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Abstract: Despite remarkable effectiveness of reperfusion and drug therapies to reduce morbidity and mortality following myocardial infarction (MI), many patients have debilitating symptoms and impaired left ventricular (LV) function highlighting the need for improved post-MI therapies. A promising concept currently under investigation is intramyocardial injection of high-water content, polymeric biomaterial gels (e.g., hydrogels) to modulate myocardial scar formation and LV adverse remodeling. We propose a degradable, bioactive hydrogel that forms a unique microstructure of continuous, parallel capillary-like channels (Capgel). We hypothesize that the innovative architecture and composition of Capgel can serve as a platform for endogenous cell recruitment and drug/cell delivery, therefore facilitating myocardial repair after MI. [Copyright &y& Elsevier]

Since the first demonstration of in vivo gene transfer into myocardium there have been a series of advancements that have driven the evolution of cardiac gene delivery from an experimental tool into a therapy currently at the threshold of becoming a viable clinical option. Innovative methods have been established to address practical challenges related to tissue-type specificity, choice of delivery vehicle, potency of the delivered material, and delivery route. Most importantly for therapeutic purposes, these strategies are being thoroughly tested to ensure safety of the delivery system and the delivered genetic material. This review focuses on the development of recombinant adeno-associated virus (rAAV) as one of the most valuable cardiac gene transfer agents available today. Various forms of rAAV have been used to deliver 'pre-event' cardiac protection and to temper the severity of hypertrophy, cardiac ischemia, or infarct size. Adeno-associated virus (AAV) vectors have also been functional delivery tools for cardiac gene expression knockdown studies and successfully improving the cardiac aspects of several metabolic and neuromuscular diseases. Viral capsid manipulations along with the development of tissue-specific and regulated promoters have greatly increased the utility of rAAV-mediated gene transfer. Important clinical studies are currently underway to evaluate AAV-based cardiac gene delivery in humans. [ABSTRACT FROM AUTHOR]

Background: The increasing worldwide prevalence of coronary artery disease (CAD) continues to challenge the medical community. Management options include medical and revascularization therapy. Despite advances in these methods, CAD is a leading cause of recurrent ischemia and heart failure, posing significant morbidity and mortality risks along with increasing health costs in a large patient population worldwide. Trial Design: The Cardiovascular Cell Therapy Research Network (CCTRN) was established by the National Institutes of Health to investigate the role of cell therapy in the treatment of chronic cardiovascular disease. FOCUS is a CCTRN-designed randomized, phase II, placebo-controlled clinical trial that will assess the effect of autologous bone marrow mononuclear cells delivered transendocardially to patients with left ventricular (LV) dysfunction and symptomatic heart failure or angina. All patients need to have limiting ischemia by reversible ischemia on single-photon emission computed tomography assessment. Results: After thoughtful consideration of both statistical and clinical principles, we will recruit 87 patients (58 cell treated and 29 placebo) to receive either bone marrow–derived stem cells or placebo. Myocardial perfusion, LV contractile performance, and maximal oxygen consumption are the primary outcome measures. Conclusions: The designed clinical trial will provide a sound assessment of the effect of autologous bone marrow mononuclear cells in improving blood flow and contractile function of the heart. The target population is patients with CAD and LV dysfunction with limiting angina or symptomatic heat failure. Patient safety is a central concern of the CCTRN, and patients will be followed for at least 5 years. [ABSTRACT FROM AUTHOR]

Several previous studies have demonstrated that administration of autologous bone marrow–derived mononuclear cells (BMMNCs) improves cardiac function in patients after acute myocardial infarction (AMI). However, optimum timing of administration has not been investigated in a clinical trial. The Cardiovascular Cell Therapy Research Network was developed and funded by the National Heart, Lung, and Blood Institute to address important questions such as timing of cell delivery and to accelerate research in the use of cell-based therapies. The TIME trial is a randomized, phase II, double-blind, placebo-controlled clinical trial. The 5 member clinical sites of the Cardiovascular Cell Therapy Research Network will enroll 120 eligible patients with moderate-to-large anterior AMIs who have undergone successful percutaneous coronary intervention of the left anterior descending coronary artery and have a left ventricular (LV) ejection fraction ≤45% by echocardiography. Participants will have bone marrow aspirations and intracoronary infusions of 150 × 106 BMMNCs or placebo on day 3 or day 7 post-AMI. Objectives of this study are (1) to evaluate effects of BMMNCs on regional and global LV function compared to placebo therapy in patients with acute AMI as assessed by cardiac magnetic resonance imaging at 6 months and (2) to assess whether effects of BMMNC infusion on global and regional LV function and safety are influenced by the time of administration. This study will provide further insight into the clinical feasibility and appropriate timing of autologous BMMNC therapy in high-risk patients after AMI and percutaneous coronary intervention. [Copyright &y& Elsevier]

The authors discuss the signs of progress in gene therapy for muscular dystrophy. The authors say that merging of adeno-associated virus (AAV)-based vectors are candidates for clinical gene transfer to muscle. They argue that it present several challenges in terms of Duchenne muscular dystrophy (DMD). They mention that DMD gene and its protein products were at the center of the study in human genetics 25 years ago that defined positional cloning.

Background: Pompe disease is a rare disorder characterised by progressive loss of muscle and respiratory function due to acid α-glucosidase deficiency. Enzyme replacement therapy with recombinant human acid α-glucosidase, alglucosidase alfa, is the first approved treatment for the disease, but some patients do not respond, and many do not show a sustained benefit. We aimed to assess the safety and efficacy of an investigational two-component therapy (cipaglucosidase alfa, a novel recombinant human acid α-glucosidase, plus miglustat, an enzyme stabiliser) for late-onset Pompe disease.Methods: We did a randomised, double-blind, parallel-group, phase 3 trial at 62 neuromuscular and metabolic medical centres in 24 countries in the Americas, Asia-Pacific, and Europe. Eligible participants were aged 18 years or older with late-onset Pompe disease, and had either been receiving alglucosidase alfa for at least 2 years or were enzyme replacement therapy-naive. Participants were randomly assigned (2:1) using interactive response technology software, stratified by 6-min walk distance and previous enzyme replacement therapy status, to intravenous cipaglucosidase alfa (20 mg/kg) plus oral miglustat or to intravenous alglucosidase alfa (20 mg/kg) plus oral placebo once every 2 weeks for 52 weeks. Patients, investigators, and outcome assessors were masked to treatment assignment. The primary endpoint was change from baseline to week 52 in 6-min walk distance, assessed using a mixed-effect model for repeated measures analysis for comparison of superiority in the intention-to-treat population (all patients who received at least one dose of study drug). This study is now complete and is registered with ClinicalTrials.gov, NCT03729362.Findings: Between Dec 3, 2018, and Nov 26, 2019, 130 patients were screened for eligibility and 125 were enrolled and randomly assigned to receive cipaglucosidase alfa plus miglustat (n=85) or alglucosidase alfa plus placebo (n=40). Two patients in the alglucosidase alfa plus placebo group did not receive any dose due to absence of genotype confirmation of late-onset Pompe disease and were excluded from analysis. Six patients discontinued (one in the alglucosidase alfa plus placebo group, five in the cipaglucosidase alfa plus miglustat group), and 117 completed the study. At week 52, mean change from baseline in 6-min walk distance was 20·8 m (SE 4·6) in the cipaglucosidase alfa plus miglustat group versus 7·2 m (6·6) in the alglucosidase alfa plus placebo group using last observation carried forward (between-group difference 13·6 m [95% CI -2·8 to 29·9]). 118 (96%) of 123 patients experienced at least one treatment-emergent adverse event during the study; the incidence was similar between the cipaglucosidase alfa plus miglustat group (n=81 [95%]) and the alglucosidase alfa plus placebo group (n=37 [97%]). The most frequently reported treatment-emergent adverse events were fall (25 [29%] patients in the cipaglucosidase alfa plus miglustat group vs 15 [39%] in the alglucosidase alfa plus placebo group), headache (20 [24%] vs 9 [24%]), nasopharyngitis (19 [22%] vs 3 [8%]), myalgia (14 [16%] vs 5 [13%]), and arthralgia (13 [15%]) vs 5 [13%]). 12 serious adverse events occurred in eight patients in the cipaglucosidase alfa plus miglustat group; only one event (anaphylaxis) was deemed related to study drug. One serious adverse event (stroke) occurred in the alglucosidase alfa plus placebo group, which was deemed unrelated to study drug. There were no deaths.Interpretation: Cipaglucosidase alfa plus miglustat did not achieve statistical superiority to alglucosidase alfa plus placebo for improving 6-min walk distance in our overall population of patients with late-onset Pompe disease. Further studies should investigate the longer-term safety and efficacy of cipaglucosidase alfa plus miglustat and whether this investigational two-component therapy might provide benefits, particularly in respiratory function and in patients who have been receiving enzyme replacement therapy for more than 2 years, as suggested by our secondary and subgroup analyses.Funding: Amicus Therapeutics. [ABSTRACT FROM AUTHOR]

The author reflects on the approval of a gene therapy orphan product by the U.S. Food and Drug Administration (FDA). He discusses the Food and Drug Administration Safety and Innovation Act (FDASIA) as well as the development of innovative medicines. He also tackles impact of the passage of the legislation on patients with rare and orphan diseases.

Highlights • Avalglucosidase alfa (neoGAA) had a well-tolerated safety profile in LOPD patients. • Respiratory and functional capacities were stable or improved in most patients. • Postinfusion, avalglucosidase alfa in plasma fell monoexponentially (t 1/2z ∼1.0 h). • AUC for avalglucosidase alfa in plasma was 5–6 × higher for the 20 vs 5 mg/kg group. • Safety/exploratory efficacy data support further avalglucosidase alfa development. Abstract This multicenter/multinational, open-label, ascending-dose study (NCT01898364) evaluated safety, tolerability, pharmacokinetics, pharmacodynamics, and exploratory efficacy of repeat-dose avalglucosidase alfa (neoGAA), a second-generation, recombinant acid α-glucosidase replacement therapy, in late-onset Pompe disease (LOPD). Patients ≥18 years, alglucosidase alfa naïve (Naïve) or previously receiving alglucosidase alfa for ≥9 months (Switch), with baseline FVC ≥50% predicted and independently ambulatory, received every-other-week avalglucosidase alfa 5, 10, or 20 mg/kg over 24 weeks. 9/10 Naïve and 12/14 Switch patients completed the study. Avalglucosidase alfa was well-tolerated; no deaths/life-threatening serious adverse events (SAEs). One Naïve patient withdrew for study drug-related SAEs (respiratory distress/chest discomfort). Infusion-associated reactions (IARs) affected 8 patients. Most treatment-emergent AEs/IARs were non-serious with mild-to-moderate intensity. At screening, 5 Switch patients tested positive for anti-avalglucosidase alfa antibodies; on-treatment, 2 Switch and 9 Naïve patients seroconverted. Post-infusion, avalglucosidase alfa plasma concentrations declined monoexponentially (t 1/2z ∼1.0 h). AUC was 5–6 × higher in the 20 vs 5 mg/kg group. Pharmacokinetics were similar between Switch and Naïve groups and over time. Baseline quadriceps muscle glycogen was low (∼6%) in most patients, generally remaining unchanged thereafter. Exploratory efficacy parameters (pulmonary function/functional capacity) generally remained stable or improved. Avalglucosidase alfa's well-tolerated safety profile and exploratory efficacy results support further avalglucosidase alfa development. [ABSTRACT FROM AUTHOR]

Objective: To evaluate whether B-cell depletion before enzyme replacement therapy (ERT) initiation can block acid alpha-glucosidase (GAA) antibody responses and improve clinical outcomes. Study design: Six subjects with Pompe disease (including 4 cross-reacting immunologic material–negative infants) aged 2-8 months received rituximab and sirolimus or mycophenolate before ERT. Four subjects continued to receive sirolimus, rituximab every 12 weeks, and intravenous immunoglobulin monthly for the duration of ERT. Sirolimus trough levels, IgG, CD3, CD4, CD8, CD19, CD20, N-terminal pro-brain natriuretic peptide, creatine kinase, creatine kinase-MB, C-reactive protein, platelets, alkaline phosphatase, gamma-glutamyl transferase, aspartate aminotransferase, and alanine aminotransferase were measured regularly. Results: Immunomodulation achieved B-cell depletion without adverse effects. After 17-36 months of rituximab, sirolimus and ERT, all subjects lacked antibodies against GAA, 4 continued to gain motor milestones, yet 2 progressed to require invasive ventilation. The absence of infusion-associated reactions allowed the use of accelerated infusion rates. Conclusion: B-cell depletion and T-cell immunomodulation in infants naïve to ERT was accomplished safely and eliminated immune responses against GAA, thereby optimizing clinical outcome; however, this approach did not necessarily influence sustained independent ventilation. Importantly, study outcomes support the initiation of immunomodulation before starting ERT, because the study regimen allowed for prompt initiation of treatment. [Copyright &y& Elsevier]

Regulation of Ca2+ flux in the heart via the blockade of the β-adrenergic receptor affects the phosphorylation state of phospholamban (PLN), a regulator of the sarcoplasmic reticulum calcium ATPase (SERCA2a). Reducing the activity of PLN enhances cardiac function in some animal models of congenital and ischemic cardiac disease. Objective: As an alternative to pharmacological regulation of PLN, the aim of this study is to use viral vectors to deliver short hairpin RNAs (shRNAs) targeting the PLN mRNA through the RNA interference pathway. We employed Adeno-associated virus serotype 1 (AAV1) and also a self-complementary version of AAV1 (scAAV1). ScAAV allows a more rapid expression of the gene of interest. The regulated expression of these PLN small interfering RNAs (siRNAs) could lead to a gene therapy for myocardial dysfunction. Methods: Two PLN-specific siRNAs, si248 and si750, and a control siRNA were designed. Co-transfection of the siRNAs in combination with a plasmid expressing PLN was performed in HEK 293 cells. Subsequently, DNA versions of small hairpins containing the three siRNAs were each cloned into AAV vectors. All vectors contained GFP in addition to one of the three siRNAs driven by the H1 promoter. Co-transfection experiments were performed using a 1:4 molar ratio of PLN:siRNA plasmids. RT-PCR was utilized for quantitation of PLN mRNA levels. Immunoblots were used to assay PLN protein levels. The siRNA plasmids were then packaged into AAV-1 capsids. These viruses were used for infection of primary ventricular neonatal cardiomyocytes. Infected myocytes were GFP positive and assessed for PLN mRNA and protein reduction. Additionally, temporal vein injections were performed into neonatal mice. Animals were assessed 4 weeks post-injection. Results: In co-transfected HEK 293 cells, si248 and si750 yielded a 55% and 33% reduction in PLN mRNA levels respectively, compared to the control siRNA. Protein levels were reduced by much higher levels (>85% for both). Similarly, a ∼40–47% reduction was seen in PLN mRNA levels 48 hours post-transfection using the PLN-targeting shRNAs expressed from the scAAV plasmids. Again, the reduction in protein levels was much higher, >80% for both. In the primary cells, levels of RNA and protein were reduced by 46% and 51% respectively after infection with scAAV virus. In addition, indirect immunofluorescence staining showed a marked reduction in PLN protein levels in cultured myocytes infected with the AAV-shRNA targeting PLN and not in cells infected with the control expressing shRNA virus. Furthermore, in vivo mRNA reduction of PLN was observed to be proportional to transduction levels in the mouse myocardium. Conclusions: AAV mediated delivery of shRNAs is a powerful tool for disrupting PLN expression. RNA interference mediated by both AAV and scAAV proved to be successful in producing targeted reduction of PLN mRNA and protein. Additional experiments are currently underway to assess the effects of in vivo RNA interference on PLN following cardiac transduction of neonatal mice.Molecular Therapy (2006) 13, S274–S274; doi: 10.1016/j.ymthe.2006.08.788 [ABSTRACT FROM AUTHOR]

The article presents the study on utilizing recombinant adeno-associated viral vector 6 (rAAV6) as potential therapy of a mouse model of familial ALS (fALS). According to the study, the rAAV serotype 6 was used to deliver small hairpin RNA (shRNA) to the muscle and motoneurons through retrograde transport to silence the expression of muSOD1 messenger RNA (mRNA) in a fALS mouse model. It also mentions that rAAV vectors are easily produce in titers.

Background A new post-myocardial infarction (MI) therapy is injection of high-water-content polymeric biomaterial gels (hydrogels) into damaged myocardium to modulate cardiac negative remodeling and preserve heart function. Methods We investigated the therapeutic potential of a novel gelatinized alginate hydrogel with a unique microstructure of uniform capillary-like channels (termed Capgel). Shortly (48 h) after induced anterior MI, Sprague Dawley rats received intramyocardial injection of Capgel directly into the antero-septal wall at the infarct border zone (n = 12) or no injection (n = 10, controls). Echocardiograms were performed at 48 h (week 0) and 4 weeks (week 4) to evaluate left ventricular function. Results Echocardiograms showed 27% improvement of left ventricular systolic function over time with gel injection: fractional shortening increased from 26 ± 3% at week 0 to 33 ± 2% at week 4 (p = 0.001). Capgel was present at the injection site after 4 weeks, but was minimal at 8 weeks. The remaining gel was heavily populated by CD68 + macrophages with CD206 + clusters and blood vessels. An in vitro experiment was performed to assess Angiotensin-(1–7) released from Capgel. Angiotensin-(1–7) was released from the Capgel in a sustained manner for 90 days. Conclusions Use of Capgel, a degradable, bioactive hydrogel composed of gelatinized capillary-alginate gel, appears safe for intramyocardial injection, is associated with improved left ventricular function after MI in rats, and may provide a long-term supply of Angiotensin-(1–7). [ABSTRACT FROM AUTHOR]

Respiratory and/or lingual dysfunction are among the first motor symptoms in Pompe disease, a disorder resulting from absence or dysfunction of the lysosomal enzyme acid α-glucosidase (GAA). Here, we histologically evaluated the medulla, cervical and thoracic spinal cords in 6 weeks old asymptomatic Pompe ( Gaa −/− ) mice to determine if neuropathology in respiratory motor regions has an early onset. Periodic acid-Schiff (PAS) staining indicated glycogen accumulation was exclusively occurring in Gaa −/− hypoglossal, mid-cervical and upper thoracic motoneurons. Markers of DNA damage (Tunel) and ongoing apoptosis (Cleaved Caspase 3) did not co-localize with PAS staining, but were prominent in a medullary region which included the nucleus tractus solitarius, and also in the thoracic spinal dorsal horn. We conclude that respiratory-related motoneurons are particularly susceptible to GAA deficiency and that neuronal glycogen accumulation and neurodegeneration may occur independently in early stage disease. The data support early therapeutic intervention in Pompe disease. [ABSTRACT FROM AUTHOR]

Pompe disease is an inherited neuromuscular disorder that affects respiratory function and leads to dependence on external ventilatory support. We studied the activation of the diaphragm using bilateral phrenic magnetic stimulation and hypothesized that diaphragm compound muscle action potential (CMAP) amplitude and evoked transdiaphragmatic pressure (Twitch P DI ) would correlate to disease severity. Eight patients with late onset Pompe disease (LOPD, aged 14–48 years) and four healthy control subjects completed the tests. Maximal Twitch P DI responses were progressively reduced in patients with LOPD compared to control subjects (1.4–17.1 cm H 2 O, p < 0.001) and correlated to voluntary functional tests ( p < 0.05). Additionally, CMAP amplitude (mA) was lower in the patients who used nighttime or fulltime ventilatory support, when compared to controls and patients who used no ventilatory support ( p < 0.005). However, the normalized (%peak) Twitch P DI and CMAP responses were similar between patients and controls. This suggests a loss of functional phrenic motor units in patients, with normal recruitment of remaining motor units. [ABSTRACT FROM AUTHOR]

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (DdcKI mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old DdcKI mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment. [ABSTRACT FROM AUTHOR]

The article presents the proceedings of the ASGCT Standardized Pathways Conference that was held in Silver Spring, Maryland, on February 20, 2014. It is mentioned that the premise of the conference was that the preclinical data and clinical outcomes from development and conduct of gene therapy clinical trials can be used to establish standardized pathways through the preclinical regulatory process.

Pompe disease is an autosomal recessive disorder caused by mutations in the acid-α glucosidase (GAA) gene. Lingual dysfunction is prominent but does not respond to conventional enzyme replacement therapy (ERT). Using Pompe (Gaa−/−) mice, we tested the hypothesis that intralingual delivery of viral vectors encoding GAA results in GAA expression and glycogen clearance in both tongue myofibers and hypoglossal (XII) motoneurons. An intralingual injection of an adeno-associated virus (AAV) vector encoding GAA (serotypes 1 or 9; 1 × 1011 vector genomes, CMV promoter) was performed in 2-month-old Gaa−/− mice, and tissues were harvested 4 months later. Both serotypes robustly transduced tongue myofibers with histological confirmation of GAA expression (immunochemistry) and glycogen clearance (Period acid-Schiff stain). Both vectors also led to medullary transgene expression. GAA-positive motoneurons did not show the histopathologic features which are typical in Pompe disease and animal models. Intralingual injection with the AAV9 vector resulted in approximately threefold more GAA-positive XII motoneurons (P < 0.02 versus AAV1); the AAV9 group also gained more body weight over the course of the study (P < 0.05 versus AAV1 and sham). We conclude that intralingual injection of AAV1 or AAV9 drives persistent GAA expression in tongue myofibers and motoneurons, but AAV9 may more effectively target motoneurons. [ABSTRACT FROM AUTHOR]

Highlights: [•] Pompe disease occurs due to mutations in the gene encoding the lysosomal enzyme acid α-glucosidase (GAA). [•] Respiratory insufficiency and tongue motor problems are common. [•] Enzyme replacement therapy targets skeletal muscle but many patients still require ventilatory assistance. [•] Emerging evidence indicates that respiratory neuron pathology contributes to motor unit dysfunction. [•] Restoration of GAA activity in respiratory muscles and neurons may be required to correct ventilatory insufficiency. [Copyright &y& Elsevier]

Pompe disease is a neuromuscular disease resulting from deficiency in acid α-glucosidase (GAA), results in cardiac, skeletal muscle, and central nervous system (CNS) pathology. Enzyme replacement therapy (ERT) has been shown to partially correct cardiac and skeletal muscle dysfunction. However, ERT does not cross the blood-brain barrier and progressive CNS pathology ensues. We tested the hypothesis that intrapleural administration of recombinant adeno-associated virus (rAAV9)-GAA driven by a cytomegalovirus (CMV) or desmin (DES) promoter would improve cardiac and respiratory function in Gaa−/− mice through a direct effect and retrograde transport to motoneurons. Cardiac magnetic resonance imaging revealed significant improvement in ejection fraction in rAAV9-GAA-treated animals. Inspiratory phrenic and diaphragm activity was examined at baseline and during hypercapnic respiratory challenge. Mice treated with AAV9 had greater relative inspiratory burst amplitude during baseline conditions when compared with Gaa−/−. In addition, efferent phrenic burst amplitude was significantly correlated with diaphragm activity in both AAV9-DES and AAV9-CMV groups but not in Gaa−/−. This is the first study to indicate improvements in cardiac, skeletal muscle, and respiratory neural output following rAAV administration in Pompe disease. These results further implicate a role for the CNS in Pompe disease pathology and the critical need to target the neurologic aspects in developing therapeutic strategies.Molecular Therapy (2013); 21 9, 1661-1667. doi:10.1038/mt.2013.96 [ABSTRACT FROM AUTHOR]

We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients. [ABSTRACT FROM AUTHOR]

Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid α-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa−/− mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C3-C4 spinal level of adult Gaa−/− mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa−/− mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa−/− mice and therefore requires further detailed evaluation in patients with Pompe disease. [ABSTRACT FROM AUTHOR]

Pompe disease is a muscular dystrophy that results in respiratory insufficiency. We characterized the outcomes of targeted delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragms of Pompe mice with varying stages of disease progression. We observed significant improvement in diaphragm contractile strength in mice treated at 3 months of age that is sustained at least for 1 year and enhanced contractile strength in mice treated at 9 and 21 months of age, measured 3 months post-treatment. Ventilatory parameters including tidal volume/inspiratory time ratio, minute ventilation/expired CO2 ratio, and peak inspiratory airflow were significantly improved in mice treated at 3 months and tested at 6 months. Despite early improvement, mice treated at 3 months and tested at 1 year had diminished normoxic ventilation, potentially due to attenuation of correction over time or progressive degeneration of nontargeted accessory tissues. However, for all rAAV2/1-treated mice (treated at 3, 9, and 21 months, assayed 3 months later; treated at 3 months, assayed at 1 year), minute ventilation and peak inspiratory flows were significantly improved during respiratory challenge. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors can significantly augment ventilatory function at initial and late phases of disease in a model of muscular dystrophy. [ABSTRACT FROM AUTHOR]

An abstract of the article "Effect of DNA-PKcs on AAV Replication," by Young-Kook Choi, Irene Zolotukhin, Barry J. Byrne and Sihong Song is presented.

Limb girdle muscular dystrophy (LGMD) describes a group of inherited diseases resulting from mutations in genes encoding proteins involved in maintaining skeletal muscle membrane stability. LGMD type-2D is caused by mutations in alpha-sarcoglycan (sgca). Here we describe muscle-specific gene delivery of the human sgca gene into dystrophic muscle using an adeno-associated virus 1 (AAV1) capsid and creatine kinase promoter. Delivery of this construct to adult sgca–/– mice resulted in localization of the sarcoglycan complex to the sarcolemma and a reduction in muscle fiber damage. Sgca expression prevented disease progression as observed in vivo by T2-weighted magnetic resonance imaging (MRI) and confirmed in vitro by decreased Evan's blue dye accumulation. The ability of recombinant AAV–mediated gene delivery to restore normal muscle mechanical properties in sgca–/– mice was verified by in vitro force mechanics on isolated extensor digitorum longus (EDL) muscles, with a decrease in passive resistance to stretch as compared with untreated controls. In summary, AAV/AAV-sgca gene transfer provides long-term muscle protection from LGMD and can be non-invasively evaluated using magnetic resonance imaging.Molecular Therapy (2007) 15 10, 1775–1781. doi:10.1038/sj.mt.6300246 [ABSTRACT FROM AUTHOR]

Pompe disease is caused by a lack of functional lysosomal acid α-glucosidase (GAA) and can ultimately lead to fatal hypertrophic cardiomyopathy and respiratory insufficiency. Previously, we demonstrated the ability of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to restore the therapeutic levels of cardiac and diaphragmatic GAA enzymatic activity in vivo in a mouse model of Pompe disease. We have further characterized cardiac and respiratory function in rAAV2/1-treated animals 1 year post-treatment. Similar to the patient population, electrocardiogram measurements (P-R interval) are significantly shortened in the Pompe mouse model. In rAAV2/1-treated mice, we show a significant improvement in cardiac conductance with prolonged P-R intervals of 39.34±1.6 ms, as compared to untreated controls (35.58±0.57 ms) (P0.05). In addition, we note a significant decrease in cardiac left ventricular mass from 181.99±10.70 mg in untreated controls to 141.97±19.15 mg in the rAAV2/1-treated mice. Furthermore, the mice displayed an increased diaphragmatic contractile force of approximately 90% of wild-type peak forces with corresponding improved ventilation (particularly in frequency, minute ventilation, and peak inspiratory flow). These results demonstrate that in addition to biochemical and histological correction, rAAV2/1 vectors can mediate sustained physiological correction of both cardiac and respiratory function in a model of fatal cardiomyopathy and muscular dystrophy.Molecular Therapy (2007) 15, 501–507. doi:10.1038/sj.mt.6300100; published online 23 January 2007 [ABSTRACT FROM AUTHOR]

Abstract: Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935–1943) found limited applications due to the system''s design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a “riboswitch,” a self-cleaving ribozyme controlled by toyocamycin in the “ON” mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes. [Copyright &y& Elsevier]

Abstract: Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications. [Copyright &y& Elsevier]

Genetically modified mice are important models for evaluation of potential gene therapies for human diseases. However, their small size often precludes the use of clinically feasible methods for vector delivery, therefore, alternative methods must be used. We have developed a gel-based method for delivery of recombinant adeno-associated virus vectors to the mouse diaphragm, an important target organ for many myopathic diseases. We hypothesized that delivery of vectors in a viscous solution would increase transduction by providing a longer exposure time to target cells. We demonstrate that gel-mediated delivery of rAAV serotypes 1, 2, and 5 results in higher transduction efficiencies than free vectors alone when administered in vivo to mouse diaphragms. We further establish greater tropism of rAAV1 vectors for the diaphragm compared to serotypes 2 and 5. This report describes a novel method for efficient delivery of rAAV vectors to the mouse diaphragm and is the first demonstration of gene transfer to the diaphragm using recombinant adeno-associated virus vectors. [Copyright &y& Elsevier]

A major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. We have developed a novel method of recombinant adeno-associated virus 2 (rAAV) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to rAAV vectors. We hypothesize that conjugation to microspheres should result in a higher effective concentration of vector as well as longer relative exposure time of vector to target cells as it moves through the tissue vasculature. In vitro experiments demonstrate that the same level of transduction seen with free vector can be achieved using 1% of vector when conjugated to microspheres. In addition, using magnetic microspheres, the region of infection can be targeted. In vivo, we demonstrate that microsphere-mediated delivery of rAAV vector results in higher transduction efficiencies than delivery with free vector alone when administered either intramuscularly or intravenously. Furthermore, we demonstrate targeting of transgene expression to specific tissues by retention of microsphere-bound vector in the capillary bed. These studies demonstrate a novel method to deliver rAAV vectors more effectively that could prove to be a successful alternative mode of virus-mediated human gene therapy. [Copyright &y& Elsevier]

Pompe disease is a lysosomal storage disease caused by the absence of acid α-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa−/− mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa−/− mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa−/− mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa−/− mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy. [Copyright &y& Elsevier]

A letter to the editor which stresses the need for an international gene therapy consortium for monogenic diseases is presented.

An abstract of the article "Sustained Correction of Glycogen Storage Disease Type II by rAAV1 Vector-Mediated Gene Therapy," by Kerry O. Cresawn and colleagues is presented.

An abstract of the article "Correction of Respiratory Function by Recombinant AAV1 Mediated Gene Therapy in a Murine Model of Glycogen Storage Disease Type II," by Cathryn Mah, Kerry O. Cresawn, Lara R. DeRuisseau, David Fuller, and Barry J. Byrne is presented.

An abstract of the article "Development of AAV-Mediated Gene Therapy for Murine Models of Genetic Diseases Affecting the Heart," by Christina A. Pacak, Cathryn Mah, Gabriel Gaidosh, Melissa Lewis, Raquel Torres, Kevin Campbell, Glenn A. Walter and Barry J. Byrne is presented.

Pompe disease is a lysosomal storage disorder resulting from deficiency of the enzyme acid alpha glucosidase (GAA). The lack of GAA is associated with glycogen accumulation in all cells. Skeletal muscle is particularly affected and diaphragm dysfunction is often life-threatening and thus necessitates mechanical ventilation. Accordingly, the first purpose of this study was to determine if breathing is affected in a mouse model of Pompe disease (the Gaa−/− knockout mouse). Using barometric plethysmography, minute ventilation (MV; mL/min), breathing frequency (F; breaths/min) and tidal volume (TV; mL/min) were measured in conscious control and Gaa−/− mice. During quiet breathing (inspired O2 = 21%; balanced N2), MV (control vs. Gaa−/−: 60 ± 6 vs. 43 ± 4), F (234 ± 11 vs. 195 ± 8), and TV (0.26 ± 0.01 vs. 0.22 ± 0.02) were attenuated in Gaa−/− mice (p<0.01). Ventilation deficits in Pompe disease patients have been attributed to respiratory muscle weakness, however, several case reports (from autopsy) suggest that the central nervous system is also affected. Therefore, we tested the hypothesis that the neural control of the respiratory muscles is impaired in Gaa−/− mice. Initially, we quantified glycogen (μg/mg wet wt) in the spinal cord of control (14.3 ± 1.8) and Gaa−/− (30.0 ± 5.0, p<0.01) mice to verify that glycogen accumulation was present in this model of Pompe disease. To directly test that a neural deficit was contributing to ventilation impairments, phrenic motor output (the final output to the diaphragm) was quantified in Gaa−/− (5.2 ± 1.2 mV) and control (49.7 ± 13.9 mV, p<0.01) mice. Our data suggest that ventilatory deficits in Pompe disease may reflect both muscular and neural mechanisms. Importantly, therapies aimed to correct breathing deficits in Pompe disease should target both the diaphragm and central nervous system. For this reason, we have initiated experiments to identify the ability of AAV serotype 1 to transduce both the diaphragm and the phrenic motoneuron pool (cervical segments C3–C5) after a single injection. AAV1-CMV-LacZ was administered via an intrathoracic injection (4.0 × 1011 particles); spinal cord (C3–C5) and diaphragm were harvested 4 weeks post injection. PCR was performed on isolated DNA using primers specific to the LacZ gene. Mice injected with AAV1-CMV-LacZ were positive for LacZ compared to negative controls in both tissues. The protein product of LacZ (betagalactosidase; rlu/mg protein above sham injected control tissue) was present in both the diaphragm (18914 ± 9346) and cervical spinal segments C3–C5 (2380 ± 792). We conclude that AAV1-CMV-LacZ is transported to the spinal cord after an intrathoracic injection. Ongoing experiments will identify if AAV1 is co-localized with retrogradely labled phrenic motoneurons. These studies contribute to our long-term goal of developing a clinically relevant method for transgene delivery to both the diaphragm and phrenic motoneuron pool for correction of respiratory insufficiency in Pompe disease.Molecular Therapy (2006) 13, S344–S344; doi: 10.1016/j.ymthe.2006.08.981 [ABSTRACT FROM AUTHOR]

The long term goal of this project is to develop a clinically relevant gene therapy approach for the treatment of Pompe Disease. Pompe Disease is a form of muscular dystrophy and metabolic myopathy caused by mutations in the acid alpha glucosidase (GAA) gene. An insufficient amount of GAA leads to the accumulation of glycogen in lysosomes and consequent cellular dysfunction. Cardio-respiratory failure typically occurs in the early onset patients within the first year of life. We have characterized the cardiac phenotype in our mouse model (gaa-/-) at various ages. Through ECG analysis we observe a shortened PR interval by 3 months of age (gaa-/-33.41±1.35ms, cont 44.95±1.58ms) mimicking the conduction phenotype in the human Pompe population. Abnormal amounts of glycogen are observed in lysosomes as demonstrated by the periodic acid shift (PAS) stain. MRI analysis shows a decrease in stroke volume (SV)(gaa-/-36.13±1.19ul, cont 51.84±3.59ul) and a decrease in cardiac output (CO)(gaa-/-7.95±0.26ml/min, cont 11.40±0.79ml/ min) at 3 months and an increase in mass (gaa-/-181.99±10.7mg, cont 140.79±5.12mg) by 12 months. This model of cardiac dysfunction is being used to develop a cardiac gene delivery technique which can be applied to many genetically inherited cardiomyopathies. Previously, we have shown that intra-venous (IV) delivery of recombinant adeno-associated virus type 1 (rAAV2/1) pseudotype capsid carrying the CMV-hgaa construct to 1 day old gaa-/- neonates restores GAA activity in mice. Also, LacZ transgene delivery using the IV administration route and rAAV2/9 pseudotype capsid resulted in 200 fold higher levels of expression in cardiac tissue than rAAV2/1. Additional experiments showed transduction following delivery to adults. We have now combined rAAV2/9 with the clinically relevant IV administration route in order to deliver the human GAA (hgaa) gene to gaa-/- mice. Neonates treated with rAAV2/9-CMV-hgaa at a range of doses (4×105vg 4×108vg 4×1010vg) have demonstrated sustained PR interval correction (39.38±2.42 ms). PAS stains as well as NMR analysis have shown less glycogen accumulation in cardiac tissue of treated gaa-/- mice as compared to untreated mice. MRI analysis shows an increase in SV and CO. Adult gaa-/- mice have also been treated using this strategy and are being assessed in order to determine if we have the ability to reverse the effects of Pompe Disease in mice already presenting the cardiac phenotype. While the focus of this project is on correction of the cardiac phenotype of Pompe Disease, our systemic delivery route, use of the CMV promoter and the fact that GAA is a secreted enzyme all promote correction throughout the body. GAA activity is observed in other tissues of treated mice including skeletal muscles and liver. These studies have demonstrated the ability of rAAV2/9 to be administered systemically using the IV delivery route, transcend the vasculature, transduce tissues throughout the body and ultimately prevent presentation of the cardiac phenotypes of Pompe Disease.Molecular Therapy (2006) 13, S12–S12; doi: 10.1016/j.ymthe.2006.08.040 [ABSTRACT FROM AUTHOR]

Glycogen storage disease type II (GSDII) is caused by a lack of functional lysosomal acid α-glucosidase (GAA) and results in massive storage of glycogen in lysosomal compartments, ultimately leading to fatal severe hypertrophic cardiomyopathy and respiratory insufficiency. Previously, we demonstrated the ability of a single intravenous administration of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to restore therapeutic levels of cardiac and diaphragmatic GAA enzymatic activity with concomitant clearance of glycogen in vivo in a mouse model of GSDII (Gaa-/-). We have further characterized both cardiac and respiratory function in rAAV2/1-treated animals one year post-treatment. Similar to the GSDII patient population, electrocardiogram (ECG) measurements (P-R interval) are significantly shortened in the mouse model. In rAAV2/1-treated mice, we show a significant improvement in cardiac conductance with prolonged P-R intervals of 39.34 ± 1.6 ms, as compared to untreated controls (35.58 ± 0.57 ms) (p≤0.05). In addition, using cardiac magnetic resonance imaging (MRI) we note a marked decrease in cardiac left ventricular mass from 181.99 ± 10.70 mg in untreated age-matched controls to 141.97 ± 19.15 mg in the rAAV2/1-treated mice. Furthermore the mice displayed increased diaphragmatic contractile force to approximately 90% of wild-type peak forces with corresponding significantly improved ventilation (particularly in frequency, minute ventilation, and peak inspiratory flow), as measured using barometric whole body plethysmography. These results demonstrate that in addition to biochemical and histological correction, rAAV2/1 vectors can mediate sustained physiological correction of both cardiac and respiratory function in a model of fatal cardiomyopathy and muscular dystrophy.Molecular Therapy (2006) 13, S158–S158; doi: 10.1016/j.ymthe.2006.08.473 [ABSTRACT FROM AUTHOR]

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder caused by a lack of functional acid α-glucosidase (GAA) and results in massive storage of glycogen in lysosomal compartments. It is characterized by progressive skeletal muscle weakness and affected patients suffer severe respiratory insufficiency, oftentimes requiring mechanical ventilation. Previously, we demonstrated that a novel gel-mediated method of delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragm in a mouse model of GSDII (Gaa-/-) could restore therapeutic levels of diaphragmatic GAA enzymatic activity with concomitant clearance of glycogen in vivo. We have further characterized the effects of this treatment method on ventilatory function. Similar to the GSDII patient population, we demonstrate in Gaa-/- mice that there is an age-related progressive weakening of diaphragm contractile strength that is accompanied by ventilation impairments. We administered 1x1011 particles rAAV2/1-CMV-GAA to diaphragms of Gaa-/- mice at 3, 9, and 21-mos of age via the gel method. Diaphragm contractile strength and respiratory function were assessed by measuring force-frequency relationships and by barometric whole-body plethysmography, respectively. For plethysmography, ventilation was measured in conscious, unrestrained mice under conditions of normoxia (FIO2:0.21, FICO2: 0.00) and hypercapnia (FIO2: 0.21, FICO2: 0.07) to assess the extended range of ventilatory capacity. In mice treated at 3 mos of age, we show a significant improvement in contractile force at 6 mos (peak force of 24.833.31 vs 16.53±0.74 N/cm2) that is sustained through 1 yr (21.59±1.59 vs 13.94±1.15 N/cm2) of age as compared to age-matched untreated controls. Under conditions of normoxia, the ratio of minute ventilation (VE; mL/min) to expired CO2 (VE/ VCO2) (18.65±0.73 vs 13.3±0.74), and peak inspiratory flow (mL/sec) (4.11±0.17 vs 3.21±0.29) were significantly improved (p≤0.05) in mice treated at 3 mos of age and tested at 6 mos as compared tountreated controls. However, these improvements did not persist to 1 yr of age. In mice treated at 9 and 21 mos of age, there was significant improvement in contractile function in treated diaphragms 3 mos post-treatment (peak force of 21.28±1.49 at 1 yr and 17.21±0.29 N/cm2 vs 12.71±0.94 N/cm2 at 2 yrs of age, respectively) as compared to age-matched untreated controls. In all rAAV2/1- treated mice, minute ventilation and peak inspiratory flows were significantly improved during hypercapnia. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors improve ventilatory function in a mouse model of muscular dystrophy.Molecular Therapy (2006) 13, S221–S222; doi: 10.1016/j.ymthe.2006.08.648 [ABSTRACT FROM AUTHOR]

INTRODUCTION: Recombinant adeno-associated virus (rAAV) vectors are capable of sustained expression of therapeutic proteins when administered to skeletal muscle. A single injection of a rAAV2 vector in mice resulted in sustained secretion of human alpha-1 antitrypsin (hAAT) at levels that would be therapeutic for prevention of lung disease in humans (11,000 nM). There is currently no definitive therapy for these patients, since protein replacement products are in short supply and require weekly intravenous infusions. Recently, we completed the ongoing preclinical testing (mouse, rabbit, and primate) of the rAAV2-hAAT vector in muscle, completed clinical grade production of rAAV2-hAAT, and initiated a phase I trial of rAAV-hAAT gene therapy in patients with AAT deficiency. MEHODS: The trial is an open-labeled, single administration study being performed in a total of 15 adult AAT-deficient patients. Five cohorts of three subjects each are being used with a dose escalation between cohorts. The dosages for the four cohorts range from 2 × 1012 vector genomes (vg) per patient to 2 × 1014 vg/patient. Subjects are screened and allowed a 28-day period for washout of protein replacement. An ultrasound-guided injection technique is used to avoid intravascular injection. The study endpoints address safety with examinations of the injection site, hematology, chemistry, coagulation studies, and pulmonary function tests. Biological activity of the vector is assessed by detection of normal M-variant hAAT protein by ELISA. Serum is also assayed for antibodies and lymphocyte proliferation responses to rAAV2 capsid and to AAT. Blood and semen DNA are also being assayed for vector DNA sequences. RESULTS: At the time of submission of this abstract, twelve subjects have been treated, and there have been no vector-related adverse events to date. All patients have demonstrated increases in anti-AAV2 capsid protein titers by ELISA, but none have shown increases in anti-hAAT antibody titers or in lymphocyte proliferation responses. Patients in the cohorts 2–4 show PCR evidence of vector DNA in the blood within the first 14 days, but without obvious ill effects. At least one subject in cohorts 3 shows evidence of M-AAT expression in the range of 50–100 nm. CONCLUSIONS: Preliminary evidence of the safety and bioactivity of rAAV2-hAAT are being generated. These support the feasibility of future clinical studies, including one recently begun with rAAV1, a serotype that shows a 500–1000-fold greater bioactivity than the current rAAV2 vector in mice.Molecular Therapy (2006) 13, S281–S281; doi: 10.1016/j.ymthe.2006.08.808 [ABSTRACT FROM AUTHOR]

An abstract of the article "Gene Therapy in a Rat Model of Pulmonary Hypertension," by Stacy L. Porvasnik and colleagues is presented.

An abstract of the article "Phase I Clinical Trial of Recombinant Adeno-Associated Virus (rAAV)-Alpha-1 Antitrypsin Vectors," by Mark L. Brantly and colleagues is presented.

An abstract of the article "Recombinant AAV1-Mediated Treatment of CNS Glycogen Accumulation in Mouse Model of Glycogen Storage Disease Type II," by Lara R. DeRuisseau, Cathryn S. Mah, Thomas J. Fraites, Jr., David D. Fuller and Barry J. Byrne is presented.

An abstract of the article "Comparison of rAAV Serotype, Promoter, and Treatment Age for the Correction of Glycogen Storage Disease Type II," by Kerry O. Cresawn and colleagues is presented.