1. Volume 13, Issue 6, Pages 1110-1120 (June 2006)
In Vivo Transduction by Intravenous Injection of a Lentiviral Vector Expressing Human ADA into Neonatal ADA Gene Knockout Mice: A Novel Form of Enzyme Replacement Therapy for ADA Deficiency 
Denise A. Carbonaro, Xiangyang Jin, Denise Petersen, Xingchao Wang, Fred Dorey, Ki Soo Kil, Melissa Aldrich, Michael R. Blackburn, Rodney E. Kellems, Donald B. Kohn 
Molecular Therapy 
Volume 13, Issue 6, Pages (June 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Map of the SMPU-R-MND-huADA, titer determination, and experimental timeline of treatment and analysis. (A) The SMPU-R-MND-huADA vector, derived from HIV-1, has a self-inactivating LTR, a minimal gag region, the central polypurine tract, and three tandem copies of the SV40 UES polyadenylation enhancer to augment polyadenylation of vector. The location of primers and probe for real-time PCR analysis are shown. (B) Lentiviral vector titer was determined by Southern blot analysis of HT29 cells transduced with dilutions of concentrated viral supernatant at 1:500, 1:5,000, or 1:50,000. A radiolabeled probe to the huADA cDNA was used, and densitometry was used to compare the vector bands to those of a standard clonal cell population. The titer of the concentrated vector preparation was determined to be 1.0 × 1010 TU/ml. (C) Experimental timeline. PB, peripheral blood.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Survival in ADA-deficient mice after receiving neonatal injections of lentiviral vector. ADA-deficient mice were either untreated (n = 7) or injected with either 1.0 × 107 TU (n = 7) or 1.0 × 108 TU (n = 10) per pup. Survival of the animals was tracked to day +180, at which time the animals were euthanized (n = 5) for tissue analysis (see Fig. 1C). Survivorship was subjected to Kaplan-Meier analysis. Of the mice receiving 1.0 × 108 TU of the vector, some mice (n = 5) had received ERT with PEG-ADA (300 U/kg weekly) until day 45 and some had not (n = 5). Three mice died, apparently with disease-related symptoms of respiratory distress and wasting (n = 2 received ERT; n = 1 received no ERT), one mouse died from apparent malocclusion (ERT), and one mouse was euthanized at day +180 but determined to have a liver tumor (no ERT) and was not analyzed.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Peripheral blood leukocyte proviral marking after neonatal injection of lentiviral vector. (A) Peripheral blood samples were collected at day 30 from mice that received 1.0 × 107 TU/pup and from mice that received 1.0 × 108 TU/pup. qPCR on leukocyte DNA was performed to determine the number of proviral copies per cell using primers and probe (see Fig. 1A) that were located in the 3′ end of the huADA cDNA and in the vector backbone. (B) Peripheral blood samples were collected at day +30 (n = 11), day +60 (n = 15), day +120 (n = 10), and day +180 (n = 7).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Proviral marking in tissues 2 and 6 months after neonatal injection of lentiviral vector. Mice were euthanized and tissues were harvested and analyzed at either 2 months (2m; n = 5) or at 6 months (6m; n = 5). Of the five animals analyzed at 6 months, two mice had received weekly ERT with 300 U/kg of PEG-ADA until day +45; three mice received no ERT. From four of the mice analyzed at 6 months (ERT n = 1; no ERT n = 3), cells were isolated using immunomagnetic separation from the thymus (CD4+CD8+) and spleen (CD19+). qPCR was performed to determine the number of proviral copies per cell. Predicted means from general linear regression is shown by the gray horizontal bars. bm, Bone marrow.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
ADA enzyme activity in ADA-deficient mice 2 months after neonatal injection of lentiviral vector. (A) Mice were euthanized, and blood, thymus, spleen, lung, and liver were harvested and analyzed at 2 months (n = 5; see legend of Fig. 4). Predicted means from general linear regression is shown by the gray horizontal bars. (B) Zymogram analysis was used to detect ADA enzyme activity. Analysis was performed on the samples from one wild-type mouse, one ADA-deficient mouse treated with ERT, and two mice that received the lentiviral vector injection as neonates and had gene marking levels in the liver of 1 and 12 copies per cell, respectively.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Immunoreactive ADA in livers and corresponding proviral copy number and ADA enzyme activity. (A) Wild-type (ADA+/+) liver section stained with an antibody specific to ADA protein. (B–F) Sections of liver from ADA-deficient (ADA–/–) mice 2 months after neonatal injection of lentiviral vector. Proviral copy numbers as determined by qPCR (Fig. 4) and corresponding ADA enzyme activities (Fig. 5) are also shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Immunoreactive ADA protein in liver and lung shows focal distribution of transduced cells. (A, B) Liver sections from an ADA-deficient mouse 2 months after neonatal injection of lentiviral vector expressing huADA were stained with an antibody specific for human ADA (C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Magnification: ×200 (A); ×400 (B). (C, D) Lung sections from an ADA-deficient mouse 2 months after neonatal injection of lentiviral vector expressing huADA were stained with an antibody specific for human ADA (C-20). Magnification: ×400 (C); ×630 (D).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

9. FIG. 8
Absolute numbers of thymocyte and splenocyte populations in ADA-deficient mice. The absolute numbers of thymocyte and splenocyte populations were determined in wild-type mice (day +60, n = 3), untreated ADA-deficient mice (day +16, n = 5), ADA-deficient mice on PEG-ADA ERT with weekly injections of 300 U/kg (day +60, n = 7), ADA-deficient mice at day +60 after receiving neonatal vector (n = 5). Fluorescence-activated cell sorting (FACS) analysis was performed on cells stained with the specific antibodies indicated. The total numbers were calculated by multiplying the total numbers of cells by the percentage of cells in each population divided by 100. Predicted means from linear regression analysis are plotted in inset graphs for each figure. There is a significant difference when the values for the predicted means differ between treatments within a cell population. (A) Whole thymus and spleen were made into a cell suspension through a 70-μm mesh and counted with a hemocytometer. (B) Thymocytes were stained with anti-CD4-PE and anti-CD8-APC and analyzed by FACS. (C) Splenocytes (s) were stained with anti-CD4-PE and anti-CD8-APC, or with anti-CD19-PE and anti-CD45-APC, and analyzed by FACS. (D) Lymphocyte proliferative function was assessed by stimulating splenic T cells with ConA for 48 hours and pulsing with [3H]thymidine for 20 hours. Cells were harvested and counted for incorporated [3H]thymidine (counts per minute, CPM). The stimulation index was calculated by dividing the CPM (in triplicate) of cells stimulated with ConA by the CPM (in triplicate) of cells not stimulated.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions