AAV and RT-PCR: True or False?
Authors: Richard O. Snyder
Journal: Molecular Therapy (2000)

Recombinant AAV vectors are capable of long-term gene expression with low toxicity in vivo (1Snyder R.O. Adeno-associated virus-mediated gene delivery.J Gene Med. 1999; 1: 166-175Crossref PubMed Google Scholar). Therapeutic and sustained protein expression and correction of animal models of disease are now being reported, and human clinical trials are in progress. Several laboratories have evaluated AAV-mediated transduction by a variety of methods, including the technique of RT-PCR (2Beck S.E. Jones L.A. Chesnut K. Walsh S.M. Reynolds T.C. Carter B.J. Askin F.B. Flotte T.R. Guggino W.B. Repeated delivery of adeno-associated virus vectors to the rabbit airway.J. Virol. 1999; 73: 9446-9455Crossref PubMed Google Scholar, 3Chatterjee S. Li W. Wong C.A. Fisher-Adams G. Lu D. Guha M. Macer J.A. Forman S.J. Wong Jr., K.K. Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors.Blood. 1999; 93: 1882-1894Crossref PubMed Google Scholar, 4During M.J. Samulski R.J. Elsworth J.D. Kaplitt M.G. Leone P. Xiao X. Li J. Freese A. Taylor J.R. Roth R.H. Sladek Jr., J.R. O'Malley K.L. Redmond Jr., D.E. In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector.Gene Ther. 1998; 5: 820-827Crossref PubMed Scopus (155) Google Scholar, 5Gardner J.P. Zhu H. Colosi P.C. Kurtzman G.J. Scadden D.T. Robust, but transient expression of adeno-associated virus-transduced genes during human T lymphopoiesis.Blood. 1997; 90: 4854-4864PubMed Google Scholar, 6Inouye R.T. Du B. Boldt-Houle D. Ferrante A. Park I.W. Hammer S.M. Duan L. Groopman J.E. Pomerantz R.J. Terwilliger E.F. Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector [published erratum appears in J. Virol.72(4):3506, 1998].J. Virol. 1997; 71: 4071-4078Google Scholar, 7Liu Y. Santin A.D. Mane M. Chiriva-Internati M. Parham G.P. Ravaggi A. Hermonat P.L. Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus.J. Interferon Cytokine Res. 2000; 20: 21-30Crossref PubMed Scopus (58) Google Scholar, 8Mi J. Chatterjee S. Wong K.K. Forbes C. Lawless G. Tobin A.J. Recombinant adeno-associated virus (AAV) drives constitutive production of glutamate decarboxylase in neural cell lines.J. Neurosci. Res. 1999; 57: 137-148Crossref PubMed Scopus (18) Google Scholar, 9Mohuczy D. Gelband C.H. Phillips M.I. Antisense inhibition of AT1 receptor in vascular smooth muscle cells using adeno-associated virus-based vector.Hypertension. 1999; 33: 354-359Crossref PubMed Google Scholar, 10Peng L. Sidner R.A. Bochan M.R. Burton M.M. Cooper S.T. Jindal R.M. Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene.J. Surg. Res. 1997; 69: 193-198Abstract Full Text PDF PubMed Scopus (11) Google Scholar, 11Ponnazhagan S. Mukherjee P. Wang X.S. Qing K. Kube D.M. Mah C. Kurpad C. Yoder M.C. Srour E.F. Srivastava A. Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: Donor variation and correlation of transgene expression with cellular differentiation.J. Virol. 1997; 71: 8262-8267Crossref PubMed Google Scholar, 12Shah R. Jindal R.M. Stable transfection of rat preproinsulin II gene into rat hematopoietic stem cells via recombinant adeno-associated virus.Life Sci. 1999; 65: 2041-2047Crossref PubMed Scopus (7) Google Scholar, 13Snyder R.O. Miao C.H. Patjn G.A. Spratt S.K. Danos O. Nagy D. Gown A.M. Winther B. Meuse L. Cohen L.K. Thompson A.R. Kay M.A. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.Nat. Genet. 1997; 16: 270-276Crossref PubMed Scopus (497) Google Scholar). However, there is a potential flaw with RT-PCR analysis in this context: Specifically, a false-positive signal can be obtained from contaminating AAV single-stranded DNA (ssDNA). Single-stranded DNA can originate from intact viral particles located intracellularly (14Bartlett J.S. Wilcher R. Samulski R.J. In fectious entry pathway of adeno-associated virus and adeno-associated virus vectors [in process citation].J. Virol. 2000; 74: 2777-2785Crossref PubMed Scopus (407) Google Scholar) or bound to the cell surface or from unpackaged genomes not yet converted to double-stranded forms (15Ferrari F.K. Samulski T. Shenk T. Samulski R.J. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors.J. Virol. 1996; 70: 3227-3234Crossref PubMed Google Scholar). During the preparation of RNA for RT-PCR analysis, there is usually a DNasel step incorporated to digest contaminating DNA prior to reverse transcription of the mRNA. This is effective for digesting double-stranded DNA; however, DNasel is unable to digest ssDNA (i.e., rAAV genomes) (16Tabor S. Struhl K. Endonucleases..in: Ausubel F.M. Brent R. Kingston R.E. In Current Protocols in Molecular Biology. Wiley, New York1989: 3.12.1-3.12.6Google Scholar, 17Boehringer Mannheim 1998 Biochemicals Catalog.Google Scholar). Annealing of the plus and minus AAV strands occurs during nucleic acid isolation which may allow a portion of the ssDNA to be digested, but some can remain (18Miao C.H. Snyder R.O. Schowalter D.B. Patjn G.A. Donahue B. Winther B. Kay M.A. The kinetics of rAAV integration in the liver [letter].Nat. Genet. 1998; 19: 13-15Crossref PubMed Scopus (148) Google Scholar, 19Vincent-Lacaze N. Snyder R.O. Gluzman R. Bohl D. Lagarde C. Danos O. Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle.J. Virol. 1999; 73: 1949-1955Crossref PubMed Google Scholar). In Ferrari et al. (15: Fig. 5, lane 1), digestion of rAAV DNA with DNasel was obtained following annealing of the single strands (R. J. Samulski, personal communication). Because the mRNA being evaluated will harbor sequences colinear with the rAAV genome, there is a likelihood of a false-positive signal. Strategies can be incorporated at each of the steps outlined in Fig. 1 to ensure detection of RNA but not AAV ssDNA, and in several of the literature references cited above precautions were taken to avoid detection of ssDNA. The time point posttransduction when the cells or tissues are harvested will influence the amount of ssDNA in the sample (18Miao C.H. Snyder R.O. Schowalter D.B. Patjn G.A. Donahue B. Winther B. Kay M.A. The kinetics of rAAV integration in the liver [letter].Nat. Genet. 1998; 19: 13-15Crossref PubMed Scopus (148) Google Scholar, 19Vincent-Lacaze N. Snyder R.O. Gluzman R. Bohl D. Lagarde C. Danos O. Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle.J. Virol. 1999; 73: 1949-1955Crossref PubMed Google Scholar), with fewer ssDNA genomes detected at later times. Messenger RNA can be purified using oligo(dT) matrices which bind mRNA and not DNA. Alternatively, total RNA can be extracted with acid guanidinium thiocyanate–phenol–chloroform which partitions dsDNA (and maybe ssDNA) to the organic phase and RNA to the aqueous phase (20Adamovicz J. Gause W.C. RNA purification..in: Dieffenbach C.W. Dveksler G.S. In PCR Primer A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1995: 113-129Google Scholar). Validating the purity of mRNA without DNA contamination is important to establish confidence in the analysis. In fact, product literature for these reagents recommends the use of DNasel to remove possible DNA contamination (TRIzol, Life Technologies Cat. No. 15596–026; MessageClean, GenHunter Corp., Cat. No. M601). Following isolation of the mRNA and DNaseI digestion, contaminating ssDNA can be digested with restriction endonucleases having activity toward ssDNA [i.e., HhaI, HinPI, MnlI, HaeIII (21New England BioLabs 1998/1999 Catalog.Google Scholar)], provided the restriction site is present in the rAAV genome. The removal or inactivation of these enzymes is needed before the samples are subjected to reverse transcription and PCR because they will digest the cDNA and double-stranded PCR products as well. Primers can be placed across a vector's intron to allow a size distinction between products from mRNA and DNA following gel electrophoresis; however, this is not a useful strategy for intronless vectors [e.g., WPRE-containing vectors (22Loeb J.E. Cordier W.S. Harris M.E. Weitzman M.D. Hope T.J. Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: Implications for gene therapy.Hum. Gene Ther. 1999; 10: 2295-2305Crossref PubMed Scopus (213) Google Scholar)]. Including a “no RT” control (i.e., carrying out the PCR on the isolated mRNA which has not been reverse transcribed) should give no signal. Finally, short extension times can reduce self-primed AAV genome amplification. RT-PCR is a sensitive method for demonstrating gene transfer and evaluating biodistribution following AAV-mediated transduction in vivo, and these steps alone or in combination can ensure that the data obtained are reliable.