Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice.

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Synapsin-caveolin-1 gene therapy preserves neuronal and synaptic morphology and prevents neurodegeneration in a mouse model of AD.

Alzheimer's disease (AD) is the most common form of neurodegeneration and cognitive dysfunction in the elderly. Identifying molecular signals that mitigate and reverse neurodegeneration in AD may be exploited therapeutically. Transgenic AD mice (PSAPP) exhibit learning and memory deficits at 9 and 11 months, respectively, with associated decreased expression of caveolin-1 (Cav-1), a membrane/lipid raft (MLR) scaffolding protein necessary for synaptic and neuroplasticity. Neuronal-targeted gene therapy using synapsin-Cav-1 cDNA (SynCav1) was delivered to the hippocampus of PSAPP mice at 3 months using adeno-associated virus serotype 9 (AAV9). Bilateral SynCav1 gene therapy was able to preserve MLRs profile, learning and memory, hippocampal dendritic arbor, synaptic ultrastructure, and axonal myelin content in 9- and 11-month PSAPP mice, independent of reducing toxic amyloid deposits and astrogliosis. Our data indicate that SynCav1 gene therapy may be an option for AD and potentially in other forms of neurodegeneration of unknown etiology.

The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1.

Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia. The sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.

Urocortin 2 Gene Transfer Improves Heart Function in Aged Mice.

Prevalence of left ventricular (LV) systolic and diastolic dysfunction increases with aging. We previously reported that urocortin 2 (Ucn2) gene transfer increases heart function in mice with heart failure with reduced ejection fraction. Here, we test the hypotheses that (1) Ucn2 gene transfer will increase LV function in aged mice and that (2) Ucn2 gene transfer given in early life will prevent age-related LV dysfunction. Nineteen-month-old (treatment study) and 3-month-old (prevention study) mice received Ucn2 gene transfer or saline. LV function was examined 3-4 months (treatment study) or 20 months (prevention study) after Ucn2 gene transfer or saline injection. In both the treatment and prevention strategies, Ucn2 gene transfer increased ejection fraction, reduced LV volume, increased LV peak -dP/dt and peak +dP/dt, and reduced global longitudinal strain. Ucn2 gene transfer-in both treatment and prevention strategies-was associated with higher levels of LV SERCA2a protein, reduced phosphorylation of LV CaMKIIa, and reduced LV α-skeletal actin mRNA expression (reflecting reduced cardiac stress). In conclusion, Ucn2 gene transfer restores normal cardiac function in mice with age-related LV dysfunction and prevents development of LV dysfunction.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Bile-duct proliferation as an unexpected side-effect after AAV2-LDLR gene transfer to rabbit liver.

Familial hypercholesterolemia (FH) is an inherited disease of lipoprotein metabolism caused by a defect in the LDL receptor (LDLR) leading to severe hypercholesterolemia, and associated with an increased risk of coronary heart disease and myocardial infarction. We have developed a gene therapy protocol for FH using AAV2, AAV9 and lentiviral vectors and tested safety and efficacy in LDL receptor deficient Watanabe Heritable Hyperlipidemic rabbits. We show that LV-LDLR produced a significant long-lasting decrease in total serum cholesterol whereas AAV9-LDLR resulted only in a transient decrease and AAV2-LDLR failed to reduce serum cholesterol levels. A significant pathological side effect, bile-duct proliferation, was seen in the liver of AAV2-LDLR rabbits associated with an increased expression of Cyr61 matricellular protein. Special attention should be given to liver changes in gene therapy applications when genes affecting cholesterol and lipoprotein metabolism are used for therapy.

Urocortin 3 Gene Transfer Increases Function of the Failing Murine Heart.

Peptide infusions of peptides the corticotropin releasing factor family, including urocortin 2, stresscopin, and urocortin 3 (UCn3), have favorable acute effects in clinical heart failure (HF), but their short half-lives make them unsuitable for chronic therapy. This study asked whether UCn3 gene transfer, which provides sustained elevation of plasma UCn3 levels, increases the function of the failing heart. HF was induced by transmural left ventricular (LV) cryoinjury in mice. LV function was assessed 3 weeks later by echocardiography. Those with ejection fractions (EF) <40% received intravenous saline or intravenous adeno-associated virus type-8 encoding murine UCn3 (AAV8.mUCn3; 1.9 × 1013 genome copies/kg). Five weeks after randomization, repeat echocardiography, assessment of LV function (+dP/dt, -dP/dt), and quantification of Ca2+ transients and sarcomere shortening in isolated cardiac myocytes were conducted, and assessment of LV Ca2+ handling and stress proteins was performed. Three weeks after myocardial infarction, prior to treatment, EFs were reduced (mean 31%, from 63% in sham-operated animals). Mice randomized to receive UCn3 gene transfer showed increased plasma UCn3 (from 0.1 ± 0.01 ng/mL in the saline group to 5.6 ± 1.1 ng/mL; n = 12 each group; p < 0.0001). Compared to mice that received saline, UCn3 gene transfer was associated with higher values for EF (p = 0.0006); LV +dP/dt (p < 0.0001), and LV -dP/dt (p < 0.0001). Cardiac myocytes from mice that received UCn3 gene transfer showed higher peak Ca2+ transients (p = 0.0005), lower time constant of cytosolic Ca2+ decline (tau, p < 0.0001), and higher rates of sarcomere shortening (+dL/dt, p = 0.03) and lengthening (-dL/dt, p = 0.04). LV samples from mice that received UCn3 gene transfer contained higher levels of SERCA2a (p = 0.0004 vs. HF) and increased amounts of phosphorylated troponin I (p = 0.04 vs. HF). UCn3 gene transfer is associated with improved Ca2+ handling and LV function in mice with HF and reduced EF.

Effects of Urocortin 2 Versus Urocortin 3 Gene Transfer on Left Ventricular Function and Glucose Disposal.

UCn2 and UCn3 peptides have recently been infused to treat patients with heart failure (HF) but are limited by their short half-lives. A 1-time intravenous injection of virus vectors encoding UCn2 or UCn3 provided sustained increases in plasma concentrations of the peptides. This was associated with increases in both systolic and diastolic left ventricular (LV) function, mediated by increased LV SERCA2a expression and Ca2+ handling. UCn2, but not UCn3, gene transfer reduced fasting glucose and increased glucose disposal. These findings support UCn2 and UCn3 gene transfer as potential treatments for HF and indicate that UCn2 may be an optimal selection in patients with diabetes and HF.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs.

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.

A novel animal model for neuroinflammation and white matter degeneration.

Small interference RNA has been widely used to suppress gene expression. Three different short hairpin RNAs (shRNAs) against dopamine D1 receptor (Drd1), driven by mouse U6 promoter in self-complementary AAV8 vector (scAAV8), were used to silence mouse striatal Drd1 expression. Transduction of mouse striatum with all three scAAV8-D1shRNA viruses, but not the control scAAV8 virus, causes extensive neuroinflammation, demyelination, and axon degeneration. RNA interference is known to be coupled to the innate immune system as a host cell defense against virus infection. Activation of the innate immune system may play a causal role in the development of neuroinflammation and white matter degeneration, providing a novel animal model for multiple sclerosis (MS) and other neuroinflammatory diseases.

Lentiviral-Mediated Transgenesis in Songbirds.

Transgenesis involves the insertion of an exogenous gene into an animal's genome, which allows the identification of the expressed phenotypes in brain function or behavior. Lentiviral-mediated transgenesis offers unique transduction potency making it possible to deliver and stably integrate transgenes into a wide variety of dividing and nondividing cells. The ability to establish long-term expression of such transgenes allows their use for transgenesis which is especially useful in organisms lacking quality pluripotent stem cell lines and which is otherwise difficult to produce via traditional pronuclear microinjection, such as songbirds. Here we describe a protocol to generate the transgenic songbird, the zebra finch, by producing and inserting lentiviral-mediated transgene into the blastoderm of freshly laid eggs. This protocol includes procedures for production of lentiviral vectors, injection of a virus into zebra finch embryos, and postinjection care. The implementation of the songbird transgenic approach provides a leap toward basic and translational neuroscience that uses an animal model for speech and language and their pathologies. Additionally, the highly quantifiable song behavior, combined with a well-characterized song circuitry, offers an exciting opportunity to develop therapeutic strategies for neurological disorders.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Lung carcinoma progression and survival versus amino- and carboxyl-parathyroid hormone-related protein expression.

PURPOSE: Expression of the carboxyl PTHrP region of parathyroid hormone-related protein (PTHrP) is a positive prognostic indicator in women with lung cancer, but amino PTHrP is a negative indicator in other lung cancer patients. This project investigated whether PTHrP could be expressed as predominantly amino PTHrP or carboxyl PTHrP in individual lung carcinomas. It also assessed domain-specific effects on cancer progression and patient survival. METHODS: PTHrP immunoreactivities were analyzed versus survival in a human lung cancer tissue microarray (TMA). Growth was compared in athymic mice for isogenic lung carcinoma xenografts differing in expression of amino and carboxyl PTHrP domains. RESULTS: In the TMA, 33 of 99 patient tumors expressed only one PTHrP domain, while 54 expressed both. By Cox regression, the hazard ratio for cancer-specific mortality (95% confidence interval) was 2.6 (1.28-5.44) for amino PTHrP (P = 0.008) and 0.6 (0-2.58) for carboxyl PTHrP (P = 0.092). Xenografts of H358 lung adenocarcinoma cells that overexpressed amino PTHrP grew twice as fast as isogenic low PTHrP tumors in athymic mice, but growth of tumors expressing amino plus carboxyl PTHrP was not significantly different than growth of the control tumors. In summary, the presence of amino PTHrP signifies worse prognosis in lung cancer patients. In mouse xenografts, this effect was abrogated if carboxyl PTHrP was also present. CONCLUSION: Amino PTHrP and carboxyl PTHrP can vary independently in different lung carcinomas. Carboxyl PTHrP may temper the stimulatory effect of amino PTHrP on cancer progression.

One-time injection of AAV8 encoding urocortin 2 provides long-term resolution of insulin resistance.

Using mice rendered insulin resistant with high fat diets (HFD), we examined blood glucose levels and insulin resistance after i.v. delivery of an adeno-associated virus type 8 encoding murine urocortin 2 (AAV8.UCn2). A single i.v. injection of AAV8.UCn2-normalized blood glucose and glucose disposal within weeks, an effect that lasted for months. Hyperinsulinemic-euglycemic clamps showed reduced plasma insulin, increased glucose disposal rates, and increased insulin sensitivity following UCn2 gene transfer. Mice with corticotropin-releasing hormone type 2-receptor deletion that were rendered insulin resistant by HFD showed no improvement in glucose disposal after UCn2 gene transfer, indicating that the effect requires UCn2's cognate receptor. We also demonstrated increased glucose disposal after UCn2 gene transfer in db/db mice, a second model of insulin resistance. UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance. UCn2 increases Glut4 translocation to the plasma membrane in skeletal myotubes in a manner quantitatively similar to insulin, indicating a mechanism through which UCn2 operates to increase insulin sensitivity. UCn2 gene transfer, in a dose-dependent manner, is insulin sensitizing and effective for months after a single injection. These findings suggest a potential long-term therapy for clinical type-2 diabetes.

Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

Intravenous AAV8 Encoding Urocortin-2 Increases Function of the Failing Heart in Mice.

Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, is costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. We previously showed that a single intravenous (IV) injection of AAV8.UCn2 increases plasma UCn2 and left ventricular (LV) systolic and diastolic function for at least 7 months in normal mice. Here we test the hypothesis that IV delivery of AAV8.UCn2 increases function of the failing heart. Myocardial infarction (MI, by coronary ligation) was used to induce heart failure, which was assessed by echocardiography 3 weeks after MI. Mice with LV ejection fraction (EF) <25% received IV delivery of AAV8.UCn2 (5×10(11) gc) or saline, and 5 weeks later echocardiography showed increased LV EF in mice that received UCn2 gene transfer (p=0.01). In vivo physiological studies showed a 2-fold increase in peak rate of LV pressure development (LV +dP/dt; p<0.0001) and a 1.6-fold increase in peak rate of LV pressure decay (LV -dP/dt; p=0.0007), indicating increased LV systolic and diastolic function in treated mice. UCn2 gene transfer was associated with increased peak systolic Ca(2+) transient amplitude and rate of Ca(2+) decline and increased SERCA2a expression. In addition, UCn2 gene transfer reduced Thr286 phosphorylation of Cam kinase II, and increased expression of cardiac myosin light chain kinase, findings that would be anticipated to increase function of the failing heart. We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart. The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy.

Intravenous adeno-associated virus serotype 8 encoding urocortin-2 provides sustained augmentation of left ventricular function in mice.

Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. Here we ask whether a single intravenous injection of adeno-associated virus type 8 encoding murine urocortin-2 (AAV8.UCn2) could provide long-term elevation in plasma UCn2 levels and increased left ventricular (LV) function. Normal mice received AAV8.UCn2 (5×10¹¹ genome copies, intravenous). Plasma UCn2 increased 15-fold 6 weeks and >11-fold 7 months after delivery. AAV8 DNA and UCn2 mRNA expression was persistent in LV and liver up to 7 months after a single intravenous injection of AAV8.UCn2. Physiological studies conducted both in situ and ex vivo showed increases in LV +dP/dt and in LV -dP/dt, findings that endured unchanged for 7 months. SERCA2a mRNA and protein expression was increased in LV samples and Ca²âº transient studies showed an increased rate of Ca²âº decline in cardiac myocytes from mice that had received UCn2 gene transfer. We conclude that a single intravenous injection of AAV8.UCn2 increases plasma UCn2 and increases LV systolic and diastolic function for at least 7 months. The simplicity of intravenous injection of a long-term expression vector encoding a gene with paracrine activity to increase cardiac function is a potentially attractive strategy in clinical settings. Future studies will determine the usefulness of this approach in the treatment of heart failure.