAV-EGFP group (by 38.2%; p0.001). TIMP-1 overexpression positively affected several pathomechanisms for the development of AV both in vitro and in vivo as compared to that in the control groups: endothelium integrity was preserved as shown by zona occludens 1 and occludin staining; MMP9 expression and activity were significantly reduced (p = 0.01); and smooth muscle cell migration was significantly reduced as smooth muscle actin positive cells predominantly remained in the aortic media in the treatment group (p = 0.001). Moreover, macrophage infiltration was markedly reduced by 49% in the AAV-TIMP-1 group (p0.001).Immediate post-harvesting allograft incubation with AAV-TIMP-1 reduces neointima formation and macrophage infiltration, constituting a possible adjunct therapeutic strategy to preserve graft function after transplantation.

Purpose: To evaluate the relation of aquaporin-1 (AQP-1) expression and scleral biomechanical strength. Method: Guinea pigs with 3 weeks old received intravitreal injection of adeno-associated viru...

Background: : Intervertebral Disc (IVD) degeneration is a major public health concern, and gene therapy seems a promising approach to delay or even reverse IVD degeneration. However, the delivery system used to transfer exogenous genes into intervertebral disc cells remains a challenge. Methods:: The MEDLINE, Web of Science, and Scopus databases were searched for English-language articles related to gene therapy for IVD degeneration articles from 1999 to May 2019. The keywords included “gene therapy” AND “intervertebral disc”. The history of the development of different delivery systems was analysed, and the latest developments in viral and non-viral vectors for IVD degeneration treatment were reviewed. Results: : Gene therapy delivery systems for IVD degeneration are divided into two broad categories: viral and non-viral vectors. The most commonly used viral vectors are adenovirus, adeno-associated virus (AAV), and lentivirus. Enthusiasm for the use of adenovirus vectors has gradually declined and has been replaced by a preference for lentivirus and AAV vectors. New technologies, such as RNAi and CRISPR, have further enhanced the advantage of viral vectors. Liposomes are the classic non-viral vector, and their successors, polyplex micelles and exosomes, have more potential for use in gene therapy for IVD degeneration. Conclusion:: Lentivirus and AAV are the conventional viral vectors used in gene therapy for IVD degeneration, and the new technologies RNAi and CRISPR have further enhanced their advantages. Nonviral vectors, such as polyplex micelles and exosomes, are promising gene therapy vectors for IVD degeneration.

Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1 , encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BK G354S ) in KCNMA1 , in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BK G354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BK G354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BK G354S channel, but not the BK WT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.

The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.

Dravet syndrome (DS) is a neurodevelopmental genetic disorder caused by mutations in the SCN1A gene encoding the α subunit of the NaV1.1 voltage-gated sodium channel that controls neuronal action potential firing. The high density of this mutated channel in GABAergic interneurons results in impaired inhibitory neurotransmission and subsequent excessive activation of excitatory neurons. The syndrome is associated with severe childhood epilepsy, autistic behaviors, and sudden unexpected death in epilepsy. Here, we compared the rescue effects of an adeno-associated viral (AAV) vector coding for the multifunctional β1 sodium channel auxiliary subunit (AAV-NaVβ1) with a control vector lacking a transgene. We hypothesized that overexpression of NaVβ1 would facilitate the function of residual voltage-gated channels and improve the DS phenotype in the Scn1a+/- mouse model of DS. AAV-NaVβ1 was injected into the cerebral spinal fluid of neonatal Scn1a+/- mice. In untreated control Scn1a+/- mice, females showed a higher degree of mortality than males. Compared with Scn1a+/- control mice, AAV-NaVβ1-treated Scn1a+/- mice displayed increased survival, an outcome that was more pronounced in females than males. In contrast, behavioral analysis revealed that male, but not female, Scn1a+/- mice displayed motor hyperactivity, and abnormal performance on tests of fear and anxiety and learning and memory. Male Scn1a+/- mice treated with AAV-NaVβ1 showed reduced spontaneous seizures and normalization of motor activity and performance on the elevated plus maze test. These findings demonstrate sex differences in mortality in untreated Scn1a+/- mice, an effect that may be related to a lower level of intrinsic inhibitory tone in female mice, and a normalization of aberrant behaviors in males after central nervous system administration of AAV-NaVβ1. The therapeutic efficacy of AAV-NaVβ1 in a mouse model of DS suggests a potential new long-lasting biological therapeutic avenue for the treatment of this catastrophic epilepsy.

Renal fibrosis leads to end-stage renal disease, but antifibrotic drugs are difficult to develop. Chronic kidney disease often results in muscle wasting, and thereby increases morbidity and mortality. In this work, adeno-associated virus (AAV)-mediated overexpressing miR-29a was hypothesized to counteract renal fibrosis and muscle wasting through muscle–kidney crosstalk in unilateral ureteral obstruction (UUO) mice. miR-29a level was downregulated in the kidney and skeletal muscle of UUO mice. The secretion of exosome-encapsulated miR-29a increased in cultured skeletal muscle satellite cells and HEK293 renal cells after stimulation with serum from UUO mice. This result was confirmed by qPCR and microRNA deep sequencing in the serum exosomes of mice with obstructed ureters. A recombinant AAV-miR-29a was generated to overexpress miR-29a and injected into the tibialis anterior muscle of the mice 2 weeks before UUO surgery. AAV-miR-29a abrogated the UUO-induced upregulation of YY1 and myostatin in skeletal muscles. Renal fibrosis was also partially improved in the UUO mice with intramuscular AAV-miR-29a transduction. AAV-miR-29a overexpression reversed the increase in transforming growth factor β, fibronectin, alpha-smooth muscle actin, and collagen 1A1 and 4A1 levels in the kidney of UUO mice. AAV-green fluorescent protein was applied to trace the AAV route in vivo, and fluorescence was significantly visible in the injected/uninjected muscles and in the kidneys. In conclusion, intramuscular AAV-miR-29a injection attenuates muscle wasting and ameliorates renal fibrosis by downregulating several fibrotic-related proteins in UUO mice.

Introduction: The development of adeno-associated virus (AAV) vectors as safe vehicles for in vivo delivery of therapeutic genes has been a major milestone in the advancement of gene therapy, enabl...

Huntington’s disease (HD) is caused by Huntingtin (Htt) gene mutation resulting in the loss of striatal GABAergic neurons and motor functional deficits. We report here an in vivo cell conversion technology to reprogram striatal astrocytes into GABAergic neurons in both R6/2 and YAC128 HD mouse models through AAV-mediated ectopic expression of NeuroD1 and Dlx2 transcription factors. We found that the astrocyte-to-neuron (AtN) conversion rate reached 80% in the striatum and >50% of the converted neurons were DARPP32+ medium spiny neurons. The striatal astrocyte-converted neurons showed action potentials and synaptic events, and projected their axons to the targeted globus pallidus and substantia nigra in a time-dependent manner. Behavioral analyses found that NeuroD1 and Dlx2-treated R6/2 mice showed a significant extension of life span and improvement of motor functions. This study demonstrates that in vivo AtN conversion may be a disease-modifying gene therapy to treat HD and other neurodegenerative disorders., In vivo reprogramming of reactive glia using transfection of a single transcription factor has been described before by these authors and applied to models of neurodegeneration. Here the authors use this procedure in the R6/2 mouse model of Huntington’s disease, targeting astrocytes in the striatum, converting them to GABAergic neurons.

Adeno-associated viruses (AAVs) are used as in vivo gene-delivery vectors in gene-therapy products and have been heavily investigated for numerous indications. Over 100 naturally occurring AAV serotypes and variants have been isolated from primate samples. Many reports have described unique properties of these variants (for instance, differences in potency, target cell or evasion of the immune response), despite high amino-acid sequence conservation. AAVhu.37 is of interest for clinical applications owing to its proficient transduction of the liver and central nervous system. The sequence identity of the AAVhu.37 VP1 to the well characterized AAVrh.10 serotype, for which no structure is available, is greater than 98%. Here, the structure of the AAVhu.37 capsid at 2.56 Å resolution obtained via single-particle cryo-electron microscopy is presented.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models. ispartof: SCIENTIFIC REPORTS vol:10 issue:1 ispartof: location:England status: published

We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4 weeks. In a Langendorff model, hypertrophic hearts were subjected to 40 min/60 min I/R or with IPostC intervention consisting of 6 cycles of 10 s reperfusion and 10 s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P

Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.

Inhibition of pulmonary fibrosis (PF) by restoring sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a) expression using targeted gene therapy may be a potentially powerful new treatment approach for PF. Here, we found that SERCA2a expression was significantly decreased in lung samples from patients with PF and in the bleomycin (BLM) mouse model of PF. In the BLM-induced PF model, intratracheal aerosolized adeno-associated virus serotype 1 (AAV1) encoding for human SERCA2a (AAV1.hSERCA2a) reduces lung fibrosis and associated vascular remodeling. SERCA2a gene therapy also decreases right ventricular pressure and hypertrophy in both prevention and curative protocols. In vitro, we observed that SERCA2a overexpression inhibits fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition induced by transforming growth factor β (TGF-β1). Thus, pro-fibrotic gene expression is prevented by blocking nuclear factor κB (NF-κB)/interleukin-6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3) activation. This effect is signaled toward an inhibitory mechanism of small mother against decapentaplegic (SMAD)/TGF-β signaling through the repression of OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1) and Forkhead box M1 (FOXM1). Interestingly, this cross-inhibition leads to an increase of SKI and SnoN expression, an auto-inhibitory feedback loop of TGF-β signaling. Collectively, our results demonstrate that SERCA2a gene transfer attenuates bleomycin (BLM)-induced PF by blocking the STAT3/FOXM1 pathway and promoting the SNON/SKI Axis. Thus, SERCA2a gene therapy may be a potential therapeutic target for PF.

Short hairpin RNAs that are delivered by recombinant adeno-associated virus (rAAV) have the potential to elicit long-term RNAi therapy for human disease. However, the discovery that short hairpin sequences can cause truncation of the rAAV genome calls into question the efficiency and gene-silencing specificity of this strategy in humans. Here, we report that embedding the guide strand of a small silencing RNA into an artificial microRNA (miRNA) scaffold derived from mouse miRNA-33 ensures rAAV genomic integrity and reduces off-targeting by 10-fold, while maintaining effective in vivo target gene repression in mice.

The medial prefrontal cortex (mPFC) is known to be critical for specific forms of long-term recognition memory, however the cellular mechanisms in the mPFC that underpin memory maintenance have not been well characterized. This study examined the importance of phosphorylation of cAMP responsive element binding protein (CREB) in the mPFC for different forms of long-term recognition memory in the rat. Adenoviral transduction of the mPFC with a dominant-negative inhibitor of CREB impaired object-in-place memory following a 6 or 24 h retention delay, but no impairment was observed following delays of 5 min or 3 h. Long-term object temporal order memory and spatial temporal order memory was also impaired. In contrast, there were no impairments in novel object recognition or object location memory. These results establish, for the first time, the importance of CREB phosphorylation within the mPFC for memory of associative and temporal information crucial to recognition.

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

Rationale: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. Methods and Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d , which when overexpressed, led to hypertrophy and heart failure, and Klf15 , which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15 , we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. Conclusions: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.

Dengue virus (DENV) remains a significant healthcare and socioeconomic burden for endemic countries. Attempts to produce a safe and effective vaccine have been unsuccessful so far, making this task one of the top priorities in the field. We have previously shown that an EDIII-based DNA vaccine is able to induce neutralizing, long-lasting and highly specific antibody responses for all four DENV serotypes in mice using gene-gun delivery technology. Here, we describe the use of recombinant Adeno-associated viral vectors as an alternative DNA delivery platform, in combination with different immunization schedules, to simplify the vaccination protocol without compromising the induction of neutralizing antibody responses. Our results demonstrate that using viral vectored-platforms to deliver genetic vaccines could potentially reduce the number of doses required to induce a sustained DENV-neutralizing response, thus facilitating the implementation and deployment of the vaccine in developing countries.

Gene therapy with adeno-associated viral (AAV) vectors has reached the clinical stage for many inherited and acquired diseases. However, due to a cargo capacity limited to5 kb, AAV-mediated treatment of diseases that require transfer of larger genes still appears elusive. This is a major drawback of a platform that has otherwise been repeatedly found to be safe and effective. Thus, great efforts have been directed toward the identification of strategies to overcome this limitation. Among the most studied approaches is the use of dual vectors, in which a transgene is split across two separate AAV vectors. Mechanisms acting at either the DNA, pre-mRNA, or protein levels have been explored to restore full-length transgene expression in infected cells. Here, we will review them as well as additional strategies developed to deliver large genes with AAV. We discuss the pros and cons of these strategies and the aspects that still need to be addressed.

International audience; Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.

Nociception is important perception that has harmful influence on daily life of humans. As to main pain management system, some descending pathways are called descending antinociceptive systems (DAS). As main pathways of DAS, it is well known that dorsal raphe (B6/B7) - rostral ventromedial medulla (B3) - spinal dorsal horn includes serotonergic system. However, possible role of supralemniscal (B9) serotonin (5-HT) cell group in pain management is still open question. In this study, we measured activities of B9 5-HT neuronal cell bodies and B9 5-HT neuron-derived axons located in the locus coeruleus (LC) and ventral tegmental area (VTA), which are also main players of pain management, using fiber photometry system. We introduced the G-CaMP6 in B9 5-HT neurons using transgenic mice carrying a tetracycline-controlled transactivator transgene (tTA) under the control of a tryptophan hydroxylase-2 (TPH2) promoter and site-specific injection of adeno associated virus (AAV-TetO(3G)-G-CaMP6). After confirmation of specific expression of G-CaMP6 in the target population, G-CaMP6 fluorescence intensity in B9 group and LC/VTA groups was measured in awake mice exposed to acute tail pinch and heat stimuli. G-CaMP6 fluorescence intensity rapidly increased by both stimuli in all groups, but not significantly reacted by nonnociceptive control stimuli. The present results clearly indicate that acute nociceptive stimuli cause a rapid increase in the activities of B9-LC/B9-VTA 5-HTergic pathways, suggesting that B9 5-HT neurons play important roles in nociceptive processing.

Joint trauma leads to post-traumatic inflammation with upregulation of inflammatory cytokines and degradative enzymes. If severe enough, this response can lead to irreversible post-traumatic osteoarthritis. Interleukin-10 (IL-10), a cytokine with potent anti-inflammatory effects, has been shown to have chondroprotective effects. A gene therapy approach using a vector to overexpress IL-10 in the joint represents a feasible method of delivering sustained high doses of IL-10 to post-traumatic joints. We hypothesized that an AAV5 vector overexpressing IL-10 would result in rapid and sustained IL-10 expression following direct intra-articular injection and that this increase would not be reflected in systemic circulation. In addition, we hypothesized that intra-articular AAV5-IL-10 injection would not induce a local inflammatory response. Twelve horses were assigned to either treatment (AAV5-IL-10-injected) or control (PBS-injected) groups. Middle carpal joints were injected with 10(12) vector genomes/joint or phosphate-buffered saline (PBS) alone (3 mL). Serial synovial fluid samples were analyzed for inflammatory changes, IL-10 concentration, and vector genome copy number. Serum samples were also analyzed for IL-10 concentration and vector genome copy number. Synovial membrane was collected on day 84. Synovial fluid IL-10 was significantly increased within 48 h of AAV5-IL-10 injection and remained increased, compared to PBS-injected joints, until day 84. Serum IL-10 was not different between groups. Vector administration did not cause a significant synovial inflammatory response. Vector genomes were detectable in the plasma, synovial fluid, and synovial membrane of AAV5-IL-10-injected horses only. IL-10 has the potential to modulate the articular inflammatory response, thereby protecting cartilage from degradation and osteoarthritis. This study demonstrates the feasibility and efficiency of intra-articular AAV5-IL-10, and future studies investigating the chondroprotective effects of IL-10 in inflamed joints in vivo are warranted.

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. Methods: As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants ex vivo. For in vivo assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). Results: We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For in vivo experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. Conclusions: Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.

目的 评价携带人凝血因子FⅧ（hFⅧ）的重组腺相关病毒（AAV）血清型8（AAV8/hFⅧ）病毒治疗血友病A（HA）小鼠效果。 方法 应用pAAV-CB-EGFP，pH22（AAV2血清型）和pfΔ6（腺病毒辅助质粒）摸索在HEK-293细胞中AAV的包装条件，pH22、pfΔ6及pAAV-CB-EGFP质粒按照1∶1∶1的比例进行转染，免疫荧光显微镜观察四个条件（包括10 cm培养皿转10 µg质粒，20 cm培养皿分别转20、30和40 µg质粒）的病毒包装效果。应用最佳包装条件在HEK-293细胞中包装AAV2-EGFP，通过冻融法获得AAV2粗提液，感染HEK-293细胞和16095细胞，应用荧光显微镜观察包装效果。应用最佳转染条件将pAAV-TTR-hFⅧ、pH28、pfΔ6按1∶1∶1的比例转染HEK-293细胞获得AAV8/hFⅧ，两轮的氯化铯梯度离心法纯化病毒，以8×1012 vg/kg剂量经尾静脉注射HA小鼠进行基因治疗，活化部分凝血活酶时间（APTT）测定分析FⅧ的活性。 结果 20 cm培养皿转染20 µg质粒的条件能够在24、48和72 h都达到最佳的转染效果，且该条件包装的AAV2粗提液在16095细胞中具有较高的感染率。HA小鼠体内AAV8/hFⅧ能够在注射后12周仍有维持在治疗水平的FⅧ活性。 结论 利用优化包装条件制备的AAV8/hFⅧ能够在HA小鼠体内有效维持治疗水平的FⅧ活性达12周。

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells., Graphical Abstract, Whereas AAV vector administration in muscle is usually immunogenic, muscle-derived delivery of an AAV1 vector in monkeys and alpha1-antitrypsin-deficient (AATD) patients can lead to persisting transgene expression. Both regulatory and exhausted T cells are involved in maintaining transgene expression.

Liver fibrosis is a chronic liver disease that could further develop to cirrhosis and liver carcinoma. Hepatic stellate cells (HSCs) are primary effector cells to initiate liver fibrosis. We aimed to explore the function and underlying mechanisms of mitochondrial fusion protein Mitofusin-2 (MFN2) in liver fibrosis. First, we utilized an alpha-smooth muscle actin promoter to overexpress MFN2 specifically in HSCs using adeno-associated virus (AAV) vector (AAV-MFN2). Overexpression of MFN2 was specifically achieved in HSC-T6 cells, but not in murine bone marrow-derived macrophages or hepatocyte AML-12 cells. We found that high expression of MFN2 induced apoptosis of HSC-T6 cells. Mechanistically, we demonstrated that high level of MFN2 inhibited TGF-β1/Smad signaling pathway, triggered downregulation of type I, type III, and type IV collagen, and antagonized the formation of factors associated with liver fibrosis. Furthermore, we found that overexpression of MFN2 using AAV-MFN2 ameliorated CCl4-induced liver fibrosis in vivo with significantly decreased immune cell infiltration. Taken together, our findings indicate that MFN2 is critical in regulating apoptosis and liver fibrosis in HSCs, which might be a useful therapeutic target to treat liver fibrosis.

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection., Graphical Abstract, A novel approach to treat glaucoma is reported that achieves reduced intraocular pressure by combining intravitreal adeno-associated virus (AAV) gene delivery to the ciliary body with selective CRISPR-Cas9-mediated disruption of Aquaporin 1. Translational viability is assessed using both human ex vivo ciliary body cultures and two experimental mouse models.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder caused by repetitive trauma to the central nervous system (CNS) suffered by soldiers, contact sport athletes, and civilians following accident-related trauma. CTE is a CNS tauopathy, with trauma-induced inflammation leading to accumulation of hyperphosphorylated forms of the microtubule-binding protein Tau (pTau), resulting in neurofibrillary tangles and progressive loss of neurons. At present, there are no therapies to treat CTE. We hypothesized that direct CNS administration of an adeno-associated virus (AAV) vector coding for an anti-pTau antibody would generate sufficient levels of anti-pTau in the CNS to suppress pTau accumulation thus interrupting the pathogenic process. Using a serotype AAVrh.10 gene transfer vector coding for a monoclonal antibody directed against pTau, we demonstrate the feasibility of this strategy in a murine CTE model in which pTau accumulation was elicited by repeated traumatic brain injury (TBI) using a closed cortical impact procedure over 5 days. Direct delivery of AAVrh.10 expression vectors coding for either of the two different anti-pTau antibodies to the hippocampus of these TBI mice significantly reduced pTau levels across the CNS. Using doses that can be safely scaled to humans, the data demonstrate that CNS administration of AAVrh.10anti-pTau is effective, providing a new strategy to interrupt the CTE consequences of TBI.

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca(2+)) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca(2+) pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLB(M)) that were engineered to compete with the binding of inhibitory wild-type PLB (PLB(WT)). Our therapeutic strategy is to relieve PLB(WT) inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLB(M) (L31A/I40A) that effectively competes with PLB(WT) binding to cardiac SERCA2a in HEK293–6E cells. We demonstrated that co-expression of PLB(M) enhances SERCA Ca-ATPase activity by increasing enzyme Ca(2+) affinity (1/K(Ca)) in PLB(WT)-inhibited HEK cell homogenates. For an initial assessment of PLB(M) physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLB(M) enhances the amplitude of SR Ca(2+) release and the rate of SR Ca(2+) re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLB(M) rescues arrhythmic Ca(2+) transients and alleviates decreased Ca(2+) transport. Thus, we propose that PLB(M) transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca(2+) transport and thus contractility in underlying systolic heart failure.

Pompe disease (glycogen storage disease type II) is caused by mutations in acid α-glucosidase (GAA) resulting in lysosomal pathology and impairment of the muscular and cardio-pulmonary systems. Enzyme replacement therapy (ERT), the only approved therapy for Pompe disease, improves muscle function by reducing glycogen accumulation but this approach entails several limitations including a short drug half-life and an antibody response that results in reduced efficacy. To address these limitations, new treatments such as gene therapy are under development to increase the intrinsic ability of the affected cells to produce GAA. Key components to gene therapy strategies include the choice of vector, promoter, and the route of administration. The efficacy of gene therapy depends on the ability of the vector to drive gene expression in the target tissue and also on the recipient's immune tolerance to the transgene protein. In this review, we discuss the preclinical and clinical studies that are paving the way for the development of a gene therapy strategy for patients with early and late onset Pompe disease as well as some of the challenges for advancing gene therapy.

Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs’ low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.

A novel, engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX (FIX) protein (BBM-H901) has been developed and is promising for haemophilia B gene therapy. We aimed to explore its safety and activity in increasing FIX concentrations and reducing bleeding frequency.We did a single-centre, single-arm, phase 1, pilot trial evaluating the safety and activity of a single intravenous infusion of BBM-H901 at the Institute of HematologyBlood Diseases Hospital, Chinese Academy of Medical SciencesPeking Union Medical College (Tianjin, China). We enrolled adult patients with haemophilia B (aged18 years) with baseline FIX coagulation activity (FIX:C) of less than 2 IU/dL, no FIX inhibitor, and low titre of neutralising antibodies (≤1:4) against vector capsid. Eligible participants were intravenously infused with a single dose of 5 × 10Between Oct 16, 2019, and Jan 13, 2021, 12 male participants were assessed, and ten Chinese participants were enrolled and infused with BBM-H901. After a median follow-up of 58 weeks (IQR 51·5-99·5), mean FIX:C reached mean 36·9 IU/dL (SD 20·5). No serious adverse events, no grade 3-4 adverse events were observed. Grade 1-2 adverse events related to BBM-H901 include pyrexia (1 [10%]) and elevation of aminotransferase(1 [10%]). No FIX inhibitors were observed. All participants developed antibodies against vector capsid after infusion. Eight (80%) participants had ALT and AST concentrations below the upper limit of normal throughout the follow-up period. Two (20%) participants had elevation of ALT and AST accompanied with decrease of FIX:C, which remained at 7 IU/dL and 11.8 IU/dL, respectively.This pilot study suggests that liver-tropic BBM-H901 is safe 1 year after infusion. Vector derived FIX:C concentration is sufficiently high to prevent bleeding events and minimise the need for replacement therapy in small populations with haemophilia B. These findings support further study.Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, National Key Research and Development Program of China, National Natural Science Foundation of China, Tianjin Municipal Science and Technology Commission Grant, and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.

Haemophilia therapy has evolved from rudimentary transfusion-based approaches to an unprecedented level of innovation with glimmers of functional cure brought by gene therapy. After decades of misfires, gene therapy has normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several clinical programmes testing adeno-associated viral (AAV) vector gene therapy are approaching completion with imminent regulatory approvals.Phase 3 studies along with multiyear follow-up in earlier phase investigations raised questions about efficacy as well as short- and long-term safety, prompting a reappraisal of AAV vector gene therapy. Liver toxicities, albeit mostly low-grade, occur in the first year in at least some individuals in all haemophilia A and B trials and are poorly understood. Extreme variability and unpredictability of outcome, as well as a slow decline in factor expression (seemingly unique to FVIII gene therapy), are vexing because immune responses to AAV vectors preclude repeat dosing, which could increase suboptimal or restore declining expression, while overexpression may result in phenotoxicity. The long-term safety will need lifelong monitoring because AAV vectors, contrary to conventional wisdom, integrate into chromosomes at the rate that calls for vigilance.AAV transduction and transgene expression engage the host immune system, cellular DNA processing, transcription and translation machineries in ways that have been only cursorily studied in the clinic. Delineating those mechanisms will be key to finding mitigants and solutions to the remaining problems, and including individuals who cannot avail of gene therapy at this time.

We transplanted RADA16-PRG self-assembled nanopeptide scaffolds (SAPNSs), bone mesenchymal stem cells (BMSCs), and a brain-derived neurotrophic factor (BDNF)-expressing adeno-associated virus (AAV) into rats subjected to acute spinal cord injury (SCI) to investigate the effects of these transplantations on acute SCI repair and explore their mechanisms. Forty-eight SCI rats were randomly divided into four groups: BBR, BR, B, and NC groups. Seven and 28 days after SCI, evoked potentials (EPs) and BBB scores were assessed to evaluate the recovery of rats’ motor behavior and sensory function after injury. HE and toluidine blue staining were performed to investigate the histological structure of the spinal cord tissue of rats from each group, and immunofluorescence staining was used to observe the red fluorescent protein (RFP) intensity of BMSCs and glial fibrillary acidic protein (GFAP) and neurofilament (NF) in the damaged area in each group. RT–PCR was utilized to detect the expression levels of the BDNF, GFAP, and neuron-specific enolase (NSE) genes in the injured area in each group. The results showed that cotransplantation of RADA16-PRG-SAPNs, BMSCs, and BDNF-AVV promoted the spinal cord’s motor and sensory function of SCI rats; increased levels of BMSCs, inhabited glial cells proliferation, and promoted neurons proliferations in the injured area; and increased NF, BDNF, and NSE levels and decreased its GFAP in the injured area. Thus, cotransplantation of RADA16-PRG-SAPNS, BMSCs, and BDNF-AAV can prolong the survival time of BMSCs in rats, reduce the postoperative scarring caused by glial proliferation, and promote the migration and proliferation of neurons in the injured area, resulting in the promotion of functional repair after acute SCI.