1. Volume 11, Issue 6, Pages 916-925 (June 2005)
Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons 
Armin Blesch, James Conner, Alexander Pfeifer, Mehdi Gasmi, Anthony Ramirez, William Britton, Ron Alfa, Inder Verma, Mark H. Tuszynski 
Molecular Therapy 
Volume 11, Issue 6, Pages (June 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Schematic drawing of the lentiviral vector constructs. A SIN lentiviral vector with a deletion in the U3 part of the LTR (ΔU3) was used for lentivirus production. (A) Constitutive expression of GFP or NGF is driven by the CMV enhancer/chicken β-actin promoter (CAG). For increased transduction and transcription the HIV central polypurine tract (cPPT) and the woodchuck posttranscriptional response element (WPRE) were included. For the NGF-coding virus the enhanced GFP cDNA sequence was replaced by the NGF cDNA. (B) For regulated GFP expression the tetracycline transactivator (tTA2) is driven by the CAG promoter, and GFP expression is controlled by the tetracycline-responsive minimal CMV promoter (CMV⁎−1). (C) For the controlled expression of NGF the human NGF cDNA followed by an internal ribosome entry site (IRES) was cloned upstream of the GFP cDNA (RSV, Rous sarcoma virus promoter/enhancer; RRE, Rev response element).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Unregulated expression of the GFP reporter gene and lesion extent. (A) Low-magnification overview of GFP labeling along the injection tract (arrows). (B) Double labeling using antibodies against NeuN (red) and GFP (green) shows numerous GFP-labeled neurons (arrows). (C) GFAP labeling to identify astrocytes (red) in combination with GFP labeling (green) shows very few transduced astrocytes (arrow) and numerous other GFP-positive cells (arrowheads) not labeled for GFAP. (D) Nissl-stained coronal section showing the extent of the unilateral fimbria–fornix lesion including a small amount of the most rostral portion of the hippocampus. Scale bar, 280 μm in (A); 47 μm in (B, C), 2.88 mm in (D).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Rescue of ChAT- and p75NTR-labeled medial septal neurons by constitutive, unregulated lentiviral NGF gene transfer. (A) Control animals injected with GFP-coding lentivirus show a loss of choline acetyltransferase (ChAT)-labeled neurons on the left lesioned side compared to the right intact side. (B) In contrast, animals injected with NGF-coding lentivirus show a significant rescue of ChAT-labeled neurons. (C) Quantification of cholinergic neurons on the lesioned and unlesioned side indicates a highly significant effect of NGF-coding lentivirus (ANOVA; P < 0.001). Labeling for the p75 neurotrophin receptor in (D) GFP and (E) NGF lentivirus-injected animals illustrates a similar, significant effect after (F) quantification of labeled neurons on the lesioned and unlesioned side (ANOVA, overall P < 0.01; NGF vs GFP P < 0.01). Animals injected with NGF virus only 1 day prior to the lesion do not differ from animals that received virus 1 week prior to the lesion (P > 0.05; NGF vs GFP P < 0.05). Arrows in A, B, D, and E indicate the midline. Remaining neurons on the lesioned side in (G) animals injected with GFP virus are smaller and have fewer neurites than p75NTR-labeled neurons in animals injected with (H) NGF virus. (I) No sprouting of p75NTR-labeled neurites can be found in animals injected with GFP virus, whereas dense sprouting occurs in animals injected with (J) NGF-coding virus. (K) Higher magnification of p75NTR-labeled fibers. Arrows in (I) and (J) indicate the needle tract. Scale bar, 177 μm in (A–E), 21 μm in (G, H), 89 μm in (I, J), 17 μm in (K).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Regulated expression of GFP in the medial septum. (A, B) Low and (C, D) high magnification of GFP immunolabeling in animals that received tetracycline-regulated lentivirus injections for the expression of GFP (HV-tet-GFP) and were (A, C) untreated (−Dox) or (B, D) treated with doxycycline in the drinking water (+Dox). Arrows in (A, B) indicate the midline. (E) Animals injected with HV-tet-NGF-IRES-GFP lentivirus show weaker GFP expression likely due to the reduced translation of GFP from the internal ribosome entry site. (F) No GFP expression can be detected in doxycycline-treated animals injected with HV-tet-NGF-IRES-GFP. Arrows in (F) indicate the needle tract. Scale bar, 424 μm in (A, B), 170 μm in (C, D), 161 μm in (E, F).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Tetracycline-regulated rescue of cholinergic neurons. (A, B) Animals that received lentivirus for the regulated expression of GFP show loss of ChAT-labeled neurons on the left lesion side when they were (A) treated with doxycycline (GFP +Dox) or (B) untreated (GFP −Dox). (C) A similar loss of labeled neurons is observed on the lesioned side in doxycycline-treated animals that received regulated NGF lentivirus (NGF +Dox). (D) In contrast, preservation of cholinergic neurons on the lesioned side can be seen in animals that received tet-NGF lentivirus and were not treated with doxycycline. (E) Quantification of cholinergic neurons indicates significant neuronal rescue in animals that received HV-tet-NGF lentivirus and were not treated with doxycycline compared to all other groups (P < 0.005). Arrows in (A–D) indicate the midline. Scale bar, 424 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Sprouting of p75NTR-labeled neurites after regulated lentiviral NGF gene transfer. (A, B) Animals injected with GFP-coding control virus show p75NTR labeling mainly around blood vessels and do not differ from doxycycline-treated animals injected with (C) HV-tet-NGF lentivirus (gene expression off). In contrast, dense sprouting occurs in animals injected with (D) HV-tet-NGF without doxycycline treatment (gene expression turned on). Inset in (D) shows higher magnification of p75NTR-labeled fibers. Scale bar, 170 μm, 27 μm for inset in (D).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
NGF ELISA after injection of lentivirus for the regulated expression of GFP or NGF. NGF levels in animals that received HV-tet-NGF lentivirus and were treated with doxycycline (NGF + Dox) are not significantly different from doxycycline-treated or untreated control animals (GFP + Dox and GFP − Dox). In contrast, significantly higher NGF levels are found in animals injected with HV-tet-NGF (NGF + Dox) that were untreated to turn NGF expression on (P < 0.02).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions