Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats

BACKGROUND: Intrathecal (IT) gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks) impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF) modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV), one of the most promising vector types for clinical use. RESULTS: Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV) vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP) for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. CONCLUSION: sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2. Experiments presented here will provide a rational basis for utilizing IT rAAV gene transfer in basic and translational studies on chronic pain

SARS-CoV-2-induced SIADH: a novel cause of hyponatremia

Background!#!Sensor-based monitoring allows continuous observations of patient mobilization after proximal femoral fractures. A wrist-worn motion tracker allows long-term observation that is low in interruption and constraints for subjects.!##!Objective!#!Description of steps development after hip fracture surgery on a specialized geriatric trauma ward and beyond.!##!Material and methods!#!In the explorative long-term field research study, an applicable motion tracker observed steps per day of 20 patients (80% female, mean age 85.2 years ± 7.86 years) for 10 weeks. Weekly mean values (days 1-7, 8-14 etc.) of steps per day formed the database for descriptive analysis (mean, SD, min, max, median).!##!Results!#!During observation weeks (ow) a positive development of steps took place. A mean increase factor of 1.285 (±0.351) occurred from ow 1 (M = 353.57 ± 310.15) to ow 10 (M = 2482.07 ± 1374.12). The highest increase by a factor of 1.8 could be reported from ow 2 (M = 556.27 ± 478.11) to ow 3 (M = 1024.86 ± 921.24) as well as from ow 6 (M = 1268.21 ± 880.47) to 7 (M = 2367.14 ± 1680.08). A slight decrease of steps occurred from ow 4 (M = 1208.27 ± 1210.45) to ow 5 (0.99-fold) and from ow 9 (M = 2689.98 ± 2339.71) to 10 (0.92-fold). High ranges and standard deviations in relation to the mean occurred constantly. The presence of several step development groups could be presumed.!##!Conclusion!#!Motion tracker and the variable steps per day can represent the ability to walk within an everyday environment, with a possible underestimation of < 10%. Differences regarding observation lengths and disruptions occurred. Cluster analysis should detect group attributes of different courses of development in subsequent studies