1. Volume 11, Issue 5, Pages 776-789 (May 2005)
Neonatal Gene Therapy of MPS I Mice by Intravenous Injection of a Lentiviral Vector 
Hiroshi Kobayashi, Denise Carbonaro, Karen Pepper, Denise Petersen, Shundi Ge, Holly Jackson, Hiroyuki Shimada, Rex Moats, Donald B. Kohn 
Molecular Therapy 
Volume 11, Issue 5, Pages (May 2005)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
(A) Map of the SMPU-R-MND-huID-IE lentiviral vector provirus. The SMPU-R vector backbone is derived from HIV-1 with a SIN LTR (ΔU3), a minimal gag region, the central polypurine tract (cPPT), and the SV40 polyadenylation enhancer added in triplicate (UESx3) to augment polyadenylation of vector transcripts, with a minimal RRE. The HIV-1 packaging sequence (ψ) is indicated. The vector contains the MND LTR U3 region (MND) as an internal promoter driving expression of a normal human α-iduronidase cDNA (IDUA) [25] followed by the EMCV internal ribosome entry site (I) followed by the enhanced green fluorescent protein (eGFP) gene. The expected vector transcript is shown by the arrow. (B) Experimental design for in vivo gene transfer studies. We injected the SMPU-R-MND-huID-IE vector via the facial vein into 1-day-old neonatal MPS I mice (FVI group) and into 8-week-old young adults via the tail vein route (TVI group), using the same weight-adjusted dosage of 1.65 × 1011 TU/kg. We also injected a second group of 8-week-old MPS I mice with a lower dosage of the vector (6.88 × 109 TU/kg). Mice were sacrificed at the same chronological age, 20 weeks, and analytic studies were performed. For long-term follow-up, three mice in the FVI group and two mice in the TVI group were studied in parallel with age-matched normal (wild type, n = 3) and MPS I disease control groups (not treated, n = 3) by X ray to evaluate the skeletal system and life span.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
In vitro analysis of SMPU-R-MND-huID-IE vector. We used the SMPU-R-MND-huID-IE vector to transduce a panel of cell lines, including HepG2 (hepatoma), SVG (SV40 transformed glioblastoma), A549 (lung carcinoma), Panc-1 (pancreas adenocarcinoma), C2C12 (murine myoblast), and GM00415 (fibroblast cell line from a patient with Hurler syndrome), at m.o.i. of 20, 2, and 0.2 TU/cell. (A) IDUA enzyme activity in transduced cell lines. IDUA enzyme activity was investigated 3 days after transduction using a fluorogenic substrate assay. Significant dose-dependent overexpression of IDUA enzyme activity was seen in all cell lines. The differences in the average IDUA enzyme activity in all cell lines transduced at each m.o.i. were significant, with P values as shown. Error bars show the standard errors of the mean (SEM). (B) eGFP expression in transduced cell lines. The percentage of cells expressing eGFP was studied 3 days after transduction, using flow cytometry. The frequency of eGFP expression was dose-dependent, with significant differences between the average percentage eGFP-positive cells at each m.o.i. Error bars show the SEM. (C) IDUA enzyme activity and eGFP expression in transduced human fibroblasts over 6 weeks. Human fibroblasts from a 5-year-old Hurler patient (GM00415) were transduced with the SMPU-R-MND-huID-IE vector at an m.o.i. of 20. The normal control cells were human fibroblasts from a normal age-matched control (GM00497). We detected stable overexpression of IDUA (10- to 30-fold) and of eGFP for up to 6 weeks. Error bars show the SEM. (D) Fluorescent microscopy of eGFP expression in transduced human fibroblasts. The transduced GM00415 fibroblasts show expression of eGFP protein by fluorescence microscopy, which was stable for at least 5 weeks.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 2
In vitro analysis of SMPU-R-MND-huID-IE vector. We used the SMPU-R-MND-huID-IE vector to transduce a panel of cell lines, including HepG2 (hepatoma), SVG (SV40 transformed glioblastoma), A549 (lung carcinoma), Panc-1 (pancreas adenocarcinoma), C2C12 (murine myoblast), and GM00415 (fibroblast cell line from a patient with Hurler syndrome), at m.o.i. of 20, 2, and 0.2 TU/cell. (A) IDUA enzyme activity in transduced cell lines. IDUA enzyme activity was investigated 3 days after transduction using a fluorogenic substrate assay. Significant dose-dependent overexpression of IDUA enzyme activity was seen in all cell lines. The differences in the average IDUA enzyme activity in all cell lines transduced at each m.o.i. were significant, with P values as shown. Error bars show the standard errors