Recombinant Adeno-associated Vector Research Articles

Abstract B053: Physical activity regulates targetable transcriptomic changes in the adipose tissue of obese PDAC mice

Abstract Background: Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a highly aggressive cancer with a low 5-year survival rate. The association between obesity and PDAC could be attributed to the inflammatory effects of obesity and its impact on the adipose microenvironment. Physical activity (PA) is a potential approach to counteract obesity and its associated inflammation. Moreover, a recent study revealed that exercise-induced interleukin (IL)-15 in tumors has an anti-tumor response. However, there is still limited understanding of the mechanisms underlying how PA impacts the adipose-tumor microenvironments in PDAC. In this study, we hypothesized that PA could upregulate anti-inflammatory signaling in the adipose tissue and that targeting some of these signaling pathways could be a novel therapy for obesity-associated PDAC. Methods: Transcriptomic analysis was performed on adipose tissue obtained from a genetically engineering mouse model of obesity-associated PDAC. The mice were fed an obesogenic high-fat diet and divided into groups with and without PA intervention for one month. An adipose-specific recombinant adeno-associated vector (rAAV) was generated and mouse IL-15 cDNA was subcloned into the vector. C57BL/6J mice were administered intraperitoneal injections of either the rAAV/empty or rAAV/IL-15 vectors. Three weeks after rAAV intraperitoneal injection, mice were orthotopically injected with mouse KRasG12D/Trp53−/−/PDX-1-CRE cells expressing firefly luciferase to monitor growth in vivo and expression of IL-15 in the adipose tissue was confirmed via qPCR. Results: PA intervention increased the expression of genes related to metabolic and inflammation-related pathways. The administration of a novel adipose-targeted rAAV resulted in the upregulation of IL-15 within the adipose tissue and not the tumor. Increasing IL-15 expression in the adipose tissue led to a notable delay in tumor growth in an orthotopic mouse model. Conclusions: Here, we show that PA modulates gene expression in the adipose tissue and the beneficial impact of adipose tissue molecular remodeling on anti-tumor effects. However, more studies are needed to characterize the full extent of the adipose-tumor interactions and how they can be effectively targeted for therapeutic interventions. Citation Format: Hsiang-Yin Hsueh, Valentina Pita-Grisanti, Ericka Vélez-Bonet, Lei Cao, Wei Huang, Zobeida Cruz-Monserrate. Physical activity regulates targetable transcriptomic changes in the adipose tissue of obese PDAC mice [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B053.

Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells.

Liver fibrosis is the common pathological process associated with the occurrence and development of various chronic liver diseases. At present, there is still a lack of effective prevention and treatment methods in clinical practice. Hepatic stellate cell (HSC) plays a key role in liver fibrogenesis. In recent years, the study of liver fibrosis targeting HSC autophagy has become a hot spot in this research field. Angiotensin-converting enzyme 2 (ACE2) is a key negative regulator of renin-angiotensin system, and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored. To investigate the effect of ACE2 on hepatic fibrosis in mice by regulating HSC autophagy through the Adenosine monophosphate activates protein kinases (AMPK)/mammalian target of rapamycin (mTOR) pathway. Overexpression of ACE2 in a mouse liver fibrosis model was induced by injection of liver-specific recombinant adeno-associated virus ACE2 vector (rAAV2/8-ACE2). The degree of liver fibrosis was assessed by histopathological staining and the biomarkers in mouse serum were measured by Luminex multifactor analysis. The number of apoptotic HSCs was assessed by terminal deoxynucleoitidyl transferase-mediated dUTP nick-end labeling (TUNEL) and immunofluorescence staining. Transmission electron microscopy was used to identify the changes in the number of HSC autophagosomes. The effect of ACE2 overexpression on autophagy-related proteins was evaluated by multicolor immunofluorescence staining. The expression of autophagy-related indicators and AMPK pathway-related proteins was measured by western blotting. A mouse model of liver fibrosis was successfully established after 8 wk of intraperitoneal injection of carbon tetrachloride (CCl4). rAAV2/8-ACE2 administration reduced collagen deposition and alleviated the degree of liver fibrosis in mice. The serum levels of platelet-derived growth factor, angiopoietin-2, vascular endothelial growth factor and angiotensin II were decreased, while the levels of interleukin (IL)-10 and angiotensin- (1-7) were increased in the rAAV2/8-ACE2 group. In addition, the expression of alpha-smooth muscle actin, fibronectin, and CD31 was down-regulated in the rAAV2/8-ACE2 group. TUNEL and immunofluorescence staining showed that rAAV2/8-ACE2 injection increased HSC apoptosis. Moreover, rAAV2/8-ACE2 injection notably decreased the number of autophagosomes and the expression of autophagy-related proteins (LC3I, LC3II, Beclin-1), and affected the expression of AMPK pathway-related proteins (AMPK, p-AMPK, p-mTOR). ACE2 overexpression can inhibit HSC activation and promote cell apoptosis by regulating HSC autophagy through the AMPK/mTOR pathway, thereby alleviating liver fibrosis and hepatic sinusoidal remodeling.

Tolerability and tropism of recombinant adeno-associated virus vectors in the African green monkey (Chlorocebus sabaeus) anterior chamber.

While many studies have investigated the use of recombinant adeno-associated vectors (rAAV) in the posterior chamber for treatment of inherited retinal diseases, fewer studies have looked at rAAV's ability to transduce cells within the anterior chamber. This study focuses on evaluating the tropism and tolerability of three rAAV serotypes-rAAV2/6, rAAV2/9, and rAAV2/2[MAX]-expressing a green fluorescent protein (GFP) reporter following intracameral injection in the non-human primate (NHP) African green monkey (Chlorocebus sabaeus) model. Injection of high dose (1 × 1012 vg/eye) rAAV vector resulted in transient inflammation characterized by aqueous flare and cellular infiltrate that resolved without intervention in all serotypes. Post-mortem histology revealed widespread expression of GFP in cells of the trabecular meshwork and iris in high dose rAAV2/6, rAAV2/9, and particularly rAAV2/2[MAX] eyes, indicating that rAAV vectors of these serotypes have broad tropism for cells of the anterior chamber and may facilitate the treatment of blinding disorders, such as glaucoma.

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.

Adeno-associated virus mediates gene transduction after static cold storage treatment in rodent lung transplantation

ObjectiveAdeno-associated virus is a clinically used gene therapy vector but has not been studied in lung transplantation. We sought to determine the efficacy of adeno-associated virus delivery during static cold storage via the airway versus the pulmonary artery before lung transplantation in a rodent model. MethodsLewis rat lung grafts were treated with a dose of 8e8 or 4e9 viral genome/μL recombinant adeno-associated virus subtype-9 vectors containing firefly luciferase genomes administered via the pulmonary artery or airway during cold storage. A control group did not receive adeno-associated virus. Recipient syngeneic rats then underwent single left lung transplantation. Animals underwent bioluminescence imaging on postoperative days 7, 14, 28, and 56. Explanted tissues were prepared as lysates to quantify luciferase activity. Immunohistochemistry was performed to evaluate cellular transgene expression patterns. ResultsControl animals with no luminescent signal produced a background radiance of 6.1e4 p/s/cm2/sr. In the airway delivery group, mean radiance was greater than the control at 4e9 viral genome/μL postoperative day 7 radiance 6.9e4 p/s/cm2/sr (P = .04). In the pulmonary artery delivery group, we observed greater in vivo luminescence in animals receiving 4e9 viral genome/μL compared with all other groups. However, analysis of tissue lysate revealed greater luminescence in the airway delivery group and suggested off-target expression in heart and liver tissue in the pulmonary artery delivery group. Immunohistochemistry demonstrated transgene staining in distal airway epithelium and alveoli but sparing of the vasculature in the airway delivery group. ConclusionsAdeno-associated virus mediates gene transduction during static cold storage in rat lung isografts when administered via the airway and pulmonary artery. Airway administration leads to robust transgene expression in respiratory epithelial cells, whereas pulmonary artery administration targets alternative cell types and increases extrapulmonary transgene expression.

Systemic Checkpoint Blockade by PD-L1 Single-Chain Antibody Confers Potent Antitumor Immunity and Long-term Survival.

Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)-programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1-mediated inhibition of CD8+ T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8+ T-cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1- CD8+ T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.

Full-length ATP7B reconstituted through protein trans-splicing corrects Wilson disease in mice

Wilson disease (WD) is a genetic disorder of copper homeostasis, caused by deficiency of the copper transporter ATP7B. Gene therapy with recombinant adeno-associated vectors (AAV) holds promises for WD treatment. However, the full-length human ATP7B gene exceeds the limited AAV cargo capacity, hampering the applicability of AAV in this disease context. To overcome this limitation, we designed a dual AAV vector approach using split intein technology. Split inteins catalyze seamless ligation of two separate polypeptides in a highly specific manner. We selected a DnaE intein from Nostoc punctiforme (Npu) that recognizes a specific tripeptide in the human ATP7B coding sequence. We generated two AAVs expressing either the 5′-half of a codon-optimized human ATP7B cDNA followed by the N-terminal Npu DnaE intein or the C-terminal Npu DnaE intein followed by the 3′-half of ATP7B cDNA, under the control of a liver-specific promoter. Intravenous co-injection of the two vectors in wild-type and Atp7b−/− mice resulted in efficient reconstitution of full-length ATP7B protein in the liver. Moreover, Atp7b−/− mice treated with intein-ATP7B vectors were protected from liver damage and showed improvements in copper homeostasis. Taken together, these data demonstrate the efficacy of split intein technology to drive the reconstitution of full-length human ATP7B and to rescue copper-mediated liver damage in Atp7b−/− mice, paving the way to the development of a new gene therapy approach for WD.

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD

BackgroundDuchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD.MethodsAll experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis.ResultsAs expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase.ConclusionsAll together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

Resistin deletion protects against heart failure injury by targeting DNA damage response.

Increased resistin (Retn) levels are associated with development of cardiovascular diseases. However, the role of Retn in heart failure (HF) is still unclear. Here we probed the functional and molecular mechanism underlying the beneficial effect of Retn deletion in HF. Wild-type (WT) and adipose tissue-specific Retn-knockout (RKO) mice were subjected to transverse aortic constriction (TAC)-induced HF. Cardiac function and haemodynamic changes were measured by echocardiography and left ventricular catheterization. Adipose tissue Retn deletion attenuated while Retn cardiac-selective overexpression, via a recombinant adeno-associated virus-9 vector, exacerbated TAC-induced hypertrophy, cardiac dysfunction, and myocardial fibrosis in WT and RKO mice. Mechanistically, we showed that Gadd45α was significantly increased in RKO HF mice while cardiac overexpression of Retn led to its downregulation. miR148b-3p directly targets Gadd45α and inhibits its expression. Retn overexpression upregulated miR148b-3p expression and triggered DNA damage response (DDR) in RKO-HF mice. Inhibition of miR148b-3p in vivo normalized Gadd45α expression, decreased DDR, and reversed cardiac dysfunction and fibrosis. In vitro Retn overexpression in adult mouse cardiomyocytes activated miR148b-3p and reduced Gadd45α expression. Gadd45α overexpression in H9C2-cardiomyoblasts protected against hydrogen peroxide- and Retn-induced DDR. These findings reveal that diminution in circulating Retn reduced myocardial fibrosis and apoptosis, and improved heart function in a mouse model of HF, at least in part, through attenuation of miR148b-3p and DDR. The results of this study indicate that controlling Retn levels may provide a potential therapeutic approach for treating pressure overload-induced HF.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (PKLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

Krüppel-like factor 7 protects retinal ganglion cells and promotes functional preservation via activating the Akt pathway after retinal ischemia-reperfusion injury

ObjectiveThe purpose of this study is to investigate the effects of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) and retinal function after retinal ischemia-reperfusion (RIR) injury in mice. MethodsMale C57BL/6J mice were intravitreally injected with recombinant adeno-associated vectors (rAAV-KLF7-EGFP or rAAV-EGFP), and subsequently used to induce RIR injury. Retinal cryosections were used to access the efficacy of virus transfection, 1, 2, 3, and 4 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rate was observed and quantified by immunofluorescent staining, 7 days after RIR injury. Meanwhile, electroretinogram (ERG) and optomotor response were used to evaluate the electrophysiological functions and visual acuity. Apoptosis was evaluated by TUNEL staining 1 day after RIR injury. Expression of KLF7, Akt, phospho-Akt, Bcl-2, and Bax were further detected by western blot to excavate the underlying mechanism. ResultsThe transfection efficiency of rAAV-KLF7-EGFP was increased in a time-dependent manner, and the number of EGFP-positive cells was increased significantly 3 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were decreased after RIR injury. With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were increased after rAAV-KLF7-EGFP transfer. The protein level of KLF7 was up-regulated after rAAV-KLF7-EGFP transfer. Up-regulation of KLF7 significantly inhibited cells apoptosis, increased phospho-Akt and Bcl-2 expression, and decreased Bax expression. There were no significant changes in Akt expression. ConclusionOverexpression of KLF7 can not only prevent the loss of RGCs, but also preserve the electrophysiological function. In addition, overexpression of KLF7 can ameliorate the retinal dysfunction after RIR injury, and ultimately improve the visual acuity of mice. The activation of Akt pathway and the suppression of the mitochondrial apoptotic pathway contribute to the neuroprotection of KLF7.

Increased Temperature Facilitates Adeno-Associated Virus Vector Transduction of Colorectal Cancer Cell Lines in a Manner Dependent on Heat Shock Protein Signature

Colorectal cancer (CRC) is one of the most common cancers in human population. A great achievement in the treatment of CRC was the introduction of targeted biological drugs and solutions of chemotherapy, combined with hyperthermia. Cytoreductive surgery and HIPEC (hyperthermic intraperitoneal chemotherapy) extends the patients' survival with CRC. Recently, gene therapy approaches are also postulated. The studies indicate the possibility of enhancing the gene transfer to cells by recombinant adeno-associated vectors (rAAV) at hyperthermia. The rAAV vectors arouse a lot of attention in the field of cancer treatment due to many advantages. In this study, the effect of elevated temperature on the transduction efficiency of rAAV vectors on CRC cells with different origin and gene profile was examined. The effect of heat shock on the penetration of rAAV vectors into CRC cells in relation with the expression of HSP and AAV receptor genes was tested. It was found that the examined cells under hyperthermia (43°C, 1 h) are transduced at a higher level than in normal conditions (37°C). The results also indicate that studied RKO, HT-29, and LS411N cell lines express HSP genes at different levels under both 37°C and 43°C. Moreover, the results showed that the expression of AAV receptors increases in response to elevated temperature. The study suggests that increased rAAV transfer to CRC can be achieved under elevated temperature conditions. The obtained results provide information relevant to the design of new solutions in CRC therapy based on the combination of hyperthermia, chemotherapy, and gene therapy.

Long-Term Metabolic Correction of Phenylketonuria by AAV-Delivered Phenylalanine Amino Lyase

Phenylketonuria (PKU) is an inherited metabolic disorder caused by mutation within phenylalanine hydroxylase (PAH) gene. Loss-of-function of PAH leads to accumulation of phenylalanine in the blood/body of an untreated patient, which damages the developing brain, causing severe mental retardation. Current gene therapy strategies based on adeno-associated vector (AAV) delivery of PAH gene were effective in male animals but had little long-term effects on blood hyperphenylalaninemia in females. Here, we designed a gene therapy strategy using AAV to deliver a human codon-optimized phenylalanine amino lyase in a liver-specific manner. It was shown that PAL was active in lysing phenylalanine when it was expressed in mammalian cells. We produced a recombinant adeno-associated vector serotype 8 (AAV8) viral vector expressing the humanized PAL under the control of human antitrypsin (hAAT) promoter (AAV8-PAL). A single intravenous administration of AAV8-PAL caused long-term correction of hyperphenylalaninemia in both male and female PKU mice (strain Pahenu2). Besides, no obvious liver injury was observed throughout the treatment process. Thus, our results established that AAV-mediated liver delivery of PAL gene is a promising strategy in the treatment of PKU.

Эффективность доставки генов в клетки мозга рыб при помощи рекомбинантных аденоассоциированных вирусов гиппокампа мыши

Viral vectors constitute a flexible system that can efficiently deliver an exogenous gene to a variety of target cells and, thus, is a potentially powerful tool for genetic manipulation. To deliver the viral genes, an injection of recombinant adeno-associated viruses was performed according to the Wallace method [1]. The use of recombinant adeno-associated vectors allows viral particles to diffuse relatively easily from the injection area and quickly reproduce themselves in the brain cells. The work objective was to study the effectiveness of infecting various brain regions of chum salmon Oncorhynchus keta aged 2 years old with mammalian viral vectors with a short-term (4 and 8 days) and a longer-term (10 weeks) time intervals with subsequent ultrastructural analysis of the nerve tissue in the injection area. We have found that the mouse hippocampal AAV-1 incorporated in vivo brains of a chum salmon Oncorhynchus keta. Injection of an adeno-associated vector into various areas of the brain was made: telencephalon, cerebellum, mesencephalic tectum, and tegmentum of Oncorhynchus keta, and led to the expression of the reporter genes in pallial (Dd and Dl) regions of the telencephalon, the periventricular region of the masencephalon, in the dorsal part of the brain stem, and in the cerebellum (valvula and corpus cerebellum). Using fluorescence microscopy, we have shown that the presence of green fluorescence is associated with the expression of green fluorescence protein (GFP) in chum salmon cells. The ultrastructural analysis confirmed the presence of subcellular virus particles in the cells of chum salmon brain and intercellular space, which indicates the ability of the adeno-associated vector to diffuse in the nervous tissue of the chum salmon brain.

Chemogenetic activation of oxytocin neurons: Temporal dynamics, hormonal release, and behavioral consequences

Chemogenetics provides cell type-specific remote control of neuronal activity. Here, we describe the application of chemogenetics used to specifically activate oxytocin (OT) neurons as representatives of a unique class of neuroendocrine cells. We injected recombinant adeno-associated vectors, driving the stimulatory subunit hM3Dq of a modified human muscarinic receptor into the rat hypothalamus to achieve cell type-specific expression in OT neurons. As chemogenetic activation of OT neurons has not been reported, we provide systematic analysis of the temporal dynamics of OT neuronal responses in vivo by monitoring calcium fluctuations in OT neurons, and intracerebral as well as peripheral release of OT. We further provide evidence for the efficiency of chemogenetic manipulation at behavioral levels, demonstrating that evoked activation of OT neurons leads to social motivation and anxiolysis. Altogether, our results will be profitable for researchers working on the physiology of neuroendocrine systems, peptidergic modulation of behaviors and translational psychiatry.

All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo

BackgroundClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.ResultsHere, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail-vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded > 35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo.ConclusionsOur findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.

AAV-8 and AAV-9 Vectors Cooperate with Serum Proteins Differently Than AAV-1 and AAV-6

Under intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders. We show that, in contrast to rAAV-1 and rAAV-6, there was a substantial similarity in protein binding patterns between mouse and human sera for these vector serotypes. To establish an in vivo role for the vector binding of these sera proteins, we chose to study platelet factor 4 (PF4), which interacts with both vectors in both mouse and human sera. Experiments using PF4-knockout mice showed that a complete lack of PF4 did not alter skeletal muscle transduction for these vectors, whereas heart transduction was moderately improved. Our results strongly support our position that the impact of serum proteins on the transduction properties of rAAV-8 and rAAV-9, already observed in mouse models, should be similar in human preclinical trials.

Depression and Stress are Highly Associated with Hypertension, Diabetes, and Cardiovascular Disease in Chilean Population

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases, both to study them and to explore therapies. However, a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study, we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV), adenoviruses and lentiviral vectors in hESC, hiPSC, and the derived cardiomyocytes. In undifferentiated cells, adenoviral and lentiviral vectors were superior, whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes, 1, 2, 6, and 9, of which 2 and 6 were superior in their transduction efficiency. Interestingly, we observed that AAVs severely diminished the viability of undifferentiated cells, an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore, we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally, adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion, adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines, whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells.

In Utero Transfer of Adeno-Associated Viral Vectors Produces Long-Term Factor IX Levels in a Cynomolgus Macaque Model

The safe correction of an inherited bleeding disorder in utero prior to the onset of organ damage is highly desirable. Here, we report long-term transgene expression over more than 6 years without toxicity following a single intrauterine gene transfer (IUGT) at 0.9G using recombinant adeno-associated vector (AAV)-human factor IX (hFIX) in the non-human primate model we have previously described. Four of six treated animals monitored for around 74 months expressed hFIX at therapeutic levels (3.9%–120.0%). Long-term expression was 6-fold higher in males and with AAV8 compared to AAV5, mediated almost completely at this stage by random genome-wide hepatic proviral integrations, with no evidence of hotspots. Post-natal AAV challenge without immunosuppression was evaluated in two animals exhibiting chronic low transgene expression. The brief neutralizing immune reaction elicited had no adverse effect and, although expression was not improved at the dose administered, no clinical toxicity was observed. This long-term surveillance thus confirms the safety of late-gestation AAV-hFIX transfer and demonstrates that postnatal re-administration can be performed without immunosuppression, although it requires dose optimization for the desired expression. Nevertheless, eventual vector genotoxicity and the possibility of germline transmission will require lifelong monitoring and further evaluation of the reproductive function of treated animals.

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%–3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%–2.5% of the target level from years 1–5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated.