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

Molecular Pain

Andreas S Beutler³  Christopher E Walsh³  John H Morrison¹  William GM Janssen¹  Douglas M McCarty²  Cheng Wang³  Michaela S Banck³  Nina M Harder³  Benjamin Storek¹ 

[1] Department of Neurosciences, Mount Sinai School of Medicine, New York, NY, USA;Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA;Department of Medicine (Hematology/Oncology), Mount Sinai School of Medicine, New York, NY, USA

Others  :  1180345
DOI  :  10.1186/1744-8069-2-4

 received in 2005-11-09, accepted in 2006-01-30,  发布年份 2006

【 摘 要 】

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.

【 授权许可】

   
2006 Storek et al; licensee BioMed Central Ltd.

【 图 表 】

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