This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1–9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase ...transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.
Decades ago, Friedmann and Roblin postulated several barriers to gene therapy, including tissue targeting, delivery across the blood⁻brain barrier (BBB), and host immune responses. These issues ...remain pertinent till today. Since then, several advances have been made in elucidating structures of adeno-associated virus (AAV) serotypes, antibody epitopes, and ways to modify antibody-binding sites. AAVs capsid has also been engineered to re-direct tissue tropism, reduce ubiquitination, and promote passage across the BBB. Furthermore, the use of high(er) dose recombinant AAV (rAAV) has been accompanied by a better understanding of immune responses in both experimental animals and early clinical trials, and novel work is being performed to modulate the immune response. While the immune responses to rAAV remains a major challenge in translating experimental drugs to approved medicine, and will likely require more than a single solution, we now better understand the hurdles to formulate and test experimental solutions to surmount them.
Mitochondrial Ca2+ Uniporter (MCU)-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex ...assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired Ca2+m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metabolism.
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•MCUR1 binds to MCU and EMRE and functions as a scaffold factor•The coiled-coil domains of both MCU and MCUR1 are essential for MCU complex assembly•Genetic deletion of MCUR1 severely impairs Ca2+m uptake and IMCU current•MCUR1 deletion impairs bioenergetics and cell migration and elicits autophagy
Tomar et al. show that genetic ablation of the MCU complex component MCUR1 perturbs MCU heterooligomeric complex and impairs mitochondrial Ca2+ uptake. MCUR1 binds to MCU and EMRE and functions as a scaffold factor that is necessary for proper Ca2+-dependent mitochondrial bioenergetics.
Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus ...(AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.
G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a ...primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Due to their efficient transduction potential, adeno-associated virus (AAV) vectors are leading candidates for gene therapy in skeletal muscle diseases. However, immune responses toward the vector or ...transgene product have been observed in preclinical and clinical studies. TLR9 has been implicated in promoting AAV-directed immune responses, but vectors have not been developed to circumvent this barrier. To assess the requirement of TLR9 in promoting immunity toward AAV-associated antigens following skeletal muscle gene transfer in mice, we compared immunological responses in WT and Tlr9-deficient mice that received an AAV vector with an immunogenic capsid, AAVrh32.33. In Tlr9-deficient mice, IFN-γ T cell responses toward capsid and transgene antigen were suppressed, resulting in minimal cellular infiltrate and stable transgene expression in target muscles. These findings suggest that AAV-directed immune responses may be circumvented by depleting the ligand for TLR9 (CpG sequences) from the vector genome. Indeed, we found that CpG-depleted AAVrh32.33 vectors could establish persistent transgene expression, evade immunity, and minimize infiltration of effector cells. Thus, CpG-depleted AAV vectors could improve outcome of clinical trials of gene therapy for skeletal muscle disease.
The hallmark of spinal muscular atrophy (SMA), an inherited disease caused by ubiquitous deficiency in the SMN protein, is the selective degeneration of subsets of spinal motor neurons. Here, we show ...that cell-autonomous activation of p53 occurs in vulnerable but not resistant motor neurons of SMA mice at pre-symptomatic stages. Moreover, pharmacological or genetic inhibition of p53 prevents motor neuron death, demonstrating that induction of p53 signaling drives neurodegeneration. At late disease stages, however, nuclear accumulation of p53 extends to resistant motor neurons and spinal interneurons but is not associated with cell death. Importantly, we identify phosphorylation of serine 18 as a specific post-translational modification of p53 that exclusively marks vulnerable SMA motor neurons and provide evidence that amino-terminal phosphorylation of p53 is required for the neurodegenerative process. Our findings indicate that distinct events induced by SMN deficiency converge on p53 to trigger selective death of vulnerable SMA motor neurons.
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•SMN deficiency induces early p53 activation in vulnerable SMA motor neurons•Inhibition of p53 prevents motor neuron degeneration in SMA mice•p53S18 phosphorylation selectively marks degenerating SMA motor neurons•Amino-terminal phosphorylation of p53 is required for motor neuron degeneration
Ubiquitous SMN deficiency causes the death of specific motor neuron pools in SMA, but the mechanisms underlying this selectivity are unknown. Simon et al. identify nuclear accumulation and phosphorylation of p53 as distinct, converging mechanisms induced by SMN deficiency that trigger selective death of vulnerable SMA motor neurons.
Background— The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function. The ...peptide βARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve βAR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term βARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results— In HF rats, we delivered βARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the β-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. βARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac βAR signaling. Addition of metoprolol neither enhanced nor decreased βARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions— Long-term cardiac AAV6-βARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, βARKct alone improves outcomes more than a β-blocker alone, whereas both treatments are compatible. These findings show that βARKct gene therapy can be of long-term therapeutic value in HF.
BACKGROUND:Immune cell–mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system ...function through β-adrenergic receptors (βARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown.
METHODS:Wild-type (WT) mice were irradiated followed by isoform-specific βAR knockout (βARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression.
RESULTS:β2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. β2ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via β-arrestin–dependent β2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to β2AR activity, and spleens from human tissue donors treated with β-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of β2AR in β2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice.
CONCLUSIONS:Immune cell–expressed β2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.
RATIONALE:Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to ...other stem cell types that have been used in recent early stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients.
OBJECTIVE:To determine if post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model.
METHODS AND RESULTS:Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia-reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n = 9) (2x10 cells total) or placebo (vehicle; VEH, n = 9) through NOGA® guided transendocardial injections. 5-ethynyl-2’deoxyuridine (EdU), a thymidine analog, containing minipumps were inserted at the time of MI induction. At 72hrs (n=8) initial injury and cell retention were assessed. At 3 Months post-MI, cardiac structure and function was evaluated by serial echocardiography, and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72hrs post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular (LV) volumes and ejection fraction were significantly more preserved in CBSC-treated hearts and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU cardiac myocytes was increased in CBSC- vs. VEH- treated animals.
CONCLUSIONS:CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves LV functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction (HFrEF).
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