of the mean (SEM). (B) eGFP expression in transduced cell lines. The percentage of cells expressing eGFP was studied 3 days after transduction, using flow cytometry. The frequency of eGFP expression was dose-dependent, with significant differences between the average percentage eGFP-positive cells at each m.o.i. Error bars show the SEM. (C) IDUA enzyme activity and eGFP expression in transduced human fibroblasts over 6 weeks. Human fibroblasts from a 5-year-old Hurler patient (GM00415) were transduced with the SMPU-R-MND-huID-IE vector at an m.o.i. of 20. The normal control cells were human fibroblasts from a normal age-matched control (GM00497). We detected stable overexpression of IDUA (10- to 30-fold) and of eGFP for up to 6 weeks. Error bars show the SEM. (D) Fluorescent microscopy of eGFP expression in transduced human fibroblasts. The transduced GM00415 fibroblasts show expression of eGFP protein by fluorescence microscopy, which was stable for at least 5 weeks.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 3
Evaluation of IDUA enzyme expression, vector biodistribution, and sGAG storage in organs from mice treated with intravenous lentiviral vector. (A) IDUA enzyme activity at 20 weeks of age in MPS I mice treated as neonates by facial vein injection of vector or by tail vein injection at 8 weeks of age. Mice were injected with the SMPU-R-MND-huID-IE vector as neonates by facial vein at 1.65 × 1011 TU/kg (FVI, n = 7) or as 8-week-old adults by tail vein at the same dosage/kg (TVI, n = 3) or as 8-week-old adults by tail vein at a lower dosage of 6.88 × 109 TU/kg (TVI-low, n = 5). They were sacrificed at 20 weeks of age and IDUA enzyme activity was measured in the listed organs and reported as a percentage of the activity compared to organs from two normal control (IDUA+/+) littermates. Error bars show the SEM. The IDUA enzyme activity was significantly higher in spleen, kidney, heart, and brain, but not liver, in the neonatal FVI group compared to the TVI group (P < 0.05, Mann–Whitney U test). (B) Biodistribution of SMPU-R-MND-huID-IE vector provirus in organs measured by real-time PCR analysis. DNA was extracted from the organs and the vector copy number per cell was determined by real-time PCR. The vector copy numbers were significantly higher in the brain of the neonatal FVI group (n = 7) compared to the adult TVI group (n = 5) (P = 0.018, Mann–Whitney U test). Error bars show the SEM. (C) Sulfated glycosaminoglycan (sGAG) storage in organs. Sulfated glycosaminoglycans were measured in the organs of MPS I mice at 20 weeks of age that had been treated as neonates by facial vein injection of the vector (FVI) or as young adults by tail vein injection using 1.65 × 1011 TU/kg (TVI) and in MPS I mice that were not treated (NT, n = 4) and in normal littermates (WT, n = 4). There was a significant reduction of sGAG storage in all five organs of the mice from the FVI group compared to the TVI and NT groups (P < 0.05 by Mann–Whitney U test). The reduction in sGAG storage in the mice treated as young adults (TVI group) did not achieve statistically significant difference compared to the levels in MPS I mice that were not treated.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 4
Morphologic assessment of lysosomal storage in MPS I mice. (A) Toluidine blue staining for lysosomal storage. Tissue samples were taken from mice at 20 weeks of age and stained with toluidine blue and photographed by microscopy (original magnification ×400). The first row shows organs from a normal wild-type (WT) mouse with no lysosomal accumulation. The second row shows organs from an untreated MPS I mouse (NT) with lysosomal inclusions in Kupffer cells (white arrows) and hepatocytes (black arrows) in the liver, macrophages (white arrows) in the red pulp in the spleen, macrophages (white arrows) in the interstitial tissue in the heart, tubular epithelial cells (black arrows) and macrophages (white arrows) in the kidney, and neurons (black arrows) in the brain. The third row shows organs from an MPS I mouse treated as a young adult by tail vein injection (TVI). Cells in those organs contain similar or slightly reduced numbers/amounts of lysosomal inclusions (macrophages indicated by white arrows; hepatocytes, renal tubular epithelial cells, and neurons indicated by black arrows) compared to the MPS I mice that were not treated (NT mice, as shown in the second row). The fourth row shows samples from an MPS I mouse treated as a neonate by facial vein injection (FVI), with marked improvement in morphology compared to NT or TVI mice and only minimal lysosomal inclusions detectable in some macrophages. (B) Electron microscopic analysis of ultrastructural features of the liver. Liver samples were prepared for electron microscopy (original magnification ×6000) from one mouse of each group (FVI, TVI, NT, and WT) that showed representative pathology (in the toluidine blue-stained sections). The upper left shows a liver sample from a normal (WT) mouse, the upper right from an MPS I mouse that was not treated (NT), the lower left from an MPS I mouse treated as a young adult (TVI), and the lower right from an MPS I mouse treated as a neonate. The liver from the untreated MPS I mouse and the MPS I mouse treated as a young adult contained numerous lysosomal inclusions in both hepatocytes (white arrowheads) and Kupffer cell (arrow). In contrast, the liver from the MPS I mouse treated as a neonate demonstrated only small, scattered collections of lysosomal inclusions in hepatocytes (white arrowhead).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 5
Immunohistochemistry of brain sections from an MPS I mouse that had received neonatal gene therapy with the SMPU-R-IE lentiviral vector. Sections were prepared from the brain of an MPS I mouse that received neonatal injection of the SMPU-R-MND-huID-IE vector (original magnification ×250). Samples were from the cortical region (images 1–6) and from the hippocampus (images 7–12) of the MPS I mouse and from the same regions of the brain of an untreated normal mouse (images 13, 14 cortex; 15, 16 hippocampus). The sections were stained with antibodies to the eGFP protein (green) and to lineage-specific markers for either terminally differentiated neurons (NeuN—red) or astrocytes (GFAP—red), and nuclei were counterstained with DAPI. The two fluorescent signals are merged in images 3, 6, 9, 12, and 13–16.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 6
Long-term effects on appearance and skeletal development of the MPS I mice. (A) Photographs of 40-week-old MPSI I mice that were not treated or were treated as neonates by facial vein injection of the SMPU-R-MND-huID-IE vector. The MPS I mice that were not treated had blunted snouts and also displayed poor grooming. In contrast, the mice treated as neonates were normal appearing. (B) X rays of the skulls of normal control and MPS mice. Examples of skull radiographs used to measure the effects of gene transfer on skeletal abnormalities. The white arrows in the leftmost image (WT) indicate the zygomatic arch at the level of the frontal suture. (C) The average width of the zygomatic arches measured at 20 weeks of age. The zygomatic arch thickness was not significantly different between the MPS I mice treated as neonates (FVI, n = 4) and the normal control mice (WT, n = 4) (P = 0.132) at 20 weeks of age. The arch thicknesses were significantly thinner in the mice treated as neonates (FVI) compared to the MPS I mice that were treated as young adults by tail vein injection (TVI, n = 2) (P = 0.023) and the MPS I mice that were not treated (NT, n = 3) (P = 0.022). (D) Longitudinal measurements of the zygomatic arch thicknesses. X rays were taken at serial time intervals to permit measurements of zygomatic arch thickness. There was a significant reduction in the zygomatic arch thickness in the MPS I mice treated as neonates (FVI group, n = 3) at 20, 30, and 40 weeks of age, compared to the MPS I mice that were not treated (NT group, n = 3). In contrast, the thickness of the arch in the MPS I mice treated as neonates (FVI group) did not differ significantly different from that of the normal control mice (WT group) (*P < 0.05 using Student t test). MPS I mice treated as young adults by tail vein injection (TVI group, n = 2) were evaluated only at 20 weeks and showed arch thickness similar to that of untreated MPS I mice. Error bars show the SEM.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

9. Fig. 7
Survival curve. The cumulative survival rate was investigated using the Kaplan–Meier method in each group of normal control mice (WT, n = 9), untreated MPS I disease control (NT, n = 11), untreated heterozygous littermates (n = 28), and MPS I mice treated in the neonatal period (FVI, n = 4). Note that at the age of 350 days, all the MPS I mice treated as newborns were alive; in contrast, the cumulative survival rate of untreated MPS I mice was 27.78%.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions