Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector.

Viral vectors have become important tools for basic research and clinical gene therapy over the past years. However, in vitro testing of vector-derived transgene function can be challenging when specific post-translational modifications are needed for biological activity. Similarly, neuropeptide precursors need to be processed to yield mature neuropeptides. SH-SY5Y is a human neuroblastoma cell line commonly used due to its ability to differentiate into specific neuronal subtypes. In this study, we evaluate the suitability of SH-SY5Y cells in a potency assay for neuropeptide-expressing adeno-associated virus (AAV) vectors. We looked at the impact of neuronal differentiation and compared single-stranded (ss) AAV and self-complementary (sc) AAV transduction at increasing MOIs, RNA transcription kinetics, as well as protein expression and mature neuropeptide production. SH-SY5Y cells proved highly transducible with AAV1 already at low MOIs in the undifferentiated state and even better after neuronal differentiation. Readouts were GFP or neuropeptide mRNA expression. Production of mature neuropeptides was poor in undifferentiated cells. By contrast, differentiated cells produced and sequestered mature neuropeptides into the medium in a MOI-dependent manner.

OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

Recombinant adeno-associated viral (rAAV) vectors for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap-expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high burst sizes per cell were achieved, combined with high infectivity rates. OneBac 2.0 represents a 2-fold advancement: First, enhanced VP1 proportions in AAV5 capsids lead to vastly increased per-particle infectivity rates. Second, collateral packaging of foreign DNA is suppressed by removal of the Rep-binding element (RBE). In this study we show that this advancement of AAV5 packaging can be translated to OneBac 2.0-derived packaging systems for alternative AAV serotypes. By removal of the RBE, collateral packaging of nonvector DNA was drastically reduced in all newly tested serotypes (AAV1, AAV2, and AAV8). However, the splicing-based strategy to enhance VP1 expression in order to increase AAV5 infectivity hardly improved infectivity rates of AAV-1, -2, or -8 compared with the original OneBac cell lines. Our results emphasize that OneBac 2.0 represents an advancement for scalable, high-titer production of various AAV serotypes, leading to AAV particles with minimal packaging of foreign DNA.

High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence.

Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from humans or nonhuman primates have so far not been proven to be causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, definition of the in vivo sites of wild-type AAV persistence and the clinical consequences of its reactivation is becoming increasingly urgent. Here, we identify the presumed cell type for AAV persistence in the human host by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic-DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV positive, predominantly AAV type 2 (AAV2) (77%), AAV5 (19%), and additional serotypes. Roughly 11% of the blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, 76% of whom were AAV positive. Of these, at least 45% displayed mixed infections. Follow-up of single blood donors over 2 years allowed repeated detection of the initial and/or additional AAV serotypes, suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3+ T lymphocytes, perceived as the putative in vivo site of AAV persistence. Moreover, infectious AAVs of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follow AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel with activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation. IMPORTANCE: Adeno-associated virus is viewed as apathogenic and replication defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes possible. Reactivation from latency can be induced by diverse stimuli, including infections, typically induced upon host immunosuppression. We show for the first time that infectious AAV is highly prevalent in human leukocytes, specifically T lymphocytes, and that AAV is strongly amplified upon immunosuppression, along with reactivation of latent human herpesviruses. In the absence of an animal model to study the AAV life cycle, our findings in the human host will advance the understanding of AAV latency, reactivation, and in vivo pathogenesis.

Very fast bulk Li ion diffusivity in crystalline Li(1.5)Al(0.5)Ti(1.5)(PO4)3 as seen using NMR relaxometry.

The realization of large powerful all-solid-state batteries is still hampered by the availability of environmentally friendly and low-cost Li ion conductors that can easily be produced on a large scale and with high reproducibility. Advanced solid electrolytes benefit from fast ion-selective transport and non-flammability, but they may have low electrochemical stability with respect to Li metal. Sol-gel-synthesized lithium titanium aluminum phosphate Li(1.5)Al(0.5)Ti(1.5)(PO4)3 (LATP), which was prepared via a new synthesis route taking advantage of an annealing step at relatively low temperatures, has the potential to become one of the major players in this field although it may suffer from reduction upon direct contact with metallic lithium. Its ion dynamics is, however, as yet poorly understood. In the present study, (7)Li nuclear magnetic resonance (NMR) spectroscopy was used to monitor the key Li jump processes on the atomic scale. NMR relaxation clearly reveals heterogeneous dynamics comprising distinct ultra-fast and slower diffusion processes. The high Li ion self-diffusion coefficients deduced originate from a rapid Li exchange with activation energies as low as 0.16 eV which means that sol-gel synthesized LATP is superior to other solid electrolytes. Our NMR results fully support recent theoretical investigations on the underlying diffusion mechanism, indicating that to rapidly jump from site to site, the ions use interstitial sites connected by low-energy barriers in LATP.

Prediction of auditory and visual p300 brain-computer interface aptitude.

OBJECTIVE: Brain-computer interfaces (BCIs) provide a non-muscular communication channel for patients with late-stage motoneuron disease (e.g., amyotrophic lateral sclerosis (ALS)) or otherwise motor impaired people and are also used for motor rehabilitation in chronic stroke. Differences in the ability to use a BCI vary from person to person and from session to session. A reliable predictor of aptitude would allow for the selection of suitable BCI paradigms. For this reason, we investigated whether P300 BCI aptitude could be predicted from a short experiment with a standard auditory oddball. METHODS: Forty healthy participants performed an electroencephalography (EEG) based visual and auditory P300-BCI spelling task in a single session. In addition, prior to each session an auditory oddball was presented. Features extracted from the auditory oddball were analyzed with respect to predictive power for BCI aptitude. RESULTS: Correlation between auditory oddball response and P300 BCI accuracy revealed a strong relationship between accuracy and N2 amplitude and the amplitude of a late ERP component between 400 and 600 ms. Interestingly, the P3 amplitude of the auditory oddball response was not correlated with accuracy. CONCLUSIONS: Event-related potentials recorded during a standard auditory oddball session moderately predict aptitude in an audiory and highly in a visual P300 BCI. The predictor will allow for faster paradigm selection. SIGNIFICANCE: Our method will reduce strain on patients because unsuccessful training may be avoided, provided the results can be generalized to the patient population.

Proprioceptive feedback and brain computer interface (BCI) based neuroprostheses.

Brain computer interface (BCI) technology has been proposed for motor neurorehabilitation, motor replacement and assistive technologies. It is an open question whether proprioceptive feedback affects the regulation of brain oscillations and therefore BCI control. We developed a BCI coupled on-line with a robotic hand exoskeleton for flexing and extending the fingers. 24 healthy participants performed five different tasks of closing and opening the hand: (1) motor imagery of the hand movement without any overt movement and without feedback, (2) motor imagery with movement as online feedback (participants see and feel their hand, with the exoskeleton moving according to their brain signals, (3) passive (the orthosis passively opens and closes the hand without imagery) and (4) active (overt) movement of the hand and rest. Performance was defined as the difference in power of the sensorimotor rhythm during motor task and rest and calculated offline for different tasks. Participants were divided in three groups depending on the feedback receiving during task 2 (the other tasks were the same for all participants). Group 1 (n = 9) received contingent positive feedback (participants' sensorimotor rhythm (SMR) desynchronization was directly linked to hand orthosis movements), group 2 (n = 8) contingent "negative" feedback (participants' sensorimotor rhythm synchronization was directly linked to hand orthosis movements) and group 3 (n = 7) sham feedback (no link between brain oscillations and orthosis movements). We observed that proprioceptive feedback (feeling and seeing hand movements) improved BCI performance significantly. Furthermore, in the contingent positive group only a significant motor learning effect was observed enhancing SMR desynchronization during motor imagery without feedback in time. Furthermore, we observed a significantly stronger SMR desynchronization in the contingent positive group compared to the other groups during active and passive movements. To summarize, we demonstrated that the use of contingent positive proprioceptive feedback BCI enhanced SMR desynchronization during motor tasks.

Psychological predictors of SMR-BCI performance.

BACKGROUND: After about 30 years of research on Brain-Computer Interfaces (BCIs) there is little knowledge about the phenomenon, that some people - healthy as well as individuals with disease - are not able to learn BCI-control. To elucidate this "BCI-inefficiency" phenomenon, the current study investigated whether psychological parameters, such as attention span, personality or motivation, could predict performance in a single session with a BCI controlled by modulation of sensorimotor rhythms (SMR) with motor imagery. METHODS: A total of N=83 healthy BCI novices took part in the session. Psychological parameters were measured with an electronic test-battery including clinical, personality and performance tests. Predictors were determined by binary logistic regression analyses. RESULTS: The output variable of the Two-Hand Coordination Test (2HAND) "overall mean error duration" which is a measure for the accuracy of fine motor skills accounted for 11% of the variance in BCI-inefficiency. The Attitudes Towards Work (AHA) test variable "performance level" which can be interpreted as a degree of concentration and a neurophysiological SMR predictor were also identified as significant predictors of SMR BCI performance. CONCLUSION: Psychological parameters as measured in this study play a moderate role for one-session performance in a BCI controlled by modulation of SMR.

Integration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome.

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.

On optimal channel configurations for SMR-based brain-computer interfaces.

One crucial question in the design of electroencephalogram (EEG)-based brain-computer interface (BCI) experiments is the selection of EEG channels. While a setup with few channels is more convenient and requires less preparation time, a dense placement of electrodes provides more detailed information and henceforth could lead to a better classification performance. Here, we investigate this question for a specific setting: a BCI that uses the popular CSP algorithm in order to classify voluntary modulations of sensorimotor rhythms (SMR). In a first approach 13 different fixed channel configurations are compared to the full one consisting of 119 channels. The configuration with 48 channels results to be the best one, while configurations with less channels, from 32 to 8, performed not significantly worse than the best configuration in cases where only few training trials are available. In a second approach an optimal channel configuration is obtained by an iterative procedure in the spirit of stepwise variable selection with nonparametric multiple comparisons. As a surprising result, in the second approach a setting with 22 channels centered over the motor areas was selected. Thanks to the acquisition of a large data set recorded from 80 novice participants using 119 EEG channels, the results of this study can be expected to have a high degree of generalizability.

A brain-computer interface controlled auditory event-related potential (p300) spelling system for locked-in patients.

Using brain-computer interfaces (BCI) humans can select letters or other targets on a computer screen without any muscular involvement. An intensively investigated kind of BCI is based on the recording of visual event-related brain potentials (ERP). However, some severely paralyzed patients who need a BCI for communication have impaired vision or lack control of gaze movement, thus making a BCI depending on visual input no longer feasible. In an effort to render the ERP-BCI usable for this group of patients, the ERP-BCI was adapted to auditory stimulation. Letters of the alphabet were assigned to cells in a 5 x 5 matrix. Rows of the matrix were coded with numbers 1 to 5, and columns with numbers 6 to 10, and the numbers were presented auditorily. To select a letter, users had to first select the row and then the column containing the desired letter. Four severely paralyzed patients in the end-stage of a neurodegenerative disease were examined. All patients performed above chance level. Spelling accuracy was significantly lower with the auditory system as compared with a similar visual system. Patients reported difficulties in concentrating on the task when presented with the auditory system. In future studies, the auditory ERP-BCI should be adjusted by taking into consideration specific features of severely paralyzed patients, such as reduced attention span. This adjustment in combination with more intensive training will show whether an auditory ERP-BCI can become an option for visually impaired patients.

Validity of the ALS-Depression-Inventory (ADI-12)--a new screening instrument for depressive disorders in patients with amyotrophic lateral sclerosis.

BACKGROUND: Depressive symptoms among patients with amyotrophic lateral sclerosis (ALS) are usually measured with conventional questionnaires. These measurements do not consider the specific circumstances of the underlying disease. The purpose of this study was to assess the validity of a new short 12 items ALS-Depression-Inventory (ADI-12). We determined convergent, criterion, and concurrent validity. The Structured Clinical Interview (SCID) for DSM-IV was used as the gold standard and the Beck Depression Inventory (BDI) and the WHO Well Being Index (WHO-5) to assess concurrent validity. METHODS: A total of 39 ALS patients in all stages of the disease were interviewed. Convergent validity was estimated by the inter-correlation between the ADI-12 and the BDI. Criterion and concurrent validity were specified with respect to sensitivity, specificity and predictive values. Receiver Operating Characteristics (ROC) and Areas Under the Curves (AUC) were calculated. RESULTS: All three depression scales showed excellent internal consistencies (Cronbach's alpha: .8-.9). The correlation between the ADI-12 and the BDI was high (r=.80). For the ADI-12 a cut-off of > or = 30 (SE=100%, SP=83%) identified all patients with a current episode of major depression. A more liberal cut-off (> or = 23) identified all patients with any depressive disorder including minor depression at the cost of specificity (60%). CONCLUSIONS: With the ADI-12 ALS patients with depressive disorders can be reliably identified. We recommend the ADI-12 for routine screening in primary care of ALS patients.