1. Volume 20, Issue 5, Pages 898-907 (May 2012)
Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats 
Katherine P Ponder, Thomas M O'Malley, Ping Wang, Patricia A O'Donnell, Anne M Traas, Van W Knox, Gustavo A Aguirre, N Matthew Ellinwood, Jason A Metcalf, Bin Wang, Emma J Parkinson-Lawrence, Meg M Sleeper, Doug A Brooks, John J Hopwood, Mark E Haskins 
Molecular Therapy 
Volume 20, Issue 5, Pages (May 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Retroviral vector (RV) diagram. hAAT-f4S-WPRE contains intact long-terminal repeats (LTRs), an extended packaging signal (ψ+), the human α1-antitrypsin (hAAT) promoter, the feline N-acetylgalactosamine 4-sulfatase (f4S) cDNA, and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The arrows indicate that transcription can initiate from the LTR or the hAAT promoter.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Serum4-sulfatase (4S) activity. Mucopolysaccharidosis (MPS) VI cats shown in the left column of the key were injected intravenously (i.v.) with 3–6 × 109 transducing units (TU)/kg of hAAT-f4S-WPRE at 2–4 days after birth, and are designated group 1 in the text and are depicted with open symbols. MPS VI cats shown in the right column were injected i.v. with 1–2 × 1010 TU/kg of a different batch of vector, and are designated group 2 in the text, and are depicted with closed symbols. Serum was tested for 4S activity using the substrate 4-methylumbelliferone-sulfate (4MU-S) at the indicated years after birth. Each line shows activity for an individual cat. Mean values ± 1 SD in adult homozygous normal cats were 8.2 ± 2.6 U/ml and those in untreated MPS VI cats were 2.6 ± 1.0 U/ml as indicated with gray and yellow boxes, respectively, in the online color version, and as indicated by the dark and light grey boxes in the print version. Individual cats are identified with different colors in the print version.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Biochemistry of liver, other organs and urine. Samples from liver of all six of the group 1 retroviral vector (RV)-treated cats were obtained at biopsy or postmortem at 4–7 months. Samples from other tissues of two RV-treated cats from group 1, 6600 and 6613, were collected at 7 and 6 months, respectively. Urine samples were collected at 0.5–2.5 years after birth. (a–c) Analysis of crude extracts of organs. The mean ± SD of 4-sulfatase (4S) activity without immunocapture (no IP), β-glucuronidase (GUSB) activity, and glycosaminoglycan (GAG) levels per mg of protein was determined as detailed in the Materials and Methods section. A large X instead of data for a particular organ indicates that an assay was not performed for that organ. Samples from homozygous normal (N = 3) and homozygous mucopolysaccharidosis (MPS) VI (N = 4) cats were age-matched. (d) Urine GAG. Urine GAGs were normalized to the amount of creatinine in the sample for four normal, four MPS VI, and six RV-treated MPS VI cats. (e) Organ 4S activity after immunocapture. Samples were immunocaptured with a feline 4S-specific antibody, and the feline 4S activity per mg wet weight of the original sample was determined with a 4-methylumbelliferone-sulfate (4MU-S) substrate. Samples from three normal, four MPS VI, and two RV-treated MPS VI cats were evaluated. For all biochemical assays in (a–e), *P of 0.01–0.05 and **P < 0.01 for comparison of the two groups linked with a bracket using ANOVA with Tukey post-hoc analysis. (f) Liver 4S protein levels. Immunoblot was performed for feline 4S protein levels using liver samples from normal, untreated MPS VI, or RV-treated MPS VI cats. After probing for feline 4S, the blot was stripped and reprobed for the liver protein C/EBPβ for normalization. Samples from RV-treated cats are organized from left to right according to lowest to highest liver 4S activity. The position of the endogenous 65 kDa feline 4S band found in normal and MPS VI cats (Endog 4S) and the 68 kDa band found in RV-treated cats (RV-4S) are indicated. (g) Liver 4S activity versus 4S protein. For individual RV-treated MPS VI cats, liver 4S activity was plotted versus liver protein levels. The latter was determined from scanning the immunoblot for feline 4S protein with normalization to the signal for C/EBPβ. The identification number for each cat is indicated at the left. A linear regression line for the data is shown with the R2 value.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Retroviral vector (RV) nucleic acid levels in RV-treated cats. (a) RV DNA levels. RV DNA levels were determined by real-time PCR for the WPRE of the RV at 4–6 months after transduction for samples from group 1 RV-treated mucopolysaccharidosis (MPS) VI cats, and normalized to the copies of β-actin to determine the copies per cell. Five samples from group 1 were evaluated from liver (values for cat 6437 are not shown as the DNA was of poor quality), 2 samples were evaluated from spleen (6600 and 6613), and 6 samples from group 1 were evaluated from white blood cells (WBC). (b) RV RNA levels. RNA was evaluated for the same samples as in a, except liver samples from cats 6437 and 6438 were not included due to poor RNA quality. RV DNA and RNA sequences were not detected for samples from normal and untreated MPS VI cats (data not shown).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Facial appearance, body weights, and skeletal radiographs. (a–d) Facial appearance. The normal cat (a) was photographed at 6 years of age. An untreated mucopolysaccharidosis (MPS) VI cat (b) has short ears and a broad face at 1 year. Two retroviral vector (RV)-treated cats from the same litter as the untreated MPS VI cat (c–d) look essentially normal at 1 year. (e) Body weight at 5 months. Body weights for the indicated number of cats in each group were determined separately for male and female animals at 5 months of age. Values in normal and RV-treated MPS VI cats were compared with those in untreated MPS VI cats of the same gender using ANOVA with Tukey post-hoc analysis; *P = 0.01–0.05, and **P < (f) Femur and C3 bone lengths. The length of the femur and height of the third cervical vertebrae (C3) were measured as detailed in the Methods section from radiographs obtained at 0.7–2 years after birth, and the ratio to normal determined for the indicated number of cats of each group and the mean ± SD determined. For the RV-treated MPS VI cats, the average serum 4-sulfatase (4S) activity in the cats that were evaluated here was 126 ± 66 U/ml (15-fold normal). (g) Cervical spine radiograph scores. Radiographs like those shown in panels h to o obtained at 2–3 years after birth were scored for abnormalities in the cervical spine, where 0 represents normal and +2 represents severely abnormal. This age was chosen as more untreated MPS VI (N = 4) and RV-treated MPS VI (N = 3) cats were available for analysis at this age than when older. Values that are statistically different between untreated MPS VI and RV-treated MPS VI cats are indicated. Values for normal cats are generally 0, but are not shown here. (h–o) Representative images of the cervical spine. Radiographs were obtained at 6 years after birth (as indicated with 6Y in the lower right corner), to illustrate better the ability of gene therapy to result in long-term bone improvements. The top panels show ventrodorsal images, while the bottom panels show lateral images. The position of the second cervical vertebrae (C2) is shown, and the cranial aspect is at the top for all images. Beaking refers to a bony proliferation of the caudoventral aspect of the vertebral body and is best seen in the lateral radiograph in panel n, where it is identified with a white arrow. Beaking is present but is more difficult to identify in panels m and o due to a reduced bone quality in these animals. Tipping refers to a rotation of one vertebral body relative to the other, so that the cranial and caudal aspects do not align; tipping is difficult to identify in these particular radiographs. Articular facet widening refers to an increased diameter of the articular facets, which are identified with the black brackets for C4 on the ventrodorsal images; widening is best appreciated in panel i. Facet fusion refers to a fusion of the bone in one articular facet with that in the adjacent one. The black arrows identify bony bridges between C2 and C3 in panels m and o. In addition, the dorsal to ventral dimensions of the laminae are markedly increased in the untreated MPS VI cat, and increased to a lesser extent in the RV-treated cats. Vertebral body shortening can be seen as the loss of vertebral body length, a feature best appreciated on the lateral image of untreated and RV-treated MPS VI cats. Widening of the vertebral bodies can also be appreciated on the ventrodorsal images of both untreated and RV-treated MPS VI cats. (p) Radiograph scores of the carpal region. Radiographs of the carpal (wrist) region were scored at 2–3 years of age for abnormalities in four untreated MPS VI and 3 RV-treated MPS VI cats, where 0 represents normal and +2 represents markedly abnormal. The carpus was scored for joint laxity, soft tissue swelling or joint effusions, articular cartilage erosion,abnormal carpal shape, bone lucency, epiphyseal dysplasia or lucency, and long bone shortening or curving. Values in normal cats are generally 0.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Magnetic resonance imaging (MRI) of the cervical spine. MRIs were obtained at the indicated age. For the retroviral vector (RV)-treated mucopolysaccharidosis (MPS) VI cats 6437 and 6598, the average serum 4-sulfatase (4S) activity is indicated. A sagittal view is shown in the left panels, with the dorsum at the top and ventrum at the bottom; the cerebellum (C), the intervertebral discs (IVD) at C2-C3 and C3–C4, and the spinal cord (SC) are indicated. For the untreated MPS VI cat, the thin white arrow identifies compression of the spinal cord due to dorsal dens angulation and ligamentous hypertrophy. The right panels show transverse images. The spinal cord (SC) and the spinous (S) and transverse (T) processes of the vertebrae are indicated.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Echocardiograms. (a–i) Representative images of echocardiograms. Echocardiograms were obtained from normal, untreated mucopolysaccharidosis (MPS) VI, or retroviral vector (RV)-treated MPS VI cats as indicated at 6 years of age, as noted by the term 6Y in the lower right corner, to allow abnormalities a maximal time to develop. (a–c) Color Flow Doppler of the aortic valve during diastole. These panels show color Flow Doppler analysis of the aortic valve during diastole. The left ventricle (LV), aorta (Ao) and a size marker for 1 cm are indicated. A regurgitant jet across the aortic valve appears multicolored and is best visualized in the untreated MPS VI cat in panel b. (d–f) Short axis 2D images of the aortic valve during diastole. The aorta is seen at the level of the sinus of Valsalva. The yellow arrows identify the coaptation of the three leaflets in some panels. For the untreated MPS VI cat in panel e, marked thickening of the leaflets is seen and the diameter of the aorta is markedly increased. (g–i) Long axis 2D images of the aorta during diastole. The inner diameter of the aorta at the sinotubular junction is indicated by the double-pointed arrow. The aorta at this position is dilated for the untreated MPS VI cat in panel h. (j) Aortic diameters. The aortic diameter was obtained at the sinus of Valsalva (SV) or the sinotubular junction (STJ) at 3 years of age, when more RV-treated cats were available for evaluation (four normal, five untreated MPS VI, and three RV-treated MPS VI cats). Values were compared in other groups with those in untreated MPS VI cats using ANOVA with Tukey post-hoc analysis. (k) Echocardiogram scores. Echocardiograms were obtained at 3 years of age, and were scored subjectively for aortic valve thickening, aortic valve regurgitation, mitral valve thickening and mitral valve regurgitation from 0 (normal) to +4 (severely abnormal); the mean ± SD are shown. Values in other groups were compared with those in untreated MPS VI cats using ANOVA on ranks; *P value 0.01–0.05 and **P value of <0.01.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions