A sensitive, real-time, RNA-specific PCR method for the detection of recombinant AAV-CFTR vector expression
| Autor: | Justine Dell’Aringa, K A Hale, W M Klump, C J Gerard |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Cystic Fibrosis
Genetic Vectors Cystic Fibrosis Transmembrane Conductance Regulator Gene Expression Biology law.invention Transduction (genetics) chemistry.chemical_compound law Complementary DNA Administration Inhalation Gene expression Genetics Animals Lung Molecular Biology Reverse Transcriptase Polymerase Chain Reaction RNA Genetic Therapy Dependovirus Macaca mulatta Molecular biology Reverse transcription polymerase chain reaction chemistry COS Cells Recombinant DNA Molecular Medicine Primer (molecular biology) DNA |
| Zdroj: | Gene Therapy. 10:1744-1753 |
| ISSN: | 1476-5462 0969-7128 |
| Popis: | Following adeno-associated virus (AAV)-mediated transduction, cellular RNA preparations can be contaminated with AAV single-stranded DNA. The single-stranded DNA genome of recombinant AAV vectors can serve as an efficient, but undesirable, template for traditional reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Consequently, recombinant AAV gene therapy presents a unique challenge to the design of sensitive and reliable methods to detect vector-derived mRNA. Several methods have been proposed to reduce the presence of single- and double-stranded vector DNA without compromising RNA specificity. For example, DNase I, although widely used, can be ineffective at completely removing the AAV single-stranded DNA genome. We have developed a sensitive real-time RNA-Specific reverse transcriptase PCR (RS-PCR) method that is independent of DNase I treatment. The RS-PCR method relies on the generation of a first-strand cDNA template using a primer with a linker sequence, X, at the 5'- end such that synthesis of second-strand cDNA incorporates the X-linker sequence into the cDNA template. The RS-PCR then utilizes forward and reverse primers targeting AAV vector sequence and the X-primer site, respectively, while a vector-specific Taqman probe makes sensitive real-time detection possible. We present data to validate the sensitivity and RNA specificity of the RS-PCR method and propose two unique endogenous control strategies by monitoring expression of both beta-glucuronidase and endogenous cystic fibrosis transmembrane conductance regulator (CFTR). Finally, we demonstrate the utility of this new RS-PCR method in detecting recombinant AAV-CFTR expression, including, an in vitro transduction assay and methods to support both preclinical and clinical trials. |
| Databáze: | OpenAIRE |
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