Volume 9, Issue 4, Pages 527-539 (April 2004) Liver-Directed Adenoviral Gene Transfer in Murine Succinate Semialdehyde Dehydrogenase Deficiency  Maneesh Gupta, Erwin E.W Jansen, Henry Senephansiri, Cornelis Jakobs, O.Carter Snead, Markus Grompe, K.Michael Gibson  Molecular Therapy  Volume 9, Issue 4, Pages 527-539 (April 2004) DOI: 10.1016/j.ymthe.2004.01.013 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 The GABA metabolic pathway (not all steps are shown). Enzymes include: (1) glutamic acid decarboxylase, EC 4.1.1.15; (2) GABA transaminase, EC 2.6.1.19; (3) one (or more) oxidoreductases converting succinic semialdehyde to γ-hydroxybutyrate; and (4) succinate semialdehyde dehydrogenase (SSADH; EC 1.2.1.24; OMIM 271980), site of the inherited defect in murine and human SSADH deficiency. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Survival in SSADH−/− mice employing various strategies. (a) Single ip injection of AD:pAD-RSV-humanSSADH (108 viral particles) administered at day 10 of life. Results from AD:pAD-RSV-humanSSADH injected into SSADH+/+ (n=11) and SSADH−/− (n=28; 39% survival; P<0.001) mice, as well as control uninjected SSADH−/− mice (n=17) are shown; n, number of mice in the study group. (b) Intraperitoneal administration of 1010 viral particles (25% survival; P<003. (c) Intraperitoneal administration of 1011 viral particles (11% survival; P<0.004). (d) Retro-orbital administration of 1011 viral particles (25% survival; P<0.02). Survival in SSADH−/− animals was taken as survival beyond day 30 of life. Statistical significance was derived using log-rank test compared with control uninjected SSADH−/− mice. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 RT-PCR analysis of human SSADH RNA expression in the liver of SSADH−/− mice treated with Ad:pAD-RSV-humanSSADH. (A) RT-PCR analysis of liver RNA at various time points following rAV ip therapy (108 viral particles) of SSADH−/− mice. Upper bands, specific primers for promoter and human SSADH cDNA; lower bands, 18S rRNA internal control; marker used, 1 kb plus molecular weight ladder; SSADH band size, 612 bp; 18S rRNA band size, 324 bp. (Lane 1) Uninjected SSADH+/+ mouse, negative control for human SSADH cDNA; (lane 2) uninjected SSADH−/− mouse, negative control for human SSADH cDNA; (lane 3) SSADH−/− mouse sham injected with AD5 null, negative control for rAV DNA; (lane 4) 293 cells transfected with rAV, positive control for rAV DNA; (lane 5) 24 h posttherapy; (lane 6) 48 h posttherapy; (lane 7) 72 h posttherapy; (lane 8) 5 days posttherapy; (lane 9) 7 days posttherapy; (lane 10) survivor (50 days of age). Each lane represents an individual mouse. (B) RT-PCR analysis of livers derived from retro-orbitally treated mice. Upper bands, SSADH-specific signal; lower bands, 18S-specific signal. (Lanes 1–7) Animals administered 1010 viral particles; (lanes 8–14) animals administered 109 viral particles. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 SSADH enzyme activity in SSADH−/− mice treated with AD:pAD-RSV-humanSSADH via ip and retro-orbital administration. (A) Liver SSADH activity in neonatal (day 0) SSADH−/− mice injected ip with rAV-SSADH (108 viral particles) and harvested at 24 and 72 h. (B) Liver SSADH activity in 10-day-old SSADH−/− mice injected ip with rAV-SSADH (108 viral particles) and harvested at 48 h, 72 h, and 5 days and surviving mice harvested at various ages (34–150 days of age). (C) Liver and brain SSADH activity in 13-day-old SSADH−/− mice injected retro-orbitally with rAV-SSADH (109 and 1010 viral particles) and harvested at 72 h; n, number of mice injected; SSADH activity expressed as nmol/h/mg protein; a% SSADH activity in treated SSADH−/− mice compared to age-matched SSADH+/+ control mice. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Histochemical staining for SSADH activity in livers of untreated SSADH+/+, untreated SSADH−/−, and treated SSADH−/− mice. (A) control medium (medium with no substrate SSA and NAD+) in untreated SSADH+/+ liver. (B) Medium M (medium with malonate) in untreated SSADH+/+ liver. (C) Medium MP (medium with malonate and parahydroxybenzaldehyde) in untreated SSADH+/+ liver. (D) Control medium with untreated SSADH−/− liver. (E) Medium M with untreated SSADH−/− liver. (F) Medium MP with untreated SSADH−/− liver. (G–I) Medium M with liver derived from three different SSADH−/− mice treated with 1010 viral particles. Original magnification 10× for all sections. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 GHB levels in tissues of SSADH−/− mice treated ip at day 0 or day 10 of life. (A) Day 0 viral administration and harvest of liver at 24 h, 72 h, and 8 days postinfusion compared to age-matched SSADH−/−controls. (B) Same as in A but brain tissue (the same animals were used for determination of liver and brain GHB levels). (C) Day 10 ip administration (108 viral particles, liver) with harvest at 48 h, 72 h, and 5 days postinfusion compared to age-matched SSADH−/− controls; GHB levels also shown for surviving SSADH−/− mice (34–150 days of age). (D) Same as C, but brain tissue. GHB expressed in μmol/100 mg protein; n, number of mice; *P<0.05, **P<0.01, ***P<0.001 compared to untreated SSADH−/− mice. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 GHB levels 72 h posttreatment in serum and tissues of SSADH−/− mice (13 days of age) treated retro-orbitally with AD:pAD-RSV-humanSSADH. (A) GHB levels in liver, brain, and kidney of SSADH−/− mice treated retro-orbitally with rAV-SSADH in comparison to untreated SSADH−/− mice (n=4). (B) Serum GHB levels from the same animals in A. GHB levels expressed in μmol/L; n, number of mice; statistical significance depicted as **P<0.01 and ***P<0.001 in comparison to SSADH−/− uninjected mutants. v.p., viral particles. Molecular Therapy 2004 9, 527-539DOI: (10.1016/j.ymthe.2004.01.013) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions