Evaluation of life cycle defective adenovirus mutants for production of adeno-associated virus vectors.

BACKGROUND: Adeno-associated virus-based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. METHODS: In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome-containing particles using a real-time polymerase chain reaction and functional units were investigated by transduction assays. RESULTS: Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication-deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild-type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. CONCLUSIONS: The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild-type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.

The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo.

The combination of exercise and respiratory training improves respiratory muscle function in pulmonary hypertension.

PURPOSE: Increased dyspnea and reduced exercise capacity in pulmonary arterial hypertension (PAH) can be partly attributed to impaired respiratory muscle function. This prospective study was designed to assess the impact of exercise and respiratory training on respiratory muscle strength and 6-min walking distance (6MWD) in PAH patients. METHODS: Patients with invasively confirmed PAH underwent 3 weeks of in-hospital exercise and respiratory training, which was continued at home for another 12 weeks. Medication remained constant during the study period. Blinded observers assessed efficacy parameters at baseline (I) and after 3 (II) and 15 weeks (III). Respiratory muscle function was assessed by twitch mouth pressure (TwPmo) during nonvolitional supramaximal magnetic phrenic nerve stimulation. RESULTS: Seven PAH patients (4 women; mean pulmonary artery pressure 45 ± 11 mmHg, median WHO functional class 3.1 ± 0.4, idiopathic/associated PAH n = 5/2) were included. The training program was feasible and well tolerated by all patients with excellent compliance. TwPmo was I: 0.86 ± 0.37 kPa, II: 1.04 ± 0.29 kPa, and III: 1.27 ± 0.44 kPa, respectively. 6MWD was I: 417 ± 51 m, II: 509 ± 39 m, and III: 498 ± 39 m, respectively. Both TwPmo (+0.41 ± 0.34 kPa, +56 ± 39 %) and 6MWD (+81 ± 30 m, +20 ± 9 %) increased significantly in the period between baseline and the final assessment (pairwise comparison: p = 0.012/<0.001; RM-ANOVA considering I, II, III: p = 0.037/<0.001). CONCLUSIONS: Exercise and respiratory training as an adjunct to medical therapy may be effective in patients with PAH to improve respiratory muscle strength and exercise capacity. Future, randomized, controlled trials should be carried out to further investigate these findings.

Properties of the adeno-associated virus assembly-activating protein.

Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.

Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly.

Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity.

A viral assembly factor promotes AAV2 capsid formation in the nucleolus.

The volume available in icosahedral virus capsids limits the size of viral genomes. To overcome this limitation, viruses have evolved strategies to increase their coding capacity by using more than one ORF while keeping the genome length constant. The assembly of virus capsids requires the coordinated interaction of a large number of subunits to generate a highly ordered structure in which the viral genome can be enclosed. To understand this process, it is essential to know which viral and nonviral components are involved in the assembly reaction. Here, we show that the adeno-associated virus (AAV) encodes a protein required for capsid formation by means of a nested, alternative ORF of the cap gene. Translation is initiated at a nonconventional translation start site, resulting in the expression of a protein with a calculated molecular weight of 23 kDa. This protein, designated assembly-activating protein (AAP), is localized in the host cell nucleolus, where AAV capsid morphogenesis occurs. AAP targets newly synthesized capsid proteins to this organelle and in addition fulfils a function in the assembly reaction itself. Sequence analysis suggests that also all other species of the genus Dependovirus encode a homologous protein in their cap gene. The arrangement of different ORFs that encode capsid proteins and an assembly factor within the same mRNA facilitates a timely coordinated expression of the components involved in the assembly process.

Diaphragmatic fatigue is counterbalanced during exhaustive long-term exercise.

Based on externally paced (repetitive) short-term trials exercise-induced diaphragmatic fatigue has been shown to manifest after rather than during exercise. The current study aimed at investigating diaphragmatic contractility and diaphragmatic fatigue during self-paced long-term exhaustive exercise at maximally tolerated loading by the use of supramaximal twitch transdiaphragmatic pressure (TwPdi). Seven trained subjects (VO(2max) 63.3+/-13.9 ml kg(-1) min(-1)) performed self-paced long-term exhaustive exercise at maximally tolerated loading (45 min+endspurt, initial workload 85% VO(2max)) followed by recovery (9 min). TwPdi (every 45 s) and ergospirometric data (continuously) were assessed throughout the protocol. Diaphragmatic contractility tended to initially increase during the exercise protocol with a slight decline and final increase during endspurt. Diaphragmatic fatigue manifested only after exercise termination (TwPdi rest 2.6+/-0.8 kPa; TwPdi exercise start/mid/end 2.9+/-0.7 kPa vs. 2.6+/-0.8 kPa vs. 2.4+/-0.6 kPa; TwPdi endspurt/recovery 2.7+/-0.8 kPa vs. 1.9+/-0.6 kPa). In conclusion, diaphragmatic contractility tends to decrease but manifestation of diaphragmatic fatigue is counterbalanced during self-paced long-term exhaustive exercise at maximally tolerated loading.

Post-exercise diaphragm shielding: a novel approach to exercise-induced diaphragmatic fatigue.

Based on the "post-exercise diaphragm shielding" hypothesis this study tested whether both diaphragmatic force-generation (DFG) and diaphragmatic fatigue (DF) remain unchanged during consecutive exercise-trials. Twelve subjects (V(O2 max) 58.4+/-6.6 ml kg(-1) min(-1)) performed three consecutive exercise-trials (T(alpha)/T(beta)/T(gamma); workload(max) 85% V(O2 max)) each followed by recovery (6 min). Twitch transdiaphragmatic pressure during supramaximal magnetic phrenic nerve stimulation (TwPdi, every 30s), ratings of perceived exertion (RPE, every 90 s) and ergospirometric data (continuously) were assessed throughout the entire protocol (46.5 min). DFG and DF did not differ among all trials (TwPdi-baseline: 2.2+/-0.7 kPa; TwPdi-peak: T(alpha)/T(beta)/T(gamma) 3.1+/-0.7 kPa vs 3.0+/-0.8 kPa vs 3.2+/-0.8 kPa; TwPdi-bottom: T(alpha)/T(beta)/T(gamma) 1.9+/-0.6 kPa vs 2.0+/-0.7 kPa vs 1.8+/-0.5 kPa, both p>0.4, RM-ANOVA). Furthermore, TwPdi revealed close relationships with RPE (r=0.91, p<0.0001) and oxygen uptake (r=0.94, p<0.0001) during exercise. In conclusion, both DFG (baseline-to-peak) and DF (baseline-to-bottom) achieve similar magnitudes during and after consecutive exercise-trials and are closely linked to RPE and oxygen uptake. This suggests that DF neither reflects impaired diaphragmatic function nor impairs exercise performance; rather it is likely to reflect post-exercise diaphragm shielding.

Independence of exercise-induced diaphragmatic fatigue from ventilatory demands.

Exercise-induced diaphragmatic fatigue (DF) manifests after - rather than during - exercise. This suggests that DF reflects post-exercise diaphragm-shielding. This study tested the physiological hypothesis that diaphragmatic force-generation undergoes similar regulations during either whole-body-exercise or controlled hyperventilation, but differs during recovery. Ten trained subjects (VO2(max) 60.3+/-6.4 ml/kg/min) performed: I, cycling exercise (maximal workload: 85% VO2(max)); II, controlled hyperventilation (exercise breathing pattern) followed by recovery. Ergospirometric data and twitch transdiaphragmatic pressure (TwPdi) were consecutively assessed. DF occurred following exercise, while hyperventilation enhanced diaphragmatic force-generation (TwPdi-rest 2.28+/-0.58 vs. 2.52+/-0.54, TwPdi-end-recovery: 1.94+/-0.32 kPa vs. 2.81+/-0.49 kPa, both p<0.05). TwPdi was comparable between the two protocols until recovery (p>0.05, RM-ANOVA) whereby it underwent a progressive increase. In conclusion, TwPdi progressively increases and is subject to similar regulations during exercise versus controlled hyperventilation, but differs markedly during recovery. Here, DF occurred after exercise while TwPdi increased subsequent to hyperventilation. Therefore, ventilatory demands regulate diaphragmatic force-generation during exercise, whereas DF must be attributed to non-ventilatory controlled feedback mechanisms.

Non-invasive ventilation applied for recovery from exercise-induced diaphragmatic fatigue.

BACKGROUND: Exercise-induced diaphragmatic fatigue (DF) is conventionally considered to reflect impaired diaphragm function resulting from load imposed on the diaphragm during exercise and is known to be reduced by the application of non-invasive ventilation (NIV) during heavy-intensity exercise testing (HEET). On that physiological condition NIV applied for diaphragm unloading during recovery from exercise should be capable of accelerating recovery from DF and therewith prolonging exercise time to exhaustion and limiting the development of DF during a subsequent HEET compared to recovery during spontaneous breathing. METHODS: Seven highly-trained subjects (V'O(2max) 62.7±7.8 ml/kg/min) performed four HEET at 85% V'O(2max) with 60 min of recovery during I spontaneous breathing and II NIV between two HEET. RESULTS: Twitch transdiaphragmatic pressure (TwPdi) during supramaximal magnetic phrenic nerve stimulation decreased (p<0.04) following first HEET and subsequently completely recovered (p>0.2) during I and II. Following second HEET TwPdi comparably decreased (I 0.24±0.21 vs II 0.32±0.29 kPa; p=0.17). Exercise time to exhaustion during second HEET was equal during I and II (I 514±49 vs II 511±92 s; p=0.88). CONCLUSIONS: In conclusion, NIV applied for diaphragm unloading during recovery following HEET does neither affect recovery from DF nor subsequent exercise performance thereby providing further evidence that DF might reflect post-exercise diaphragm shielding rather than impaired diaphragm function.

Impairment of respiratory muscle function in pulmonary hypertension.

It has been suggested that impaired respiratory muscle function occurs in patients with PH (pulmonary hypertension); however, comprehensive investigations of respiratory muscle function, including the application of non-volitional tests, needed to verify impairment of respiratory muscle strength in patients with PH have not yet been performed. In the present study, respiratory muscle function was assessed in 31 patients with PH (20 females and 11 males; mean pulmonary artery pressure, 51+/-20 mmHg; median World Health Organization class 3.0+/-0.5; 25 patients with pulmonary arterial hypertension and six patients with chronic thromboembolic PH) and in 31 control subjects (20 females and 11 males) well-matched for gender, age and BMI (body mass index). A 6-min walking test was performed to determine exercise capacity. Volitionally assessed maximal inspiratory (7.5+/-2.1 compared with 6.2+/-2.8 kPa; P=0.04) and expiratory (13.3+/-4.2 compared with 9.9+/-3.4 kPa; P<0.001) mouth pressures, sniff nasal (8.3+/-1.9 compared with 6.6+/-2.2 kPa; P=0.002) and transdiaphragmatic (11.3+/-2.5 compared with 8.7+/-2.5 kPa; P<0.001) pressures, non-volitionally assessed twitch mouth (1.46+/-0.43 compared with 0.97+/-0.41 kPa; P<0.001) and transdiaphragmatic (2.08+/-0.55 compared with 1.47+/-0.72 kPa; P=0.001) pressures during bilateral anterior magnetic phrenic nerve stimulation were markedly lower in patients with PH compared with control subjects. Maximal inspiratory mouth (r=0.58, P<0.001) and sniff transdiaphragmatic (r=0.43, P=0.02) pressures were correlated with the 6-min walking distance in patients with PH. In conclusion, the present study provides strong evidence that respiratory muscle strength is reduced in patients with PH compared with well-matched control subjects. Furthermore, the 6-min walking distance is significantly linked to parameters assessing inspiratory muscle strength.

New physiological insights into exercise-induced diaphragmatic fatigue.

Data on the dynamic process and time-point of manifestation of exercise-induced diaphragmatic fatigue (DF) are lacking. Therefore, this study was aimed assessing dynamic changes of diaphragmatic strength during exercise and determining the time-point of DF manifestation. Fourteen trained subjects (maximal oxygen uptake (VO2(max)) 59.3+/-5.5 ml/min/kg) performed standardized exercise protocols (maximal workload: 85% VO2(max)) followed by recovery (6 min). Ergospirometric data and twitch transdiaphragmatic pressure (TwPdi) were consecutively assessed. DF was induced (TwPdi-rest: 2.34+/-0.26 versus TwPdi-end-recovery 2.01+/-0.21 kPa, p<0.01). TwPdi progressively increased during exercise (TwPdi-rest: 2.34+/-0.26 versus TwPdi-maximal-workload: 3.28+/-0.38 kPa, p<0.001). DF was detectable immediately after exercise-termination (TwPdi-maximal-workload: 3.28+/-0.38 versus TwPdi-early-recovery 2.55+/-0.34 kPa, p<0.001). TwPdi during exercise was highly correlated to workload, VO2(max) and dyspnea (r=0.96/r=0.92/r=0.97; all p<0.0001). In conclusion, diaphragmatic strength progressively increases with increasing workload, and DF manifests after - rather than during - exercise. In addition, TwPdi is highly correlated to key-measures of ergospirometry, approving the physiological thesis that muscle strength is progressively enhanced and escapes fatiguing failure during high-intensity exercise performance.

Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus.

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A(2) catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.

Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity.

Adeno-associated virus type 2 (AAV2) capsids show 12 pores at the fivefold axes of symmetry. We mutated amino acids which constitute these pores to investigate possible functions of these structures within the AAV2 life cycle. Mutants with alterations in conserved residues were impaired mainly in genome packaging or infectivity, whereas few mutants were affected in capsid assembly. The packaging phenotype was characterized by increased capsid-per-genome ratios. Analysis of capsid-associated DNA versus encapsidated DNA revealed that this observation was due to reduced and not partial DNA encapsidation. Most mutants with impaired infectivity showed a decreased capability to expose their VP1 N termini. As a consequence, the activation of phospholipase A2 (PLA2) activity, which is essential for efficient infection, was affected on intact capsids. In a few mutants, the exposure of VP1 N termini and the development of PLA2 activity were associated with enhanced capsid instability, which is obviously also deleterious for virus infection. Therefore, PLA2 activity seems to be required on intact capsids for efficient infection. In conclusion, these results suggest that the pores at the fivefold axes function not only as portals for AAV2 single-stranded DNA packaging but also as channels for presentation of the PLA2 domain on AAV2 virions during infection.