Cardiac Gene Transfer Research Articles

Controlling the Inhibition of the Sarcoplasmic Ca2+-ATPase by Tuning Phospholamban Structural Dynamics

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.

A Dual-Targeted Lentiviral Vector Homing in on Prostate Bone Metastases

A Dual-Targeted Lentiviral Vector Homing in on Prostate Bone Metastases

Abstract 404: Catheter Based Delivery of scAAV6 Leads to High Level, Global Gene Transfer to the Canine Heart Superior to scAAV8, scAAV9, and ssAAV9

Background: Heart disease is the leading cause of morbidity and mortality, and its prevalence continues to rise. As a result, novel therapies are needed. Adeno-associated virus (AAV) mediated gene transfer may be an effective strategy since the low immunogenicity of AAV favors persistent transgene expression. In addition, the recently developed self-complementary AAV (scAAV) vectors have demonstrated higher expression than traditional, single stranded AAV (ssAAV) vectors. The goal of this study was to compare cardiac gene transfer efficiency of four AAV vectors using non-invasive, catheter based delivery in the dog. Methods: Four AAV vectors expressing GFP under control of the CB promoter were evaluated in this study–ssAAV9, scAAV9, scAAV8, scAAV6 –so that comparison could be made between ss and sc vectors as well as among serotypes 9, 8, and 6. Dogs (5–10kg, n=4 per group) underwent cardiac catheterization with introduction of an injection catheter into the LV under fluoroscopy. The interventricular septum (IVS) and LV free wall (LVFW) were targeted from base to apex with 40 injections of 250 ul. The total viral dose was either 2x10 13 gc/kg (ss) or 5x10 11 gc/kg (sc). At 7–10 days post-injection, hearts were explanted and divided into four equal sections from base to apex. GFP expression was quantified in four representative areas from IVS and LVFW in each section. TaqMan PCR was used to track biodistribution. Results: GFP expression in the scAAV6 group was approximately 10-fold higher than in the scAAV8 and scAAV9 groups, and GFP expression in the scAAV9 group was approximately 10-fold higher than in the ssAAV9 group, both in terms of total positive cells and total fluorescence (p<0.01). Biodistribution studies revealed that vector genome copies were at least 10 times more abundant in the heart than in any other organ for scAAV6 (p≤0.05). Conclusion: scAAV is superior to ssAAV for cardiac gene transfer, and AAV6 has a higher cardiac tropism than AAV8 and AAV9 in the canine. Catheter based delivery of scAAV6 is a safe, effective method for achieving high level, global cardiac gene transfer to the LV. scAAV6 has great potential for transferring therapeutic transgenes to the failing canine heart and may be the vector of choice for clinical trials of cardiac gene transfer.

Is Phospholamban or Troponin I the “Prima Donna” in β-Adrenergic Induced Lusitropy?

See related articles, pages 377–386 In normal myocardium, acute β-adrenergic stimulation augments both systolic and diastolic performance via protein kinase A (PKA)–mediated phosphorylation of key proteins governing Ca2+ handling and the contractile machinery. Twenty-five years ago Kranias and Solaro identified troponin I (TnI) and phospholamban (PLN) as the 2 major cardiac proteins which were nearly simultaneously phosphorylated by β-adrenergic stimulation of the beating heart in synchrony with the agonist effect of augmenting contractility (inotropy) and rate of relaxation (lusitropy).1 Numerous subsequent studies have mechanistically examined the role of these proteins in inotropy and lusitropy. It is well established that enhanced Ca2+ availability during systole is the major, though perhaps not the only, driver of enhanced inotropy with β- adrenergic stimulation.2–4 The effect on Ca2+ dynamics is primarily mediated by phosphorylation of PLN, though increased ionic current through the L-type calcium channel contributes to Ca2+ loading. When PLN is phosphorylated by PKA, the “brake” imposed by PLN on sarcoplasmic reticulum (SR) Ca2+ ATPase is relieved, resulting in an increase in the activity of the latter that leads to a faster sequestration of Ca2+ into the SR, enhancing cardiac relaxation and re-loading the SR with Ca2+ to increase Ca2+ release in subsequent beats. Yet, PLN phosphorylation by PKA is not the sole mechanism implicated in enhancing relaxation. In particular, phosphorylation of TnI by PKA has long been proposed to have a role in diastole because it desensitizes the myofilament to Ca2+, increases the off rate of Ca …

Cardiac Transgenic and Gene Transfer Strategies Converge to Support an Important Role for Troponin I in Regulating Relaxation in Cardiac Myocytes

Elucidating the relative roles of cardiac troponin I (cTnI) and phospholamban (PLN) in beta-adrenergic-mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that beta-adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic (Tg) mice bearing near complete replacement of native cTnI with a beta-adrenergic phospho-mimetic of cTnI whereby tandem serine codons 23/24 were converted to aspartic acids (cTnI S23/24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro-carbon fibers. At baseline, cTnI S23/24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation (Iso) had only a small effect to further speed relaxation in cTnI S23/24D myocytes (delta Iso: 7.2 ms) relative to the maximum Iso effect (31.2 ms) in control. The Ca(2+) transient decay rate was similarly accelerated by Iso in Tg and nontransgenic (Ntg) myocytes. Gene transfer of cTnI S23/24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/24 converted to alanines (cTnI S23/24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild-type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance.

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.

Targeted gene transfer increases contractility and decreases oxygen cost of contractility in normal rat hearts

The aim of this study was to examine how global cardiac gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) can influence left ventricular (LV) mechanical and energetic function, especially in terms of O(2) cost of LV contractility, in normal rats. Normal rats were randomized to receive an adenovirus carrying the SERCA2a (SERCA) or beta-galactosidase (beta-Gal) gene or saline by a catheter-based technique. LV mechanical and energetic function was measured in cross-circulated heart preparations 2-3 days after the infection. The end-systolic pressure-volume relation was shifted upward, end-systolic pressure at 0.1 ml of intraballoon water volume was higher, and equivalent maximal elastance, i.e., enhanced LV contractility, was higher in the SERCA group than in the normal, beta-Gal, and saline groups. Moreover, the LV relaxation rate was faster in the SERCA group. There was no significant difference in myocardial O(2) consumption per beat-systolic pressure-volume area relation among the groups. Finally, O(2) cost of LV contractility was decreased to subnormal levels in the SERCA group but remained unchanged in the beta-Gal and saline groups. This lowered O(2) cost of LV contractility in SERCA hearts indicates energy saving in Ca(2+) handling during excitation-contraction coupling. Thus overexpression of SERCA2a transformed the normal energy utilization to a more efficient state in Ca(2+) handling and superinduced the supranormal contraction/relaxation due to enhanced Ca(2+) handling.

Skeletal muscle gene transfer and regulated expression of IGF‐I increases function of the failing heart

Current methods of cardiac gene transfer include intramuscular injection (im) into heart muscle or coronary delivery. When applied to rodents with heart failure, these surgical approaches are associated with high mortality, an impediment that would also complicate clinical studies. To circumvent this, we selected a transgene, Insulin-like Growth Factor-I (IGF-I), which may have favorable effects on heart function when secreted from a remote site. This study examines the feasibility of skeletal muscle injection of adeno-associated virus-5 encoding IGF-I under tet regulation (AAV5.IGFI-tet). Left coronary ligation was performed in rats and MI confirmed 1wk later by echocardiography. Rats with MI then received 2×1012 vp of AAV5.IGFI-tet in the anterior tibialis (im). Four wks later they were randomly assigned to receive doxycycline in the drinking water (IGF-On; n=5) or not (IGF-Off; n=3). Ten wks post MI anesthetized rats underwent a final echo and terminal study in which a 1.8F pressure-volume transducer was placed in the left ventricle (LV). IGF-On rats showed an increased in LV fractional area change (p=0.04), ejection fraction (p=0.04), and velocity of circumferential fiber shortening (p=0.04), and had smaller LV end-diastolic (p=0.002) and end-systolic (p=0.003) dimensions. LV function was improved with dobutamine infusion (Table; IGF-On, n=4; IGF-Off, n=3). Serum IGF-I was increased 5 wk after activation (IGF-On: 218±7 ng/ml; IGF-Off: 158±24 ng/ml; p=0.046). These data indicate that intramuscular injection of AAV5.IGFI-tet increases function of the failing heart.

Adenylylcyclase Gene Transfer Increases Function of the Failing Heart

A persistent question in cardiovascular gene transfer concerns whether an exogenously delivered gene can increase function of the failing heart. Here we test the hypothesis that intracoronary delivery of adenovirus encoding adenylylcyclase type VI (Ad.ACVI) in the setting of active heart failure will increase function of the failing heart. As a model of heart failure, we used transgenic mice with dilated and poorly functioning hearts resulting from cardiac-directed expression of Galphaq.Galphaq mice with equivalent pretreatment impairment in left ventricular (LV) function (echocardiography) received 2.5x1010 viral particles of Ad.ACVI or Ad.EGFP (enhanced green fluorescent protein), or saline, by indirect intracoronary delivery. Serial echocardiograms obtained before and 14 days after gene transfer showed that Ad.ACVI increased LV ejection fraction (p<0.01) and velocity of circumferential fiber shortening (p<0.03). Detailed measurements in isolated hearts showed that ACVI gene transfer increased LV positive dP/dt (p=0.02) and LV negative dP/dt (p=0.01). Gene transfer was confirmed by polymerase chain reaction. These data show that, in an animal model that mimics key aspects of clinical congestive heart failure, cardiac gene transfer of ACVI increases function of the failing heart.

Remodeling of the Cardiac Pacemaker L-Type Calcium Current and Its β-Adrenergic Responsiveness in Hypertension After Neuronal NO Synthase Gene Transfer

Hypertension is associated with abnormal neurohumoral activation. We tested the hypothesis that beta-adrenergic hyperresponsiveness in the sinoatrial node (SAN) of the spontaneously hypertensive rat occurs at the level of the L-type calcium current because of altered cyclic nucleotide-dependent signaling. Furthermore, we hypothesized that NO, a modulator of cGMP and cAMP, would normalize the beta-adrenergic phenotype in the hypertensive rat. Chronotropic responsiveness to norepinephrine (NE), together with production of cAMP and cGMP, was assessed in isolated atrial preparations from age-matched hypertensive and normotensive rats. Right atrial/SAN pacemaking tissue was injected with adenovirus encoding enhanced green fluorescent protein (control vector) or neuronal NO synthase (nNOS). In addition, L-type calcium current was measured in cells isolated from the SAN of transfected animals. Basal levels of cGMP were lower in hypertensive rat atria. These atria were hyperresponsive to NE at all of the concentrations tested, with elevated production of cAMP. This was accompanied by increased basal and norepinephrine-stimulated L-type calcium current. Using enhanced green fluorescent protein, we observed transgene expression within both tissue sections and isolated pacemaking cells. Adenoviral nNOS increased right atrial nNOS protein expression and cGMP content. NE-stimulated cAMP concentration and L-type calcium current were also attenuated by adenoviral nNOS, along with the chronotropic responsiveness to NE in hypertensive rat atria. Decreased calcium current after cardiac nNOS gene transfer contributes to the normalization of beta-adrenergic hyperresponsiveness in the SAN from hypertensive rats by modulating cyclic nucleotide signaling.

Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8

It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 × 1011 particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF121 (AdVEGF121) versus maximum medical treatment

The demonstration that angiogenic growth factors can stimulate new blood vessel growth and restore perfusion in animal models of myocardial ischemia has led to the development of strategies designed for the local production of angiogenic growth factors in patients who are not candidates for conventional revascularization. The results of recent clinical trials of proangiogenesis gene therapy have been disappointing; however, significant limitations in experimental design, in particular in gene transfer strategies, preclude drawing definitive conclusions. In the REVASC study cardiac gene transfer was optimized by direct intramyocardial delivery of a replication-deficient adenovirus-containing vascular endothelial growth factor (AdVEGF121, 4 x 10(10) particle units (p.u.)). Sixty-seven patients with severe angina due to coronary artery disease and no conventional options for revascularization were randomized to AdVEGF121 gene transfer via mini-thoracotomy or continuation of maximal medical treatment. Exercise time to 1 mm ST-segment depression, the predefined primary end-point analysis, was significantly increased in the AdVEGF121 group compared to control at 26 weeks (P=0.026), but not at 12 weeks. As well, total exercise duration and time to moderate angina at weeks 12 and 26, and in angina symptoms as measured by the Canadian Cardiovascular Society Angina Class and Seattle Angina Questionnaire were all improved by VEGF gene transfer (all P-values at 12 and 26 weeks < or =0.001). However, if anything the results of nuclear perfusion imaging favored the control group, although the AdVEGF121 group achieved higher workloads. Overall there was no significant difference in adverse events between the two groups, despite the fact that procedure-related events were seen only in the thoracotomy group. Therefore, administration of AdVEGF121 by direct intramyocardial injections resulted in objective improvement in exercise-induced ischemia in patients with refractory ischemic heart disease.

Transmural Gradients of Repolarization and Excitation–Contraction Coupling in Mouse Ventricle

See related article, pages 1306–1313 The effects of changes in action potential waveform on excitation–contraction coupling in mammalian ventricle were first identified almost 40 years ago.1 Many important details concerning the relationship between action potential duration and tension development have been elucidated.2 Recent work has demonstrated significant effects on the intracellular Ca transient and excitation–contraction coupling of early repolarization of the action potential, which is strongly regulated by a Ca2+-independent transient outward K+ current denoted I to.3–6 The molecular physiology and some aspects of the pharmacology of this current are now quite well understood, based, in part, on a series of comprehensive articles from the Nerbonne et al,7,8 Strauss and Campbell,9 and others.6 Interestingly, a somewhat similar transient outward K+ current is expressed in neurons10 and in selected regions near the intraventricular septum of mammalian hearts.11–13 One of the most striking features of the transient outward K+ current in mammalian ventricle is the difference in its transmural expression, with significantly higher expression levels in epi- than in endocardium14 (see also references 8, 9, 11). The basis for this transmural heterogeneity, and also for the higher levels of expression in the right ventricle compared with the left, continues to be a topic of intense investigation. Significant new information and plausible working hypotheses for the way in which this heterogeneity …

Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors

Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMV(enh)/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Intravenous injections of AAV-2 vectors harboring the CMV(enh)/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Combining transcriptional targeting by the CMV(enh)/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction.

236. Ultrasound_Targeted Microbubble Destruction Augments Cardiac AAV_Mediated Gene Transfer after Systemic Administration in Adult Rats

Top of pageAbstract Systemic gene transfer using AAV-6 serotype-vectors has been shown to be an efficient route of application in mice. However, systemic application requires high doses of vector or pre-treatment with VEGF to allow vascular permeabilization. The aim of our study was to analyse whether ultrasound-targeted microbubble destruction can augment virus uptake in the heart after intravenous administration. Lipid stabilized microbubbles loaded with 4|[times]|10(10) genomic particles of AAV6 encoding for a luciferase reporter gene under the control of a CMV-enhanced 1.5 kb myosin-light chain promoter were infused into the jugular vein of adult (250g) Sprague-Dawley rats. During the infusion high mechanical index ultrasound was administered to the heart. After 4 weeks, organs were harvested and analyzed for reporter gene expression. In contrast to low cardiac expression after systemic transfer of the vector solution without ultrasound, ultrasound-mediated destruction of microbubbles augmented cardiac reporter activities by more than one order of magnitude. Specificity of gene transfer was also increased since background activities in extracardiac organs such as liver and lung were similar with or without ultrasound. In conclusion, ultrasound-targeted microbubble destruction augments cardiac gene transfer with low titer AAV-6 serotype vectors in rats. This approach may be suitable also for increasing efficiency and specificity of AAV-mediated gene transfer in larger species and holds promise for human gene therapy.

Dominant-negative c-Jun inhibits rat cardiac hypertrophy induced by angiotensin II and hypertension

Cardiac activator protein-1 (AP-1), composed of c-Jun, is significantly activated by hypertension or angiotensin II (AngII). This study was undertaken to elucidate whether c-Jun could be the potential target for treatment of cardiac hypertrophy. We constructed recombinant adenovirus carrying dominant-negative mutant of c-Jun (Ad.DN-c-Jun). Using catheter-based technique of adenoviral gene transfer, we achieved global myocardial transduction of DN-c-Jun in rats, to specifically inhibit cardiac AP-1. (1) AngII (200 ng/kg/min) infusion in rats caused cardiac hypertrophy, increased cardiac p70S6 kinase activity by 1.3-fold (P<0.05) and enhanced the gene expression of cardiac hypertrophic markers. Ad.DN-c-Jun, which was transferred to the heart 2 days before AngII infusion, prevented cardiac hypertrophy (P<0.01), decreased p70S6 kinase phosphorylation (P<0.05), and suppressed cardiac gene expression of brain natriuretic peptide, collagen I, III, and IV, monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) (P<0.01). (2) In genetically hypertensive rats with cardiac hypertrophy, cardiac gene transfer of Ad.DN-c-Jun, without affecting hypertension, regressed cardiac hypertrophy (P<0.05), and suppressed p70S6 kinase phosphorylation by 20% (P<0.05) and suppressed the enhanced expression of collagen I, III, and IV, MCP-1 and PAI-1. These results provided the first evidence that in vivo blockade of cardiac c-Jun inhibits pathologic cardiac hypertrophy.

PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury

Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). However, less is known about the chronic effects of Akt activation in the heart, and, paradoxically, Akt is activated in samples from patients with chronic heart failure. We generated Tg mice with cardiac-specific expression of either activated (myristoylated [myr]) or dominant-negative (dn) Akt and assessed their response to IRI in an ex vivo model. While dn-Akt hearts demonstrated a moderate reduction in functional recovery after IRI, no function was restored in any of the myr-Akt-Tg hearts. Moreover, infarcts were dramatically larger in myr-Akt-Tg hearts. Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of PI3K activity through both proteasome-dependent degradation of insulin receptor substrate-1 (IRS-1) and inhibition of transcription of IRS-1 as well as that of IRS-2. To test the functional significance of these signaling changes, we performed in vivo cardiac gene transfer with constitutively active PI3K in myr-Akt-Tg mice. Restoration of PI3K rescued function and reduced injury after IRI. These data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection and suggest a mechanism by which chronic Akt activation can become maladaptive.

909. Sustained Correction of Glycogen Storage Disease Type II by rAAV1 Vector-Mediated Gene Therapy

Top of pageAbstract Glycogen storage disease type II (GSDII), is a lysosomal storage disease caused by a partial to complete deficiency in the lysosomal hydrolase, acid |[alpha]|-glucosidase (GAA). From gestation, affected individuals store glycogen in their lysosomal compartments, resulting in severe hypertrophic cardiomyopathy and respiratory insufficiency. Previous studies with intrahepatic and intramuscular delivery of recombinant adeno-associated viral (rAAV) vectors to GSDII mice resulted in a variable anti-GAA immune response and insufficient levels of circulating protein in immune-responsive mice. In this study, we evaluated the potential for intravenously delivered rAAV vectors to correct the biochemical, histological and functional phenotype in GSDII mice over a sustained period of time. Neonatal GSDII mice were intravenously administered 5 |[times]| 1010 particles of rAAV serotype 1 vector encoding human GAA under control of the CMV promoter and analyzed at 2.5, 6 and 11 months post-injection (PI). GAA levels in the heart and diaphragm peaked at 6 months PI with averages of 42 |[plusmn]| 23-fold and 1.4 |[plusmn]| 0.6-fold normal respectively. At 11 months PI, cardiac levels dropped but remained therapeutic at 6.4 |[plusmn]| 1.9-fold normal and these levels were concomitant with glycogen clearance in the heart. Additionally, at 11 months, PI, contractile function of hind-limb muscles was significantly improved and the observed mobility of the treated animals was improved compared to age-matched mice. Six of the 26 treated mice formed anti-GAA antibodies between 4 and 10 weeks PI that quickly dropped to background levels by 15 weeks PI. Based on historical data, the therapeutic target for GAA restoration is 20-30% of normal. In this study, we achieved long-term therapeutic levels of correction from a single intravenous delivery in the two most critically affected muscles, the heart and diaphragm, with functional correction in the skeletal muscle. These results demonstrate the utility of intravenously administered rAAV1 vectors for both skeletal and cardiac muscle gene transfer in a model of fatal cardiomyopathy.

46. In Vivo Adenoviral Gene Transfer of Activated PI3-Kinase Rescues Cardiac Dysfunction and Injury Induced by Ischemia-Reperfusion after Chronic Cardiac Akt Activation

Acute activation of the serine-threonine kinase, Akt, is cardioprotective and reduces both infarction and dysfunction after ischemia-reperfusion injury (IRI). However, Akt is paradoxically and chronically activated in human advanced heart failure suggesting it may become maladaptive. To test the effects of chronic Akt activation, we generated transgenic mice (TG) with cardiac specific expression of either activated Akt (myr-Akt) or dominant-negative (dn) Akt and studied their response to global IRI in the ex vivo Langendorff perfusion model. We have previously reported that after IRI (20min Ischemia, 40min Reperfusion), none of the myr-Akt TG hearts recovered any function while littermate control (NTG) hearts recovered to 53.5|[plusmn]|3.5% of the baseline LV developed pressure (LVDP) (n=11 each, p<0.01). dn-Akt manifested a moderate reduction in functional recovery (34.4|[plusmn]|7.5%, n=5, p<0.001), consistent with a cardioprotective role for endogenous Akt. TTC staining and CPK release into the perfusate demonstrated dramatically increased infarct size in myr-Akt TG hearts (p<0.01). Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of upstream activation of PI3Kinase (PI3K) through both proteasome-dependent degradation of insulin-receptor substrate-1 (IRS-1) and inhibition of IRS transcription. QRT-PCR demonstrated mRNA levels for both IRS-1 and IRS-2 were significantly reduced in myr-Akt TG hearts (68.7%, p<0.001 and 59.9%, p<0.005, respectively). To understand the functional significance of this, we performed in vivo cardiac gene transfer of constitutively active PI3K (Ad.BD110) that does not depend on IRS-1 for activation or control virus in myr-Akt TG mice. Three days after transfection, expression of BD110 was documented by immunoblotting and associated with a significant increase in PI3K activity (2.6 fold, p<0.05). No functional recovery was seen in myr-Akt TG mice transduced with control virus (Ad.GFP) after IRI. However, after Ad.BD110 infection, myr-Akt TG mice demonstrated significant functional recovering achieving a maximal LVDP of 34.5|[plusmn]|1.8% (n=7, p<0.01 compared to myr-Akt TG without BD110). CPK release from myr-Akt TG hearts was also dramatically reduced after IRI (0.015 fold, p<0.01 vs myr-Akt TG alone, p=NS vs NTG). Taken together, these data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection. These findings demonstrate a mechanism by which chronic Akt activation can become maladaptive in heart failure and suggest the importance of therapeutic strategies that target multiple PI3K-dependent pathways simultaneously.

One year transgene expression with adeno-associated virus cardiac gene transfer

Background Adeno-associated virus (AAV) has shown promise as a vector for cardiac gene transfer given its ability to stably integrate into the host genome and its lack of immune reactivity. This study examined the feasibility of AAV-mediated myocardial gene transfer in mice, the animal which, because of transgenic technology, has become the disease model of choice for cardiovascular research. Methods AAV encoding the cytomegalovirus promoter driven LacZ reporter gene (10 7 LacZ-forming units per animal) or vehicle control was injected into the hearts of young adult C57Bl/6 mice by a transdiaphragmatic approach. At one, two, three, six, and twelve months post-injection, cardiac function was assessed by transthoracic echocardiography and hearts were assayed by X-gal histochemical staining. Results Echocardiography revealed normal left ventricular function in both AAV and control groups at all time points. X-gal staining of cryostat sections of hearts revealed uniform LacZ expression at all time points. There were minimal signs of immunologic infiltration by hematoxylin and eosin staining. Conclusions AAV-mediated myocardial gene transfer by transdiaphragmatic injection can be conducted safely and results in long-term expression of the LacZ gene for at least one year without causing significant inflammatory response or adversely affecting LV systolic function.