1. Volume 12, Issue 4, Pages 763-771 (October 2005)
Oncogenesis Following Delivery of a Nonprimate Lentiviral Gene Therapy Vector to Fetal and Neonatal Mice 
Mike Themis, Simon N. Waddington, Manfred Schmidt, Christof von Kalle, Yoahe Wang, Faisal Al-Allaf, Lisa G. Gregory, Megha Nivsarkar, Matthew Themis, Maxine V. Holder, Suzanne M.K. Buckley, Niraja Dighe, Alaine T. Ruthe, Ajay Mistry, Brian Bigger, Ahad Rahim, Tuan H. Nguyen, Didier Trono, Adrian J. Thrasher, Charles Coutelle 
Molecular Therapy 
Volume 12, Issue 4, Pages (October 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Schematic representations of HIV vectors HR′SIN-cPPT-S-FIX-W, RRLsin.cPPT.CMV.eGFP.WPRE, RRLsin.cPPT.CMV.hFIX.WPRE, and RRLsin.cPPT.mTTR.hFIX.WPRE and EIAV vectors SMART 2 and SMART 3 provirus vector genomes. All vectors contain self-inactivating (SIN) LTRs, a central polypurine tract (cPPT), and versions of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The HR′SIN-cPPT-S-FIX-W SIN HIV-1-based vector [19] uses an internal SFFV promoter and its WPRE sequence is mutated at the X gene promoter and start codon to prevent transcription of truncated X protein (designated mutated WPRE). RRLsin.cPPT.CMV.eGFP.WPRE and RRLsin.cPPT.CMV.hFIX.WPRE vectors [24,30] use the CMV promoter to drive transgene expression and contain an unmutated WPRE that is not modified at the X gene promoter or start codon (designated WPRE). The RRLsin.cPPT.mTTR.hFIX.WPRE vector uses a liver-specific murine transthyretin promoter and the unmutated WPRE (details of the construction of pRRLsin.cPPT.mTTR.hFIX.WPRE are provided in the supplementary materials and methods). All SMART vectors use internal CMV promoters to drive gene expression and also contain the unmutated WPRE. SMART 2Z [14] carries a non-nuclear-localized β-galactosidase gene (LacZ) and SMART 2hFIX carries the human factor IX gene (details of the construction of pSMART 2hFIX are provided in the supplementary material and methods). To determine the clonality of tumors and provirus copy number in each tumor by Southern analysis in mice following SMART 2Z and SMART 2hFIX administration a HindIII restriction site (denoted H) on the vector genome was used. Restriction digestion of the integrated provirus by this enzyme allows 3′ vector sequences including the WPRE sequence, the vector LTR, and the neighboring genomic DNA to be visualized using an [α-32P]CTP-labeled NotI (denoted N) fragment excised from the parent pSMART 2 plasmid vector. SMART 2 vectors contain the largest proportion of the EIAV wild-type genome. The accessory genes are point mutated or partially deleted, including the S2 gene (the function of which is poorly understood). SMART 2 also carries fragments of the EIAV envelope gene. SMART 3NZ carries a nuclear-localized β-galactosidase gene (NLSLacZ) and lacks the EIAV sequences downstream of the reporter gene cassette in SMART 2. This region includes the remaining sequences from S2, rev, and part of EIAV env. Rev-responsive elements (RRE) are present in SMART 2 and hFIX HIV vector genomes, but not in SMART 3.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Representative photomicrographs of liver tumor tissue from EIAV treated mice. The liver and other organs were removed from isoflurane-euthanized mice and fixed in 10% formaldehyde solution, paraffin embedded, sectioned, and counterstained with neutral red. Images are of SMART 2hFIX lentiviral fetally infected adult mice and control uninfected livers. (A) Mouse sacrificed at 299 days of age, early stage of hepatocellular carcinoma following vector injection showing dysplastic cells around central vein (H&E staining, original magnification ×200). (B) Mouse sacrificed at 154 days of age, cross section of liver showing a tumor nodule with well-defined border (H&E staining, original magnification ×40). (C) High magnification of tumor nodule from B, showing a trabecular architecture of tumor cells with pronounced cellular polymorphism; black arrows indicate abnormal mitosis in tumor cells (H&E staining, original magnification ×200). (D) Representation of normal liver tissue from an untreated 386-day-old mouse shows fatty degeneration and hepatocyte morphology (H&E staining, original magnification ×200). (E) Normal liver tissue from a 192-day-old mouse shows normal structure and cellular morphology (H&E staining, original magnification ×200).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions