We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca2+ cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLBM) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLBM in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLBM and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLBM for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.
SERCA; PLB; HEK; FRET; Competition
Impaired myocardial sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) activity is a
hallmark of failing hearts, and SERCA2a gene therapy improves cardiac function in
animals and patients with heart failure (HF). Deregulation of microRNAs has been
demonstrated in HF pathophysiology. We studied the effects of therapeutic AAV9.SERCA2a
gene therapy on cardiac miRNome expression and focused on regulation, expression, and
function of miR-1 in reverse remodelled failing hearts.
Methods and results
We studied a chronic post-myocardial infarction HF model treated with AAV9.SERCA2a gene
therapy. Heart failure resulted in a strong deregulation of the cardiac miRNome. miR-1
expression was decreased in failing hearts, but normalized in reverse remodelled hearts
after AAV9.SERCA2a gene delivery. Increased Akt activation in cultured cardiomyocytes
led to phosphorylation of FoxO3A and subsequent exclusion from the nucleus, resulting in
miR-1 gene silencing. In vitro SERCA2a expression also rescued miR-1 in
failing cardiomyocytes, whereas SERCA2a inhibition reduced miR-1 levels. In
vivo, Akt and FoxO3A were highly phosphorylated in failing hearts, but
reversed to normal by AAV9.SERCA2a, leading to cardiac miR-1 restoration. Likewise,
enhanced sodium–calcium exchanger 1 (NCX1) expression during HF was normalized by
SERCA2a gene therapy. Validation experiments identified NCX1 as a novel functional miR-1
SERCA2a gene therapy of failing hearts restores miR-1 expression by an
Akt/FoxO3A-dependent pathway, which is associated with normalized NCX1 expression and
improved cardiac function.
FoxO3A; Gene therapy; Heart failure; MicroRNA-1; SERCA2a; NCX1
Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs.
Calcium (Ca2+) handling proteins are known to play a pivotal role in the pathophysiology of cardiomyopathy. However little is known about early changes in the diabetic heart and the impact of insulin treatment (Ins).
Zucker Diabetic Fatty rats treated with or without insulin (ZDF ± Ins, n = 13) and lean littermates (controls, n = 7) were sacrificed at the age of 19 weeks. ZDF + Ins (n = 6) were treated with insulin for the last 6 weeks of life. Gene expression of Ca2+ ATPase in the cardiac sarcoplasmatic reticulum (SERCA2a, further abbreviated as SERCA) and phospholamban (PLB) were determined by northern blotting. Ca2+ transport of the sarcoplasmatic reticulum (SR) was assessed by oxalate-facilitated 45Ca-uptake in left ventricular homogenates. In addition, isolated neonatal cardiomyocytes were stimulated in cell culture with insulin, glucose or triiodthyronine (T3, positive control). mRNA expression of SERCA and PLB were measured by Taqman PCR. Furthermore, effects of insulin treatment on force of contraction and relaxation were evaluated by cardiomyocytes grown in a three-dimensional collagen matrix (engineered heart tissue, EHT) stimulated for 5 days by insulin. By western blot phosphorylations status of Akt was determed and the influence of wortmannin.
SERCA levels increased in both ZDF and ZDF + Ins compared to control (control 100 ± 6.2 vs. ZDF 152 ± 26.6* vs. ZDF + Ins 212 ± 18.5*# % of control, *p < 0.05 vs. control, #p < 0.05 vs. ZDF) whereas PLB was significantly decreased in ZDF and ZDF + Ins (control 100 ± 2.8 vs. ZDF 76.3 ± 13.5* vs. ZDF + Ins 79.4 ± 12.9* % of control, *p < 0.05 vs control). The increase in the SERCA/PLB ratio in ZDF and ZDF ± Ins was accompanied by enhanced Ca2+ uptake to the SR (control 1.58 ± 0.1 vs. ZDF 1.85 ± 0.06* vs. ZDF + Ins 2.03 ± 0.1* μg/mg/min, *p < 0.05 vs. control). Interestingly, there was a significant correlation between Ca2+ uptake and SERCA2a expression. As shown by in-vitro experiments, the effect of insulin on SERCA2a mRNA expression seemed to have a direct effect on cardiomyocytes. Furthermore, long-term treatment of engineered heart tissue with insulin increased the SERCA/PLB ratio and accelerated relaxation time. Akt was significantly phosphorylated by insulin. This effect could be abolished by wortmannin.
The current data demonstrate that early type 2 diabetes is associated with an increase in the SERCA/PLB ratio and that insulin directly stimulates SERCA expression and relaxation velocity. These results underline the important role of insulin and calcium handling proteins in the cardiac adaptation process of type 2 diabetes mellitus contributing to cardiac remodeling and show the important role of PI3-kinase-Akt-SERCA2a signaling cascade.
Diabetic heart; Insulin; SERCA expression; Relaxation velocity
To investigate regulation of SERCA1a and SERCA2a calcium pump isoforms by phospholamban (PLB), the proteins were fused to fluorescent protein tags and their interactions were quantified by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a or SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 angstroms. The data indicate the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.
phospholamban; SERCA1a; SERCA2a; calcium ATPase; affinity; FRET; calcium handling; membrane proteins
Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at approximately two weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with siRNAs against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.
adeno-associated virus; DNA repair; gene therapy; miR-24; MRN; permissivity
We have developed a quantitative immunoblot method to measure the mole fraction of phospholamban (PLB) phosphorylated at Ser16 (Xp) in biological samples. In cardiomyocytes, PLB phosphorylation activates the sarcoplasmic reticulum calcium ATPase (SERCA), which reduces cytoplasmic Ca++ to relax the heart during diastole. Unphosphorylated PLB (uPLB) inhibits SERCA at low [Ca++] and phosphorylated PLB (pPLB) is less inhibitory, so myocardial physiology and pathology depend critically on Xp. Current methods of Xp determination by immunoblot provide moderate precision but poor accuracy. We have solved this problem using purified uPLB and pPLB standards, produced by solid-phase peptide synthesis. In each assay, a pair of blots is performed with identical standards and unknowns, using antibodies partially selective for uPLB and pPLB, respectively. When performed on mixtures of uPLB and pPLB, the assay measures both total PLB (tPLB) and Xp with accuracy of 96% or better. We assayed pig cardiac SR and found that Xp varied widely among four animals, from 0.08 to 0.38, but there was remarkably little variation in the ratios of Xp/tPLB and uPLB/SERCA, suggesting that PLB phosphorylation is tuned to maintain homeostasis in SERCA regulation.
Phospholamban; phosphorylation; SERCA; heart failure; western blot; XpPhospholamban phosphorylation and heart disease
The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays a central role in cardiac contraction, shows decreased expression in heart failure (HF). Increasing SERCA2a expression in HF models improves cardiac function. We used direct cardiac delivery of adeno-associated virus encoding human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety, efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were analyzed for hSERCA2a expression (Western blot) and immune responses (histology and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks and then declined (to ∼50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05) whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a developed neutralizing antibodies (titer ≥1:120) and cardiac cellular infiltration. Immunosuppression dramatically reduced immune responses (reduced inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a expression and improves cardiac function. However, the hSERCA2a protein level is reduced by host immune responses. Immunosuppression alleviates immune responses and sustains transgene expression, and may be an important adjuvant for clinical gene therapy trials.
Zhu and colleagues employ direct cardiac delivery of adeno-associated virus encoding the human calcium pump SERCA2a (AAV6-hSERCA2a) in heart failure and normal canine models in order to study the safety and efficacy of the approach as well as the effects of concomitant immunosuppressant treatment. Tachycardic-paced dogs injected with AAV6-hSERCA2a via the left ventricle wall displayed hSERCA2a expression and improved cardiac function, although hSERCA2a protein levels were reduced by host immune responses. Immunosuppression dramatically reduced inflammation and neutralizing antibody titers while maintaining hSERCA2a expression.
Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca2+ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.
AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca2+ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.
Increased Ca2+ leak and cytosolic Ca2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca2+ cycling.
The cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a critical role in maintaining the intracellular calcium homeostasis during cardiac contraction and relaxation. It has been well documented over the years that altered expression and activity of SERCA2a can lead to systolic and diastolic dysfunction. The activity of SERCA2a is regulated by two structurally similar proteins, phospholamban (PLB) and sarcolipin (SLN). Although, the relevance of PLB has been extensively studied over the years, the role SLN in cardiac physiology is an emerging field of study. This review focuses on the advances in the understanding of the regulation of SERCA2a by SLN and PLB. In particular, it highlights the similarities and differences between the two proteins and their roles in cardiac patho-physiology.
SERCA2a; sarcolipin; phospholamban; calcium; regulation
Phospholamban (PLB) or the sarcoplasmic reticulum Ca2+-ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB fused to yellow fluorescent protein (YFP). The expressed fluorescently tagged proteins were imaged using epifluorescence and total internal reflection fluorescence microscopy. YFP fluorescence was selectively bleached by a focused laser beam. CFP fluorescence at the targeted site increased after YFP photobleaching, indicating fluorescence resonance energy transfer between CFP-SERCA/CFP-PLB and YFP-PLB. The increased donor fluorescence relaxed back toward baseline as a result of donor diffusion and exchange of bleached YFP-PLB for unbleached YFP-PLB, which restored fluorescence resonance energy transfer. Requenching of CFP donors, termed Förster transfer recovery (FTR), was quantified as an index of the rate of PLB subunit exchange from the PLB:SERCA and PLB:PLB membrane complexes. PLB subunit exchange from the PLB:SERCA regulatory complex was rapid, showing diffusion-limited FTR (τ=1.4 second). Conversely, PLB:PLB oligomeric complexes were found to be stable on a much longer time scale. Despite free lateral diffusion in the membrane, they showed no FTR over 80 seconds. Mutation of PLB position 40 from isoleucine to alanine (I40A-PLB) did not abolish PLB:PLB energy transfer, but destabilization of the PLB:PLB complex was apparent from an increased FTR rate (τ=8.4 seconds). Oligomers of I40A-PLB were stabilized by oxidative crosslinking of transmembrane cysteines with diamide. We conclude that PLB exchanges rapidly from its regulatory complex with the SERCA pump, whereas subunit exchange from the PLB oligomeric complex is slow and does not occur on the time scale of the cardiac cycle.
phospholamban; SERCA; FRET; TIRF; crosslinking
In this paper, the authors identify a modified adeno-associated viral (AAV) vector, self-complementary AAV (scAAV) that, in contrast to the conventional AAV, transduces the trabecular meshwork of living rats and monkeys. scAAV confers long-term expression to the trabecular meshwork (>3.5 months in rats and >2.35 years in monkeys) without clinical adverse effects.
AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals.
Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays.
No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys.
The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.
After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2).
We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents.
RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.
Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.
We evaluated the feasibility of self-complementary adeno-associated virus (scAAV) vector-mediated knockdown of the pyruvate dehydrogenase complex using small interfering RNAs directed against the E1α subunit gene (PDHA1). AAV serotype 8 was used to stereotaxically deliver scAAV8-si3-PDHA1-Enhanced Green Fluorescent Protein (knockdown) or scAAV8-EGFP (control) vectors into the right striatum and substantia nigra of rats. Rotational asymmetry was employed to quantify abnormal rotation following neurodegeneration in the nigrostriatal system. By 20 weeks after surgery, the siRNA-injected rats exhibited higher contralateral rotation during the first 10 min following amphetamine administration and lower 90-min total rotations (p≤0.05). Expression of PDC E1α, E1β and E2 subunits in striatum was decreased (p≤0.05) in the siRNA-injected striatum after 14 weeks. By week 25, both PDC activity and expression of E1α were lower (p≤0.05) in siRNA-injected striata compared to controls. E1α expression was associated with PDC activity (R2=0.48; p=0.006) and modestly associated with counterclockwise rotation (R2=0.51;p=0.07). The use of tyrosine-mutant scAAV8 vectors resulted in ~17-fold increase in transduction efficiency of rat striatal neurons in vivo. We conclude that scAAV8-siRNA vector-mediated knockdown of PDC E1α in brain regions typically affected in humans with PDC deficiency results in a reproducible biochemical and clinical phenotype in rats that may be further enhanced with the use of tyrosine-mutant vectors.
PDC, pyruvate dehydrogenase complex; scAAV, self-complementary adeno-associated virus; EGFP, enhanced green fluorescent protein; siRNA, small interfering ribonucleic acid; animal model; rare disease
To demonstrate the high efficiency and rapidity of allotopic expression of a normal human ND4 subunit of complex I in the vertebrate retina using a self-complementary adeno-associated virus (scAAV) vector for ocular gene delivery to treat acute visual loss in Leber hereditary optic neuropathy (LHON).
The nuclear-encoded human ND4 subunit fused to the P1 isoform of subunit C of adenosine tri-phosphate synthase (ATPc) mitochondrial targeting sequence and FLAG epitope was packaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavities of mice. The contralateral eyes were injected with scAAV–green fluorescent protein (GFP). One week later, pattern electroretinograms and gene expression of the human ND4 subunit and GFP were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed relative to Thy1.2-labeled retinal ganglion cells (RGCs).
Pattern electroretinogram amplitudes remained normal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG, and GFP. Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mount. In contrast, scAAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG–positive RGCs was 91% and that of ssAAV-ND4FLAG–positive RGCs was 51%.
Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complex I, mutated in most cases of LHON, when delivered by an scAAV vector.
Unlike most retinal degenerations that result in slowly progressive loss of vision over many years, LHON due to mutated mitochondrial DNA results in apoplectic, bilateral severe and usually irreversible visual loss. For rescue of acute visual loss in LHON, a highly efficient and rapid gene expression system is required.
Heart failure is a progressive, debilitating disease that is characterized by inadequate contractility of the heart. With an aging population, the incidence and economic burden of managing heart failure are anticipated to increase substantially. Drugs for heart failure only slow its progression and offer no cure. However, results of recent clinical trials using recombinant adeno-associated virus (AAV) gene delivery offer the promise, for the first time, that heart failure can be reversed. The strategy is to improve contractility of cardiac muscle cells by enhancing their ability to store calcium through increased expression of the sarco(endo)plasmic reticulum Ca2+-ATPase pump (SERCA2a). Preclinical trials have also identified other proteins involved in calcium cycling in cardiac muscle that are promising targets for gene therapy in heart failure, including the following: protein phosphatase 1, adenylyl cyclase 6, G-protein-coupled receptor kinase 2, phospholamban, SUMO1, and S100A1. These preclinical and clinical trials represent a “quiet revolution” that may end up being one of the most significant and remarkable breakthroughs in modern medical practice. Of course, a number of uncertainties remain, including the long-term utility and wisdom of improving the contractile performance of “sick” muscle cells. In this regard, gene therapy may turn out to be a way of buying additional time for actual cardiac regeneration to occur using cardiac stem cells or induced pluripotent stem cells.
Diabetes is a cause of cardiac dysfunction, reduced myocardial perfusion, and ultimately heart failure. Nerve growth factor (NGF) exerts protective effects on the cardiovascular system. This study investigated whether NGF gene transfer can prevent diabetic cardiomyopathy in mice. We worked with mice with streptozotocin-induced type 1 diabetes and with nondiabetic control mice. After having established that diabetes reduces cardiac NGF mRNA expression, we tested NGF gene therapies with adeno-associated viral vectors (AAVs) for the capacity to protect the diabetic mouse heart. To this aim, after 2 weeks of diabetes, cardiac expression of human NGF or β-Gal (control) genes was induced by either intramyocardial injection of AAV serotype 2 (AAV2) or systemic delivery of AAV serotype 9 (AAV9). Nondiabetic mice were given AAV2–β-Gal or AAV9–β-Gal. We found that the diabetic mice receiving NGF gene transfer via either AAV2 or AAV9 were spared the progressive deterioration of cardiac function and left ventricular chamber dilatation observed in β-Gal–injected diabetic mice. Moreover, they were additionally protected from myocardial microvascular rarefaction, hypoperfusion, increased deposition of interstitial fibrosis, and increased apoptosis of endothelial cells and cardiomyocytes, which afflicted the β-Gal–injected diabetic control mice. Our data suggest therapeutic potential of NGF for the prevention of cardiomyopathy in diabetic subjects.
To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection.
ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity.
GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced.
When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.
Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson’s disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9wks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.
neurodegeneration; RNAi; substantia nigra; gene therapy; tyrosine hydroxylase
Cardiac gene transfer is a potentially useful strategy for cardiovascular diseases. The adeno-associated virus (AAV) is a common vector to obtain transgene expression in the heart. Initial studies conducted in rodents used indirect intracoronary delivery for cardiac gene transfer. More recently AAV vectors with so-called cardiac tropism have enabled significant cardiac transgene expression following intravenous injection. However, a direct comparison of intravenous versus intracoronary delivery with rigorous quantification of cardiac transgene expression has not been conducted. In the present study we tested the hypothesis that intracoronary AAV delivery would be superior to intravenous delivery vis-à-vis cardiac transgene expression. We compared intravenous and intracoronary delivery of AAV5, AAV6, and AAV9 (5×1011 genome copies per mouse). Using enhanced green fluorescent protein as a reporter, we quantified transgene expression by fluorescence intensity and Western blotting. Quantitative polymerase chain reaction (PCR) was also performed to assess vector DNA copies, employing primers against common sequences on AAV5, AAV6, and AAV9. Intracoronary delivery resulted in 2.6- to 28-fold higher transgene protein expression in the heart 3 weeks after AAV injection compared to intravenous delivery depending on AAV serotype. The highest level of cardiac gene expression was achieved following intracoronary delivery of AAV9. Intracoronary delivery of AAV9 is a preferred method for cardiac gene transfer.
There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA).
Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA.
Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.
RNA interference; Adeno-associated virus 9; Dorsal root ganglia; Blood–nerve barrier
The purpose of this study is to demonstrate that the expression of rhodopsin can be down regulated in vivo by AAV-delivered siRNA. This is the first step in an RNA replacement strategy for the allele-independent treatment of Autosomal Dominant Retinitis Pigmentosa (ADRP).
HEK 293 cells were co-transfected with a plasmid carrying mouse RHO cDNA driven by the CMV promoter and a chemically synthesized siRNA duplex of 21 nucleotides. Reduction of RHO mRNA was confirmed by RT-PCR. One active siRNA and a control siRNA were embedded in a small hairpin RNA (shRNA) and cloned in Adeno-associated virus (AAV) vector under regulation of the H1 promoter and containing a GFP reporter. AAV5 expressing either active siRNA or an irrelevant siRNA were subretinaly injected into the right eyes of wild-type or RHO+/− heterozygote mice at postnatal day 16. At 1 and 2 months post injection, animals were analyzed by electroretinography (ERG). Animals were then sacrificed, and retinas were examined by western blot, RT-PCR, histology and immunohistochemistry.
All of the siRNAs tested in HEK 293 cells caused degradation of RHO mRNA, although the efficiency varied from 25% to 80%. In vivo siRNA delivery to the retina led to more than 40% reduction of scotopic a-and b-wave amplitudes in RHO+/− heterozygotes. Although the reduction of RHO mRNA was estimated at 30% compared to control animals, western blots revealed 60% decrease in rhodopsin content. Histological analysis showed significant reduction in the thickness of the ONL, ranging between 53% and 86%.
AAV-siRNA delivery into the subretinal space resulted in the reduction of retinal function caused by diminished RHO mRNA and protein content. This level of reduction may permit the replacement of endogenous mRNA with siRNA-resistant mRNA encoding wild-type RHO.
retina; retinitis pigmentosa; gene therapy; rhodopsin; RNA interference; Adeno-associated virus
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.
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.
gene therapy; heart failure; neurohormones; cardiac remodeling, ventricular
We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehy-drogenase E1α subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72 h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3-to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1α protein expression were determined 10 days post-transduction. Both vectors increased E1α expression 40–60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1α. Exposure for 24 h to 5 mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1α protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.
Pyruvate dehydrogenase deficiency; AAV vector; Gene therapy; Dichloroacetate