Volume 19, Issue 2, Pages 243-250 (February 2011) Sustained Enzymatic Correction by rAAV-Mediated Liver Gene Therapy Protects Against Induced Motor Neuropathy in Acute Porphyria Mice  Carmen Unzu, Ana Sampedro, Itsaso Mauleón, Manuel Alegre, Stuart G Beattie, Rafael Enríquez de Salamanca, Jolanda Snapper, Jaap Twisk, Harald Petry, Gloria González-Aseguinolaza, Julio Artieda, María Sol Rodríguez-Pena, Jesús Prieto, Antonio Fontanellas  Molecular Therapy  Volume 19, Issue 2, Pages 243-250 (February 2011) DOI: 10.1038/mt.2010.210 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Therapeutic efficacy of the rAAV2/8-hPBGD vector in AIP mice. Porphyrin precursor excretion [(a) females, (b) males] and motor coordination [(c) females, (d) males] were determined in mice before (baseline) and after biochemical induction of porphyrin precursors with increasing doses of phenobarbital for four consecutive days. Motor coordination was determined by the time that mice can stay on a rotating dowel turning at a positive acceleration. The first bar is baseline; the second bar represents measurements performed 15 days after the administration of a respective dose of rAAV2/8 vector and after phenobarbital administration. The third and forth bars show the same procedure performed 28 and 90 days after respective dose of gene therapy, respectively. Panels c and d have extra bars corresponding to baseline values from noninjected wild-type mice. The Wilcoxon signed-rank test was used for comparison of means before and after phenobarbital administrations. *P < 0.05; **P < 0.01; ***P < 0.001 versus baseline values in each group. AIP, acute intermittent porphyria; ALA, δ-aminolevulinic acid; PBG, porphobilinogen; rAAV, recombinant adeno-associated virus; WT, wild type. Molecular Therapy 2011 19, 243-250DOI: (10.1038/mt.2010.210) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Dose-dependent increase of functionally active hepatic hPBGD 3 months after the administration of rAAV2/8-hPBGD. The figure shows the expression of PBGD obtained after different gene doses of recombinant PBGD; measured as enzyme activity in (a) female and (b) male, by (c) immunoblot assay and (d) immunohistochemistry. A polyclonal antibody against PBGD, generated in our laboratory, recognized both human and murine PBGD. Both proteins show a different pattern of migration when separated on a 12% sodium dodecyl sulfate–polyacrylamide gel. It allows for differentiation between endogenous mouse and exogenous human PBGD, as illustrated in c. The micrographs illustrated in d, are representative for immunochemical analysis of livers from animals injected with increasing doses of rAAV2/8-hPBGD (and controls). Values in the upper left corner represent the mean percentage (±SD) of brown stained PBGD-positive cells calculated in each group of mice (additional detail in Supplementary Materials and Methods). All the analyses are performed in mice 3 months after gene therapy. The results obtained with different rAAV2/8 doses are statistically compared to the noninjected AIP mice (&&P < 0.01; &&&P < 0.01) and also to the wild type animals (**P < 0.01; ***P < 0.001) using the Mann–Whitney test. AIP, acute intermittent porphyria; URO, uroporphyrin; rAAV, recombinant adeno-associated virus; WT, wild type. Molecular Therapy 2011 19, 243-250DOI: (10.1038/mt.2010.210) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Number and caliber of axons in the sciatic nerve from wild-type and AIP mice injected with different doses of rAAV2/8-hPBGD. The number of degenerated axons [(a) female, (b) males] and the mastocyte counts [(c) females, (d) male] were measured in light micrographs sections (see Supplementary Materials and Methods) of sciatic nerves from mice 90 days after respective dose of gene therapy. Mastocyte cells were readily differentiated based upon morphological criteria and the presence of metachromatic granules. (e) Axon density and percentage of large axons were also measured in the same animals. The results obtained with different rAAV2/8 doses are statistically compared to the baseline level in the AIP group (&P < 0.05; &&P < 0.01) and also to the wild-type animals (*P < 0.05; **P < 0.01) using the nonparametric Mann–Whitney test. AIP, acute intermittent porphyria; rAAV, recombinant adeno-associated virus; WT, wild type. Molecular Therapy 2011 19, 243-250DOI: (10.1038/mt.2010.210) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 The impact on sciatic nerve function caused by high levels of porphyrin precursors. (a) The compound-muscle action potentials evoked by proximal stimulation of the sciatic nerve (left, latency; right, amplitude) were measured before and after seven consecutive attacks (induced biweekly by the administration of four increasing doses of phenobarbital). AIP animals at different ages are compared to wild-type and to those AIP mice receiving rAAV2/8-hPBGD, using the Mann–Whitney test (&P < 0.05; &&&P < 0.001 versus old AIP mice and *P < 0.05; **P < 0.01; ***P < 0.001 versus wild-type animals). (b) The figure shows the impact on the sciatic nerve function (left, latency; right, amplitude) caused solely by PBGD deficiency, by intravenous (i.v.) administration of ALA (3 mmol/kg/day for 4 consecutive days) or by high levels of porphyrin precursors induced by phenobarbital (after four consecutive doses). The compound-muscle action potentials were measured in the two hind legs of each of the animals. Comparisons are performed between animals with increased levels of porphyrin precursors induced by phenobarbital and those receiving ALA i.v., using the Wilcoxon signed-rank test. AIP, acute intermittent porphyria; ALA, δ-aminolevulinic acid; rAAV, recombinant adeno-associated virus; WT, wild type. Molecular Therapy 2011 19, 243-250DOI: (10.1038/mt.2010.210) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Full protection against phenobarbital-induced attacks of porphyria in AIP mice injected with therapeutic rAAV2/5 or rAAV2/8 vectors. (a) Porphyrin precursor excretion in 24-hour urine samples before and after an acute attack induced with phenobarbital 3 months after the rAAV injection. (b) PBGD activity measured in the livers of female mice 3 months after the (separate) administration of two different rAAV vector serotypes. (c) Hepatic PBGD activity in male AIP animals obtained 1, 2, and 3 months after the administration of different therapeutic vectors. The Wilcoxon signed-rank test was used for comparison of porphyrin excretion before and after phenobarbital inductions. **P < 0.01 versus baseline values in each group. The nonparametric Mann–Whitney test was used for comparison of hepatic PBGD activity. ALA, δ-aminolevulinic acid; AIP, acute intermittent porphyria; cohpbgd, codon-optimized cDNA of human PBGD protein; Luc, luciferase; rAAV, recombinant adeno-associated virus; PBG, porphobilinogen; URO, uroporphyrin; WT, wild type. Molecular Therapy 2011 19, 243-250DOI: (10.1038/mt.2010.210) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions