1. Volume 4, Issue 6, Pages 559-566 (December 2001)
Immediate and Long-Term Safety of Recombinant Adeno-associated Virus Injection into the Nonhuman Primate Muscle 
David Favre, Nathalie Provost, Véronique Blouin, Gilles Blancho, Yan Chérel, Anna Salvetti, Philippe Moullier 
Molecular Therapy 
Volume 4, Issue 6, Pages (December 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Structure of rAAV vector plasmids. AAVcm-ET(LTR) and AAVcm-ET(CAG) vectors encode the cynomolgus erythropoietin cDNA (cmEpo) under the control of the doxycyclin-inducible tetO-CMV promoter and the rtTA chimeric protein under the control of the retroviral murine LTR or the CAG promoter, respectively. The rAAVcm-ET term alone designates both types of vectors. Thin arrows indicate the start transcription site; block arrows indicate the position of the primers used for the detection of rAAVcm-ET sequences. The size of the expected PCR product is indicated.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Detection of AAV genome in body fluids. (A) Sensitivity of rAAVcm-ET vector sequence detection by PCR in biological fluids. Known amounts of rAAVcm-ET(LTR) genome particles, as determined by dot blot, were added either in medium or in biological samples as indicated, and viral DNA extracted. PCR was carried out using primers that amplified a 648-bp region between the tetO-CMV promoter and the cmEpo cDNA. After PCR amplification, reaction products were separated on a 2% agarose gel (top) and then transferred to a nylon membrane and hybridized to a cmEpo probe (bottom). The number of genome particles added is indicated at the top of each figure; (c+) refers to a positive control using 25 pg of the pAAVcm-ET vector plasmid. (B) Detection of rAAV genome particles in the serum. The serum of each animal was collected starting from 30 min (30‘) until 9 days (9 d) post rAAVcm-ET(LTR) or rAAVcm-ET(CAG) administration (Table 1). Numbers on the top of each figure indicate at which time the sample was collected.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Detection of infectious AAV by mRCA. (A) Detection of rAAVcm-ET infectious particles in biological fluids. The sensitivity of the mRCA in biological fluids (S, serum; F, feces; U, urine) was compared with the result obtained when rAAV was incubated in medium (M). The total number of infectious particles introduced in the well is indicated (top). (B) Detection of rAAVcm-ET(LTR) infectious particles in the serum of group II animals. The sera correspond to Mac 3 and Mac 4. mRCA was performed before rAAV delivery (0) and 30 minutes (30‘) and 1, 2, and 5 days (1 d, 2 d, 5 d) after rAAV injection.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Kinetics of rAAVcm-ET infectious particles in the serum of all eight animals. The number of infectious particles/ml of serum is indicated for each animal at the different time points analyzed. All animals were tested at each time point (30‘, 6 h, 1 d, 2 d), except where indicated by an asterisk (*).
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Detection of rAAVcm-ET sequence in PBMC and bone marrow cells. Genomic DNA was extracted from group II animals PBMC (A) and bone marrow cells from group III animals (B) and analyzed by PCR (top) and Southern blot (lower) as indicated in the legend of Fig. 2. The number at the top of each lane indicates the time, in months, following rAAV injection at which the sample was harvested. (C) PCR results after fractionation of PBMC, from Mac 6 and Mac 8 at 5 and 2 months after rAAV injection, respectively. Each DNA sample was also positive for the amplification of cytochrome B sequence (data not shown). PCR performed using 25 pg of pAAVcm-ET vector plasmid or water served as positive (c+) and negative (c-) controls, respectively.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
PCR analysis of rAAVcm-ET vector in distant organs. Genomic DNA was extracted from each tissue of Mac 3 at the time of sacrifice, 18 months after rAAV injection. As a control, each sample was also used for the amplification of a 350-bp cytochrome-B DNA fragment.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions