AAV Vectors Research Articles

Neoantigen-augmented iPSC cancer vaccine combined with radiotherapy promotes antitumor immunity in poorly immunogenic cancers

Although irradiated induced-pluripotent stem cells (iPSCs) as a prophylactic cancer vaccine elicit an antitumor immune response, the therapeutic efficacy of iPSC-based cancer vaccines is not promising due to their insufficient antigenicity and the immunosuppressive tumor microenvironment. Here, we found that neoantigen-engineered iPSC cancer vaccines can trigger neoantigen-specific T cell responses to eradicate cancer cells and increase the therapeutic efficacy of RT in poorly immunogenic colorectal cancer (CRC) and triple-negative breast cancer (TNBC). We generated neoantigen-augmented iPSCs (NA-iPSCs) by engineering AAV2 vector carrying murine neoantigens and evaluated their therapeutic efficacy in combination with radiotherapy. After administration of NA-iPSC cancer vaccine and radiotherapy, we found that ~60% of tumor-bearing mice achieved a complete response in microsatellite-stable CRC model. Furthermore, splenocytes from mice treated with NA-iPSC plus RT produced high levels of IFNγ secretion in response to neoantigens and had a greater cytotoxicity to cancer cells, suggesting that the NA-iPSC vaccine combined with radiotherapy elicited a superior neoantigen-specific T-cell response to eradicate cancer cells. The superior therapeutic efficacy of NA-iPSCs engineered by mouse TNBC neoantigens was also observed in the syngeneic immunocompetent TNBC mouse model. We found that the risk of spontaneous lung and liver metastasis was dramatically decreased by NA-iPSCs plus RT in the TNBC animal model. Altogether, these results indicated that autologous iPSC cancer vaccines engineered by neoantigens can elicit a high neoantigen-specific T-cell response, promote tumor regression, and reduce the risk of distant metastasis in combination with local radiotherapy.

Adeno-associated virus (AAV) 6 provides efficient gene transfer upon intramyocardial injection in vivo

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Innovation Council Eurostars Background Cardiac gene transfer utilizing adeno-associated virus (AAV) is a promising approach for the treatment of various cardiovascular diseases. Intramyocardial injection is an efficient delivery route, and is particularly relevant for ischemic disease or pacemaker and conduction system abnormalities, where local treatment is favoured. However, which AAV variants provide efficient cardiac gene transfer after intramyocardial injection remains to be tested. Aim This study aims to compare several cardiotropic AAV variants in order to identify their relative potency in the setting of direct intramyocardial injection. Methods A green fluorescent protein (GFP)-coding sequence was packaged in the two benchmark AAV capsids, AAV6 and AAV9, and a selection of recently reported AAV9 capsid variants which are named MyoAAVs. These AAV vectors were added to neonatal rat ventricular cardiomyocytes (NRVM) and their transduction efficiency was evaluated by flow cytometry and quantitative PCR. AAV6, AAV9 and MyoAAV4A vectors were then delivered into the left ventricle of FVB mice via intramyocardial injection. AAV6 and AAV9 carrying GFP in a different expression cassette were also intramyocardially injected in FVB and Bl6 mice. Direct fluorescence, transgene mRNA, and viral genomes were quantified to determine the transduction efficiency 4 weeks post injection. Picrosirius red staining was used to assess whether AAV-mediated GFP delivery causes cardiac fibrosis. We further studied GFP encoding AAV6 and AAV9 vectors upon intramyocardial injection into the left ventricle of pigs and determined the transduction efficiency by quantitative PCR. Results NRVM transduction experiment revealed that among the tested capsid variants, AAV6 is the most efficient in transducing cardiomyocytes, followed by MyoAAV4A. In mice, AAV6 is most effective in transducing myocardium upon intramyocardial injection, resulting in approximately 10-fold higher transgene expression than that obtained with AAV9 vector and 2-fold higher than that with MyoAAV4A vector. Histology revealed no vector-related cardiac fibrosis. The use of different expression cassettes and animal strains consistently showed that AAV6 outperforms AAV9 in cardiac transduction efficiency. Transgene expression in liver was only observed in Bl6 mice injected with AAV9. In porcine myocardium, AAV6 led to robust transgene expression after intramyocardial injection and this expression level is higher than that of AAV9. Conclusion Among the AAV variants tested, AAV6 appears to be the most efficient for cardiac transduction by intramyocardial injection. These results suggest that AAV6 represents an attractive vector for direct cardiac gene transfer and for future capsid engineering in the context of local transgene delivery.

AAV-mediated gene delivery to the cardiac conduction system

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO open technology program Biopace #18485 Background Gene therapy for cardiac arrhythmias has so far mostly focused on modifying the arrhythmogenic substrate in working myocardium or on creating biological pacemakers with viral vectors that did not selectively target the cardiac conduction system (CCS). As a result, CCS targeting by viral vectors is a relatively unexplored area. Aim In the current project, we explored the basic requirements for CCS targeting using adeno-associated virus (AAV) vectors. Methods Regular AAV9 and AAV-Myo4A capsid-pseudotyped AAV2 vectors (hereafter called AAV2/9 and AAV2/Myo4A vectors, respectively) expressing enhanced green fluorescent protein (eGFP) from a CMV or TNNT2 promoter, respectively, and an AAV2/9 vector expressing eGFP from a human GJA5 regulatory element were systemically injected in mice. After 4 weeks, hearts were harvested, fixed with paraformaldehyde and stained for TNNT2, eGFP, HCN4 and DNA to determine CCS transduction. Results Transduction of the sinoatrial and atrioventricular (AV) node using AAV2/9 vectors was very poor, while transduction was visible in the atrioventricular (i.e. His) bundle and working myocardium. In contrast, the transduction rate of the AV node and bundle using AAV2/Myo4A vectors were similar to that of the working myocardium. The human GJA5 regulatory element was able to restrict eGFP expression by AAV2/9 vectors to the AV bundle in 2/3 animals but the transduction efficiency was low. A third animal showed no transduction of any tissue, either indicative of the low transduction efficiency or of a failed injection. Conclusion This study not only provides proof-of-principle for CCS-targeted gene therapy but also shows that CCS transduction can be improved through AAV capsid engineering and be restricted to (parts of) the CCS by using specific transcriptional control elements.

Poor in vivo transduction of myocardium by lentiviral vectors is likely caused by a post-entry block of transduction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Stichting LSH-TKI Introduction In cardiac gene therapy, two prototypic viral vectors are typically considered for long-term transgene expression, i.e. lentivirus (LV) and adeno-associated virus (AAV) vectors. Compared to AAV vectors, LV vectors have the advantages of a larger insert capacity and the ability to confer permanent transgene expression due to integration of the vector genome in the host cell’s chromosomal DNA . However, standard VSV G protein-pseudotyped LV vectors (VSV G-LV vectors) has shown suboptimal transduction of cardiac myocytes in vivo. Purpose In this study, we aimed to improve LV-mediated transduction of cardiomyocytes in vivo by evaluating LV vectors carrying various different envelopes and compared the most potent variants with AAV1 capsid-pseudotyped AAV2 vectors (AAV2/1 vectors). Methods We made a panel of LV vectors pseudotyped with envelope proteins from 76 different viruses carrying an expression cassette for enhanced green fluorescent protein (eGFP). Transduction efficiencies in neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocyte progenitor cells (CMPCs) were determined by fluorescence-activated cell sorting (FACS) as percentage eGFP-positive cells. Male NOD -SCID mice aged 3-6 months were injected directly into the left ventricular myocardium with eGFP-encoding VSV G-LV and AAV2/1 vectors at doses of 1.4 x 10^7 infectious units and 2.0 x 10^10 genome copies, respectively, or PBS as a control. Mice were sacrificed one and four weeks after surgery. Per heart, left ventricular and eGFP-positive area was determined from 40 – 50 slides and the ratio was used as a measure of transduction efficiency. Results Of the panel of pseudotyped LVs none was able to consistently outperform VSV G-LV vectors, which reached transduction efficiencies in vitro of 58% in NRVMs to 96% in hiPSC-CMs and 100% in CMPCs . In AAV2/1 vector-injected hearts, the eGFP expressing area in the left ventricle increased from 1.07% in week 1 to 5.42% in week 4. In contrast, eGFP expression in LV-injected hearts was 0.31% and 0.16% after 1 week and 4 weeks, respectively (figure 1). Conclusion In vitro screening of a panel of pseudotyped LV vectors showed standard VSV G-LV vectors as top performer. Despite encouraging in vitro results and previously reported findings, VSV G-LV vectors was unable to provide for robust in vivo transduction. The observation that none of the 76 different envelopes yielded a meaningful improvement of lentiviral transduction rates together with the discrepancy between in vitro and in vivo transduction efficiencies, suggest that a post-entry block of transduction likely represents an important component of the poor in vivo performance.Figure 1in vivo transduction

Widespread correction of brain pathology in feline alpha-mannosidosis by dose escalation of intracisternal AAV vector injection

Alpha-mannosidosis is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions in the brain and other tissues. Enzyme replacement therapy is available but is not approved for treating the CNS since the enzyme does not penetrate the blood-brain barrier. However, intellectual disability is a major manifestation of the disease thus a complimentary treatment is needed. While enzyme replacement therapy into the brain is technically feasible, it requires ports and frequent administration over time that are difficult to manage medically. Infusion of adeno-associated viral vectors into the cerebrospinal fluid is an attractive route for broadly targeting brain cells. We demonstrate here the widespread post-symptomatic correction of the globally distributed storage lesions by infusion of a high dose of AAV1-fMANB into the CSF via the cisterna magna in the gyrencephalic alpha-mannosidosis cat brain. Significant improvements in clinical parameters occurred and widespread global correction was documented pre-mortem by non-invasive magnetic resonance imaging. Post-mortem analysis demonstrated high levels of MANB activity and reversal of lysosomal storage lesions throughout the brain. Thus, CSF treatment by adeno-associated viral vector gene therapy appears to be a suitable complement to systemic enzyme replacement therapy to potentially treat the whole patient.

Comparison of different promoters to improve AAV vector-mediated gene therapy for neuronopathic Gaucher disease.

Gaucher Disease (GD) is an inherited metabolic disorder caused by mutations in the GBA1 gene. It can manifest with severe neurodegeneration and visceral pathology. The most acute neuronopathic form (nGD), for which there are no curative therapeutic options, is characterised by devastating neuropathology and death during infancy. In this study, we investigated the therapeutic benefit of systemically delivered AAV9 vectors expressing the human GBA1 gene at two different doses comparing a neuronal-selective promoter with ubiquitous promoters. Our results highlight the importance of a careful evaluation of the promoter sequence used in gene delivery vectors, suggesting a neuron-targeted therapy leading to high levels of enzymatic activity in the brain but lower GCase expression in the viscera, might be the optimal therapeutic strategy for nGD.

Synergism of dual AAV gene therapy and rapamycin rescues GSDIII phenotype in muscle and liver.

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.

FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, yet effective treatment is lacking. Moreover, the underlying pathomechanisms of ALS remain unclear, with impaired mitophagy function being increasingly recognized as a contributing factor. FUN14 domain-containing protein 1 (FUNDC1) is an autophagy receptor localized to the outer mitochondrial membrane and a mitochondrial membrane protein that mediates mitophagy and therefore considered as important factor in neurodegenerative diseases. However, its specific role in ALS is not yet clear. Therefore, this study aimed to investigate the regulatory role of FUNDC1 in ALS and determine its regulatory mechanisms. ALS transgenic mice were obtained and maintained under standard conditions. Cell lines were generated by stable transfection with hSOD1G93A or control vectors. Mice received intrathecal injections of AAV9 vectors expressing FUNDC1 or EGFP. Motor function was assessed through behavioral tests, and histological and immunostaining analyses were performed. Colocalization analysis was conducted in transfected cells, and protein expression was evaluated via western blotting. We first observed that FUNDC1 was significantly downregulated in the spinal cord tissues of SOD1G93A mice. FUNDC1 overexpression considerably improved locomotor activity and prolonged survival time in SOD1G93A mice. Mechanistically, reduced expression of FUNDC1 resulted in decreased mitophagy, as indicated by decreased recruitment through LC3 in SOD1G93A mice and cellular models. Consequently, this led to increased mitochondrial accumulation and cell apoptosis, exacerbating the ALS phenotype. Furthermore, we identified transcription factor FOXD3 as an essential upstream factor of FUNDC1, resulting in reduced transcription of FUNDC1 in ALS lesions. This study suggests a novel strategy of targeting FUNDC1-mediated mitophagy for developing therapeutic interventions to mitigate disease progression and improve outcomes for ALS patients.

LncRNA Oip5-as1 inhibits excessive mitochondrial fission in myocardial ischemia/reperfusion injury by modulating DRP1 phosphorylation

BackgroundAberrant mitochondrial fission, a critical pathological event underlying myocardial ischemia/reperfusion (MI/R) injury, has emerged as a potential therapeutic target. The long non-coding RNA (lncRNA) Oip5-as1 is increasingly recognized for its regulatory roles, particularly in MI/R injury. However, its precise mechanistic role in modulating mitochondrial dynamics remains elusive. This study aims to elucidate the mechanistic role of Oip5-as1 in regulating mitochondrial fission and evaluate its therapeutic potential against MI/R injury.MethodsTo simulate in vitro MI/R injury, HL-1 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R). Lentiviral vectors were employed to achieve overexpression or knockdown of Oip5-as1 in HL-1 cells by expressing Oip5-as1 or shRNA targeting Oip5-as1, respectively. The impact of Oip5-as1 on mitochondrial dynamics in HL-1 cells was assessed using CCK-8 assay, flow cytometry, immunofluorescence staining, and biochemical assays. MI/R injury was induced in mice by ligating the left anterior descending coronary artery. Conditional knockout mice for Oip5-as1 were generated using the CRISPR/Cas9 genome editing technology, while overexpression of Oip5-as1 in mice was achieved via intramyocardial administration of AAV9 vectors. In mice, the role of Oip5-as1 was evaluated through echocardiographic assessment, histopathological staining, and transmission electron microscopy. Furthermore, Western blotting, RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation assays were conducted to investigate Oip5-as1’s underlying mechanisms.ResultsThe expression levels of Oip5-as1 are significantly decreased in MI/R-injured HL-1 cells and myocardium. In HL-1 cells undergoing H/R injury, overexpression of Oip5-as1 attenuated excessive mitochondrial fission, preserved mitochondrial functionality, and reduced cellular apoptosis, while knockdown of Oip5-as1 exhibited the opposite effects. Furthermore, in a mouse model of MI/R injury, overexpression of Oip5-as1 diminished mitochondrial fission, myocardial infarct size and improved cardiac function. However, knockout of Oip5-as1 exacerbated myocardial injury and cardiac dysfunction, which were significantly reversed by treatment with a mitochondrial division inhibitor-1 (Mdivi-1). Mechanistically, Oip5-as1 selectively interacts with AKAP1 and CaN proteins, inhibiting CaN activation and subsequent DRP1 dephosphorylation at Ser637, thereby constraining DRP1’s translocation to the mitochondria and its involvement in mitochondrial fission.ConclusionsOur study underscores the pivotal role of Oip5-as1 in mitigating excessive mitochondrial fission during MI/R injury. The findings not only enhance our comprehension of the molecular mechanisms underlying MI/R injury but also identify Oip5-as1 as a potential therapeutic target for ameliorating MI/R injury.Graphical

Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Adeno-associated virus-based gene therapy delivering combinations of two growth-associated genes to MPS IVA mice

Mucopolysaccharidosis type IVA (MPS IVA) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme responsible for the degradation of specific glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, hypoplasia, tracheal obstruction) and several symptoms in other organs. To date, no treatment is effective for patients with bone abnormalities. To improve bone pathology, we propose a novel combination treatment with the AAV vectors expressing GALNS enzyme and a C-type natriuretic peptide (CNP; NPPC gene) as a growth-promoting agent for MPS IVA. In this study, an MPS IVA mouse model was treated with an AAV vector expressing GALNS combined with another AAV vector expressing NPPC gene, followed for 12 weeks. After the combination therapy, bone growth in mice was induced with increased enzyme activity in tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP-treated mice with increased CNP activity in plasma. Delivering combinations of CNP and GALNS gene therapies enhanced bone growth in MPS IVA mice more than in GALNS gene therapy alone. Enzyme expression therapy alone fails to reach the bone growth region; our results indicate that combining it with CNP offers a potential alternative.

PLA2G12A protects against diet-induced obesity and insulin resistance by enhancing energy expenditure and clearance of circulating triglycerides.

Secreted phospholipase A2s are involved in the development of obesity, type 2 diabetes mellitus (T2DM) and cardiovascular disease, which have become serious and growing health concerns worldwide. Integration of genome-wide association study and gene co-expression networks analysis showed that the secreted phospholipase A2 group XIIA (PLA2G12A) may participate in hepatic lipids metabolism. Nevertheless, the role of PLA2G12A in lipid metabolism and its potential mechanism remain elusive. Here, we used AAV9 vector carrying human PLA2G12A gene to exogenously express hPLA2G12A in the liver of mice. We demonstrated that the overexpression of hPLA2G12A resulted in a significant decrease in serum lipid levels in wild-type mice fed with chow diet or high-fat diet (HFD). Moreover, hPLA2G12A treatment protected against diet-induced obesity and insulin resistance in mice fed a HFD. Notably, we found that hPLA2G12A treatment confers protection against obesity and hyperlipidemia independent of its enzymatic activity, but rather by increasing physical activity and energy expenditure. Furthermore, we demonstrated that hPLA2G12A treatment induced upregulation of ApoC2 and Cd36 and downregulation of Angptl8, which contributed to the increase in clearance of circulating triglycerides and hepatic uptake of fatty acids without affecting hepatic de novo lipogenesis, very low-density lipoprotein secretion, or intestinal lipid absorption. Our study highlights the potential of PLA2G12A gene therapy as a promising approach for treating obesity, insulin resistance and T2DM.

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

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

Optimization of AAV vectors for transactivator-regulated enhanced gene expression within targeted neuronal populations

Optimization of AAV vectors for transactivator-regulated enhanced gene expression within targeted neuronal populations

Chronic Intermittent Hypoxia Alters GABAergic Inhibition in Median Preoptic Nucleus (MnPO) Neurons of Rats

Chronic intermittent hypoxia (CIH) is an animal model simulating the hypoxemia associated with obstructive sleep apnea (OSA) in humans. Male rats exposed to CIH exhibit increased sympathetic tone and persistent elevations in blood pressure similar to that observed in patients with. In MnPO neurons, CIH reduces GABAa inhibition and, in some neurons, GABAa activation produces excitation. Here, loose patch and perforated patch recordings were conducted in MnPO neurons in response to optogenetic stimulation of GABAergic SFO (efferent) and MnPO (local) neurons to assess the role of GABAergic signaling in these nuclei on MnPO excitability. Our working hypothesis was that CIH would diminish the inhibitory effects of optogenetic stimulation of GABA neurons. This hypothesis was tested using an AAV vector that selectively expresses channel rhodopsin in GABA neurons Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received infusions (0.2 μL) of pAAV-mDlx-ChR2-mCherry-Fishell-3 in the SFO or MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hr) or normoxia (Norm). CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For in vitro recordings, rats were anesthetized with isoflurane (2-3%), and sagittal or coronal slices (300 μm) containing MnPO were cut using standard slice procedures. Voltage-clamp loose patch or perforated patch recordings using gramicidin (100 ug/ml in 140 mM K-gluconate) were collected in MnPO neurons in response to optogenetic stimulation of GABAergic neurons (2 ms pulse, 20 Hz, 1 sec). MnPO neurons from CIH-treated rats showed differing responses to GABAergic stimulation while MnPO neurons from Norm-treated rats predominantly exhibited GABAergic inhibition. MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH-treated rats show variable mIPSC frequencies and heterogeneous responses to GABAergic neuronal activation in the SFO and MnPO. The results demonstrate that CIH alters the Cl− homeostasis in some MnPO neurons altering their response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH. Supported by R01 HL155977. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 139: Contribution Of Platelet Granule Cargo Uptake And Secretion To The Initiation And Progression Of Aortic Aneurysms

Aortic aneurysms present as aortic widening and often catastrophic rupture. While precipitated by a thrombo-inflammatory environment, underlying pathological mechanisms are unclear and adequate treatment is lacking. Antithrombotics have been implied as therapy, but few studies have defined how platelets contribute to aortic aneurysms. We studied the role of platelet granule cargo uptake and secretion in aortic aneurysm formation. Aneurysms were induced in mice defective in platelet α-granule biogenesis (Nbeal2 -/- , Serglycin -/- ), endocytosis (Arf6 -/- , VAMP2/3 Δ ), and exocytosis (Munc13-4 Jinx ) via: i.p. injection of an AAV vector with a mouse PCSK9 gain-of-function mutation; western diet; and continuous s.c. infusion of angiotensin II. Thoracic and abdominal aneurysm formation was assessed with ultrasound. At 4 and 12 weeks, or after rupture, aortas were harvested, analyzed, and prepared for histology. During the study, 30% (3/10) of Nbeal2 -/- and 21.4% (3/18) of Munc13-4 Jinx mice suffered aortic rupture, compared to 0% (0/26) of wildtype mice. In the survivors, the inner diameter of neither the thoracic nor abdominal aorta of Nbeal2 -/- mice changed over time. Ex vivo , Serglycin -/- mice showed a clear, more distributed, widening of the thoracic and descending aorta instead of local aneurysm formation with an increased maximum outer diameter of the ascending region. Munc13-4 Jinx mice had an all-or-nothing phenotype; either early-stage massive aneurysm formation or rupture, or a more diffused phenotype. Mice with platelet endocytosis defects survived and at early stages, the differential increase in the inner diameter of both the thoracic and abdominal aorta was higher in female Arf6 -/- mice. Similarly, VAMP2/3 Δ mice had the largest inner thoracic diameter increase after 28 days. Arf6 -/- and VAMP2/3 Δ mice showed a smaller maximum outer diameter of the descending aorta as wildtype. Preliminary histology shows reduced elastin breaks in all genetic strains, especially in Nbeal2 -/- and VAMP2/3 Δ mice. Our data show that platelet granule cargo exerts significant but perhaps contrasting effects on aortic aneurysm formation. By comparing phenotypes, more detailed mechanistic insights into the role of platelets in aortic aneurysms are possible.

Orexin 1 Receptors in The Paraventricular Nucleus of The Hypothalamus (PVN) Facilitate The Peripheral Chemoreflex

Increased sensitivity of the peripheral chemoreflex (PCR) to hypoxia is associated with cardiovascular-related diseases such as hypertension and heart failure. We previously showed that systemic blockade of both orexin 1 (Ox1R) and orexin 2 receptors reduced the PCR-mediated hypoxic ventilatory response (HVR), and that hypoxia activates PVN-projecting orexin neurons. The effect of OxR blockade on the HVR was strongest in the active phase of the circadian cycle and was associated with decreased numbers of activated neurons in the nucleus of the solitary tract (nTS) and nTS-projecting corticotropin-releasing neurons in the PVN. While the projection from the PVN to the nTS contributes to the PCR, the neuronal mechanism(s) activating this projection are unclear. We hypothesized that orexin acting at Ox1R in the PVN facilitates the PCR. To test this hypothesis, we first performed IHC to confirm the expression of Ox1R in the PVN. We then assessed the degree to which specific Ox1R blockade reduced the HVR (n=6; tested first with vehicle (100% DMSO), then Ox1R antagonist SB-334867, 1 mg/kg, i.p). To assess the contribution of PVN Ox1Rs to the PCR as well as the associated activation of neurons in the PVN and nTS, we specifically knocked down Ox1R expression in the PVN via nanoinjection of an Ox1R shRNA-GFP expressing AAV vector. Rats were exposed to graded hypoxia during the active phase, with inspired O2 taken from 21% to 11% over a ~5 min period. Rats were kept near-isocapnic by slightly increasing the inspired CO2. Subsets of rats were exposed to hypoxia to assess the effects of Ox1R on neuronal activation using c-Fos expression as a surrogate marker. Systemic Ox1R blockade significantly reduced the HVR compared to the control response (by ~30%; p=0.0027). Knockdown of Ox1R in the PVN also reduced the HVR (by ~20%; p=0.049) and reduced the number of c-Fos expressing PVN neurons in response to hypoxia (p=0.0232). More specifically, hypoxia induced c-Fos expression in fewer PVN neurons transfected with Ox1R shRNA (confirmed by GFP expression) than neurons from control rats transfected with scrambled RNA (p=0.0083). Ox1R knockdown in the PVN also significantly reduced the hypoxia-induced increase in number of c-Fos expressing tyrosine-hydroxylase (TH) nTS neurons (p=0.0303), suggesting that PVN Ox1Rs mediate hypoxia-induced activation of TH neurons in the nTS. These findings suggest that in the PVN, orexin acting at Ox1Rs mediate the facilitation of the PCR and the associated activation of the PVN and nTS neurons. Ox1R in the PVN could be a novel target to treat diseases associated with increased PCR sensitivity. Grants: 5R01HL098602 (to DK, EH and KC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3032: Novel GLP-Grade E-Selectin/AAV2 Gene Therapy Optimal Dosage For Vascular Regeneration

Introduction: Chronic Limb Threatening Ischemia (CLTI) represents a major public health burden with with an estimated 15 to 20% CLTI patients undergoing major limb amputation facing a 50% mortality risk at 5 years post amputation. We previously demonstrated the pro-angiogenic actions of a research-grade E-Selectin/AAV gene therapy in a hindlimb gangrene murine model. In this study, a dosage study utilizing GLP grade codon-optimized and DeNovo-synthesized E-selectin/AAV2 was conducted dosage optimization and determination of the necessary scale-up for subsequent clinical trials. Methods: codon-optimized and de novo synthesized human and murine E-selectin AAV vectors under GLP conditions was evaluated in vitro by cell transduction assays. E-selectin/AAV2 i.m. injection in three cohorts of FVB/NJ mice (9-12 weeks old, N=6/group) with GFP/AAV2 (9-12 weeks old, N=6/group) groups, was performed in vivo in escalating log doses ranging from 2x10^9 VG to 2x10^11 VG. Hindlimb ischemia and gangrene were subsequently induced through left femoral artery/vein ligation/excision (FAVLE). Limb perfusion recovery and function was assessed postoperatively by Laser Doppler Imaging (LDI), Faber hindlimb ischemia scores, treadmill exhaustion test, whole-body Dil perfusion Results: Treatment with high dose (2x10^11 VG) of E-selectin/AAV2 significantly improve perfusion indices compared to control groups (p=.0019, N=6/group) and lower doses (p=.0006 and p=.0082, N=6/group) with decreased Faber’s scores. A significant increase in neovascularization was also observed compared to GFP control (0.41 ±0.14 to 0.95±0.18; p=.0080, N=5/group) and lower doses (p=.0026 and p=.009, N=5/group). Running stamina was also significantly improve by VRGT with a 2x10^11 VG dosage treatment compared to GFP (p=0.0047) control and lower doses, establishing a clear dose-dependent response. Conclusion: VGRT improves recovery of ischemic hindlimb perfusion and function at an optimal dose of 2x10 11 VG. This pre-clinical study paves the way towards further studies using the same AAV construct manufactured under cGMP conditions and subsequent Investigational New Drug (IND) process for clinical pilot studies and disease burden relief.

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

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

Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF

BackgroundThe advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting.ResultsHere, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA.ConclusionsOur data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.