Volume 7, Issue 5, Pages 588-596 (May 2003) Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin  Alison S Bienemann, Enca Martin-Rendon, Anna S Cosgrave, Colin P.J Glover, Liang-Fong Wong, Susan M Kingsman, Kyriacos A Mitrophanous, Nicholas D Mazarakis, James B Uney  Molecular Therapy  Volume 7, Issue 5, Pages 588-596 (May 2003) DOI: 10.1016/S1525-0016(03)00069-8 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Schematic representation of the structure and processing of the AVP gene in a normal and a Brattleboro rat. In normal animals the three exons encode a prepropeptide consisting of a signal peptide (SP), the AVP hormone, the neurophysin (NPH) carrier protein, and a carboxy-terminal glycopeptide (CP). The prohormone is posttranslationally modified within secretory vesicles en route to the posterior pituitary where it is stored until secreted. In the Brattleboro rat a single base deletion in exon 2 causes a frameshift and the altered prohormone cannot be processed in the endoplasmic reticulum to produce functional AVP. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Schematic representation of the third-generation EIAV (pSMART-2) vector genomes generated in this study. All vectors contain a self-inactivating (SIN) LTR, the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a central polypurine tract (cPPT). The hatched box represents part of the EIAV packaging signal and the dotted box represents the EIAV RRE region. Expression of the EGFP (SMART2EGFP) and AVP (SMART2AVP) transgenes is under the control of the hCMV promoter. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 EIAV-mediated transfection of magnocellular neurons within the SON. (A and D) Confocal fluorescence micrographs of sections taken through the SON following transfection with the EIAV-CMV-EGFP vector two weeks after injection. (B and E) Serial sections of EIAV-CMV-AVP-transfected neurons immunocytochemically stained with anti-PS41 (which recognizes AVP-producing magnocellular neurons) and anti-GFAP (a glial cell-specific marker), respectively. The images taken in A and B and in D and E are shown overlaid in C and F, respectively. The EIAV vector mediated EGFP expression only in neurons of the SON (C); no EGFP expression was seen in glial cells (F). Scale bars represent 50 μm. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 In situ hybridization analysis of EIAV vector-mediated AVP expression. A coronal section through the hypothalamus probed for AVP mRNA is shown. EIAV-derived AVP mRNA is shown 1 month after the injection into a single hemisphere in the region of the substantia innominata. Endogenous, magnocellular AVP is also evident bilaterally in the SON. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Immunocytochemical detection of the functional AVP precursor protein in control and Brattleboro rats following injection of EIAV-CMV-AVP. EIAV-CMV-EGFP and EIAV-CMV-AVP vectors were injected into Brattleboro rats and coronal sections taken and immunocytochemically stained with the PS41 antibody, which will recognize only the (nonmutated) NPH carrier protein within a functional AVP precursor protein. Photomicrographs of the immunocytochemically stained sections were taken using a Leica DMRB microscope and image capture system. (A) Following the injection of the EIAV-CMV-EGFP vector into Brattleboro rats no NPH-positive immunostaining was detected, while many large “ghost” magnocellular cells were evident. (B) Sections taken from a Long Evans control rat shows the intense NPH-positive immunostaining in the dense population of magnocellular cells of the SON. (C) Low-power and (D) high-power photomicrographs of numerous darkly staining NPH-positive magnocellular cells in the SON of Brattleboro rats injected with 2 μl of EIAV-CMV-AVP at 2 and 8 months postinjection, respectively. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Measurements of water intake and urine volume following injection of EIAV-CMV-AVP gene therapy in AVP-deficient Brattleboro rats. (A) These results show daily urine production and daily water consumption values taken from an individual rat every week for 1 year before (pretreatment measure) and after the bilateral injection of either the EIAV-CMV-EGFP (gray bars) or the EIAV-CMV-AVP (black bars) vector. The results demonstrate that in an EIAV-CMV-AVP-treated rat there was a dramatic decrease in daily urine production and daily water consumption 1–2 days after the injection (compared to the values obtained 2 days prior to the injection of the construct and compared to an animal injected with the EIAV-CMV-EGFP vector). The results further demonstrate that the recovery in water homeostasis is maintained for 52 weeks. For comparison, measurements of daily urine production and daily water consumption taken from Long Evans control rats are also shown (continuous bold line). (B) Graphs show daily water intake and daily urine output values averaged from measurements taken every week from Brattleboro rats injected bilaterally with the EIAV-CMV-AVP vector (n = 5) or injected with the EIAV-CMV-EGFP vector (n = 4). These results demonstrate that there is a maximal decrease in urine production and water consumption 4 days postinjection of the AVP-expressing lentiviral vector. This recovery in water homeostasis was shown to be unaltered 1 year after the initial injection. Comparing values obtained from Brattleboro rats injected with the AVP-expressing lentiviral vector (black bars) with Long Evans control rats (continuous bold line) demonstrates a complete (100%) reversal of the diabetes insipidus phenotype. Statistical analysis showed that urine volume and water intake values taken from EIAV-CMV-AVP-treated rats over the 1-year measured period differed significantly from the pretreatment measures and the EIAV-CMV-EGFP-treated groups. Two-way analysis of variance gave the following values: water intake values from the EIAV-CMV-AVP group compared to values from the EIAV-CMV-EGFP group, F(1,6) = 120.2, P < 0.0001. Urine output values from the EIAV-CMV-AVP group compared to values from the EIAV-CMV-EGFP group, F(1,6) = 124.4, P < 0.0001. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Measurements of water intake and urine volume following injection of EIAV-CMV-AVP gene therapy in AVP-deficient Brattleboro rats. (A) These results show daily urine production and daily water consumption values taken from an individual rat every week for 1 year before (pretreatment measure) and after the bilateral injection of either the EIAV-CMV-EGFP (gray bars) or the EIAV-CMV-AVP (black bars) vector. The results demonstrate that in an EIAV-CMV-AVP-treated rat there was a dramatic decrease in daily urine production and daily water consumption 1–2 days after the injection (compared to the values obtained 2 days prior to the injection of the construct and compared to an animal injected with the EIAV-CMV-EGFP vector). The results further demonstrate that the recovery in water homeostasis is maintained for 52 weeks. For comparison, measurements of daily urine production and daily water consumption taken from Long Evans control rats are also shown (continuous bold line). (B) Graphs show daily water intake and daily urine output values averaged from measurements taken every week from Brattleboro rats injected bilaterally with the EIAV-CMV-AVP vector (n = 5) or injected with the EIAV-CMV-EGFP vector (n = 4). These results demonstrate that there is a maximal decrease in urine production and water consumption 4 days postinjection of the AVP-expressing lentiviral vector. This recovery in water homeostasis was shown to be unaltered 1 year after the initial injection. Comparing values obtained from Brattleboro rats injected with the AVP-expressing lentiviral vector (black bars) with Long Evans control rats (continuous bold line) demonstrates a complete (100%) reversal of the diabetes insipidus phenotype. Statistical analysis showed that urine volume and water intake values taken from EIAV-CMV-AVP-treated rats over the 1-year measured period differed significantly from the pretreatment measures and the EIAV-CMV-EGFP-treated groups. Two-way analysis of variance gave the following values: water intake values from the EIAV-CMV-AVP group compared to values from the EIAV-CMV-EGFP group, F(1,6) = 120.2, P < 0.0001. Urine output values from the EIAV-CMV-AVP group compared to values from the EIAV-CMV-EGFP group, F(1,6) = 124.4, P < 0.0001. Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Measurements of AVP-mediated water homeostasis in Brattleboro rats 1 year after EIAV-CMV-AVP treatment. (A) Urine was collected from untreated Long Evans rats, untreated Brattleboro rats, and Brattleboro rats treated with EIAV-CMV-EGFP or EIAV-CMV-AVP vectors. Urine osmolarity (U.O.) was then measured using an osmometer (Reisling). The results show mean ± SD of urine osmolarity values obtained from Brattleboro rats 1 year after the injection of the EIAV-CMV-AVP vector (n = 12) and those obtained from EIAV-CMV-EGFP (n = 12)-treated animals. (B) Measurements of urine volume and osmolarity in EIAV-CMV-EGFP control Brattleboro rats (black bars), EIAV-CMV-AVP-treated Brattleboro rats (gray bars), and Long Evans rats (light gray bars) deprived of water 1 year after EIAV injection. EIAV-CMV-AVP-treated Brattleboro rats were able to maintain significantly lower urine production and higher osmolarity levels 1 year after the initial treatment. ***P < 0.00001 compared to EIAV-CMV-EGFP-treated animals by Student t test. *P < 0.01 when EIAV-CMV-AVP-treated animals were compared to EIAV-CMV-EGFP-treated animals (data analyzed by ANOVA and post hoc Bonferroni tests). +P < 0.01 when urine osmolarity values measured after 8 h water deprivation were compared to those taken after 2 h of water deprivation (analyzed by Students t test). Molecular Therapy 2003 7, 588-596DOI: (10.1016/S1525-0016(03)00069-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions