AAV-mediated gene therapy for metabolic diseases: dosage and reapplication studies in the molybdenum cofactor deficiency model
© Hahnewald et al; licensee BioMed Central Ltd. 2009
Received: 20 March 2009
Accepted: 18 June 2009
Published: 18 June 2009
In a mouse model for molybdenum cofactor deficiency as an example for an inherited metabolic disease we have determined the dosage of recombinant AAV necessary to rescue the lethal deficiency phenotype. We demonstrated long-term expression of different expression cassettes delivered in a chimeric AAV capsid of serotype 1/2 and compared different routes of application. We then studied the effect of double and triple injections at different time points after birth and found a short neonatal window for non-response of the immune system. Exposition with rAAV capsids within this window allows transgene expression after a second rAAV transduction later. However, exposition within this window does not trigger immunotolerance to the viral capsid, which limits rAAV-mediated refurbishment of the transgene to only one more application outside this permissive window.
In mammals, molybdenum cofactor (MoCo) is essential for the activity of sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase . The gene products of the human genes MOCS1, MOCS2, MOCS3 and GEPH are required for the biosynthesis of MoCo . A mutational block of these genes leads to MoCo deficiency (OMIM #252150) associated with a progressive neuronal damage and death before adolescence in affected patients. The majority of patients suffer from type A deficiency and harbour mutations in the gene MOCS1 .Mocs1 knockout-mice show no detectable residual Mocs1 mRNA levels and display a severe phenotype reflecting the biochemical characteristics of human MoCo-deficient patients .
Recently, we described the phenotypical rescue of Mocs1-deficient mice by intrahepatic injection of a recombinant adeno-associated virus (rAAV) vector carrying an expression cassette for the human MOCS1 cDNA . The MOCS1 expression cassette has been describe before and essentially contains a hybrid promoter consisting of a cytomegalovirus (CMV) enhancer, a human β-actin promoter, exons 1 through 10 of the human MOCS1 gene, a deleted intron 9, which allows for alternative splicing leading to the gene products MOCS1A and MOCS1B and a bovine growth hormone (BGH) polyadenylation (poly A)-signal. MOCS1A and MOCS1B together produce the relatively stable intermediate cPMP, which is further processed to active MoCo by the products of the genes MOCS2, MOCS3 and GEPH.
Transfer of the MOCS1 gene was primarily aimed at transduction of hepatocytes, since the liver is the primary organ involved in detoxification of sulfite to sulphate by sulfite oxidase . In the meantime, mice rescued by the intrahepatic rAAV-MOCS1 reached a lifespan of up to 666 days. To study the long-term expression profile after AAV transduction, we here analyzed wild-type mice, which had received an intrahepatic injection of AAV encoding the green fluorescent protein (AAV-EGFP) on day 6 after birth. The EGFP expression cassette contains the coding sequence for EGFP instead of the MOCS1 cDNA.
As a further approach to the treatment of patients, we investigated the efficacy of systemic AAV delivery. Comparative studies 1 month after rAAV-EGFP application showed similar tissue transduction after either intrahepatic or intravenous injection . Using the MOCS1 expression cassette in an AAV 1/2 capsid, we here studied the effect of systemic delivery by tail vein injections. For this application we used mice with a minimum body weight of around 15 g corresponding to an age of approximately 40 days. Untreated Mocs1 deficient mice are unable to build cPMP, the first intermediate in the MoCo biosynthesis, and die on average 7.5 days after birth . We pretreated Mocs1-deficient mice until day 40 with periodic intrahepatic injections of purified cPMP from Escherichia coli  to achieve a suitable size for tail vein injection.
Considering the lower dosage of 4 × 109 tu AAV-MOCS1 for systemic delivery, as compared to 1 × 1010 tu for the intrahepatic injections described previously  and above, the results described here indicate a similar efficacy for both application schemes. All five above described mice had been mated and were fertile. The offspring (n = 64) died on average on day 5.35 after birth, which corresponds to the lifespan of untreated homozygous Mocs1 knockout mice from matings of heterozygous mice. This is indirect evidence that the intravenous tail vein injections did not result in germ line transmission of the vector genome.
To estimate the necessary dosage for the treatment of humans, we determined the minimal dosage required to rescue the deficiency phenotype via the intravenous route. 20 neonatal Mocs1-deficient mice were pretreated with purified cPMP as described above. At day 40 after birth, the animals obtained a single intravenous tail vein injection containing various amounts of AAV-MOCS1 in phosphate-buffered saline. First, we investigated the effect of a thirty-fold reduced dosage of AAV-MOCS1 as compared to the experiments described above, i.e. 1.5 × 108 tu (n = 8). Mice of this group died on average 28.75 ± 6.5 days after the AAV-MOCS1 injection and discontinuation of cPMP substitution (figure 2, red line). This reduced dosage apparently is not sufficient to rescue the lethal phenotype.
Next, we studied the effect of an intermediate dosage of 4 × 108 tu AAV-MOCS1 on day 40 after cPMP pre-treatment (n = 12). The mice of this group died on average 238.5 ± 124.4 days after AAV-MOCS1 injection and cPMP withdrawal at day 40 after birth (figure 2, green line). All animals of this group were mated and all but one were fertile. Again, the offspring died within the range of untreated animals (data not shown). The observed high variance of the life span suggests that the intermediate dosage of 4 × 108 tu AAV-MOCS1 represents a borderline result and indicates a range for the minimal dosage required for abolishing the MoCo deficiency phenotype. With a maximum body weight of 40 g for the mice used here this would correspond to 1 × 1010 tu per kg body weight. A one year old child with a body weight of 10 kg thus would require 1 × 1011 tu of AAV-MOCS1, which is within the range of GMP production facilities.
Although one single injection could abolished the phenotype of the MoCo-deficiency, our murine model allows a prediction only for the natural life span of mice, i.e. 2 to 3 years. In contrast to long-lasting expression in mice, rats, hemophilic dogs and nonhuman primates, expression at therapeutic levels in humans was limited to a period of around 8 weeks [9–14]. This difference was mainly attributed to prior infection of the human patients with natural AAVs in combination with helper adenovirus . This leads to formation of memory CD8+ T cells and their activation upon reexposure to the AAV capsid.
Thus, the possibility of repeated vector administrations in the treatment of patients from an immunological point of view is an important issue to be addressed. To this end, we investigated the feasibility of successful rAAV re-administration at different time points in the MoCo deficient mouse model and compared the reapplication possibilities in different developmental stages. AAV serotype 1/2 (in a 50:50 ratio) was used throughout.
Studies on hemophilia B mice showed that in utero or neonatally AAV-treated mice do not develop antibodies to the AAV capsid after the first injection . They demonstrated that it is possible to establish tolerance to the transgene product human factor IX by these early injections and to obtain long-term therapeutic levels in immunocompetent mice. Here, the transgene products of the MOCS1 expression cassette are localized intracellular and thus not accessible for antibodies. We therefore concentrated on the existence of a "window of opportunity" to induce tolerance against the viral capsid in repeated exposures.
Since the products of the MOCS1 and the EGFP expression cassette do not share cross-reacting epitopes, we could investigate the potential of early injections to induce an immune tolerance against the viral capsid by triple injections. Two wild-type mice obtained a first intrahepatic injection of 1 × 109 tu AAV-MOCS1 on day 1 after birth and a second injection with 1 × 109 tu AAV-MOCS1 on day 10. After two months they received a third injection of 1 × 109 tu AAV-EGFP. A positive control for the AAV-EGFP injections obtained only a single injection of 1 × 109 tu AAV-EGFP. Two months after AAV-EGFP injections, all mice were perfused with 4% paraformaldehyde. Here, the rAAV-EGFP injections did not lead to an EGFP expression (figure 4d), even though the first exposure to AAV1/2 capsid occurred on day 1 after birth (compare figure 4c). While the role of a cytotoxic T-cell response in mice remains unclear, the immune system clearly built neutralizing antibodies (nABs) [17, 18] against the viral vector after the second injection of viral vector. Thus, the early exposure of the immune system to viral vector capsid allows a successful second application but does not induce an immunotolerance against the capsid proteins.
An important factor in nAB response is the time point of viral vector administration. The group of Petry et al . showed that the efficacy of readministration is dependent on the titer of nAB and that the level of nABs is proportional to the virus dose used for the first injection. Since repeated AAV treatment in adolescence leads to immune responses, future experiments will have to show whether the combination of early first exposure, a lower dosage of virus and/or temporary immunosuppression (e.g. with cyclosporine) facilitates more successful rAAV reapplications.
We thank Günter Schwarz (Köln) for providing cPMP and Sebastian Kügler (Göttingen) for rAAVs. This work was supported by the Deutsche Forschungsgemeinschaft (RE 768/12).
- Schwarz G, Mendel RR: Molybdenum cofactor biosynthesis and molybdenum enzymes. Annu Rev Plant Biol. 2006, 57: 623-647. 10.1146/annurev.arplant.57.032905.105437.View ArticlePubMedGoogle Scholar
- Reiss J: Genetics of molybdenum cofactor deficiency. Hum Genet. 2000, 106 (2): 157-163. 10.1007/s004390051023.View ArticlePubMedGoogle Scholar
- Reiss J, Johnson JL: Mutations in the molybdenum cofactor biosynthetic genes MOCS1, MOCS2, and GEPH. Hum Mutat. 2003, 21 (6): 569-576. 10.1002/humu.10223.View ArticlePubMedGoogle Scholar
- Lee HJ, Adham IM, Schwarz G, Kneussel M, Sass JO, Engel W, Reiss J: Molybdenum cofactor-deficient mice resemble the phenotype of human patients. Hum Mol Genet. 2002, 11 (26): 3309-3317. 10.1093/hmg/11.26.3309.View ArticlePubMedGoogle Scholar
- Kugler S, Hahnewald R, Garrido M, Reiss J: Long-term rescue of a lethal inherited disease by adeno-associated virus-mediated gene transfer in a mouse model of molybdenum-cofactor deficiency. Am J Hum Genet. 2007, 80 (2): 291-297. 10.1086/511281.PubMed CentralView ArticlePubMedGoogle Scholar
- Garrett RM, Bellissimo DB, Rajagopalan KV: Molecular cloning of human liver sulfite oxidase. Biochim Biophys Acta. 1995, 1262 (2–3): 147-149.View ArticlePubMedGoogle Scholar
- Hauck B, Chen L, Xiao W: Generation and Characterisation of Chimeric Recombinant AAV Vectors. Mol Ther. 2003, 7 (3): 419-425. 10.1016/S1525-0016(03)00012-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwarz G, Santamaria-Araujo JA, Wolf S, Lee HJ, Adham IM, Grone HJ, Schwegler H, Sass JO, Otte T, Hanzelmann P, Mendel RR, Engel W, Reiss J: Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli. Hum Mol Genet. 2004, 13 (12): 1249-1255. 10.1093/hmg/ddh136.View ArticlePubMedGoogle Scholar
- Snyder RO, Miao C, Meuse L, Tubb J, Donahue BA, Lin HF, Stafford DW, Patel S, Thompson AR, Nichols T, Read MS, Bellinger DA, Brinkhous KM, Kay MA: Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med. 1999, 5 (1): 64-70. 10.1038/13518.View ArticlePubMedGoogle Scholar
- Manno CS, Pierce G, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo M, Hoots K, Blatt P, Konkle B, Dake M, Kaye R, Razavi M, Zajko A, Zehnder J, Rustagi PK, Nakai H, Chew A, Leonard D, Wright JF, Lessard RR, Sommer JM, Tigges M, Sabatino D, Luk A, Jiang H, Mingozzi F, Couto L, Ertl HC, High KA, Kay MA: Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. nature medicine. 2006, 12 (3): 342-347. 10.1038/nm1358.View ArticlePubMedGoogle Scholar
- Niemeyer GP, Herzog RW, Mount J, Arruda VR, Tillson DM, Hathcock J, van Ginkel FW, High KA, Lothrop CDJ: Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy. blood. 2009, 113: 797-806. 10.1182/blood-2008-10-181479.PubMed CentralView ArticlePubMedGoogle Scholar
- Mount JD, Herzog RW, Tillson DM, Goodman SA, Robinson N, McCleland ML, Bellinger D, Nichols TC, Arruda VR, Lothrop CD, High KA: Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy. Blood. 2002, 99 (8): 2670-2676. 10.1182/blood.V99.8.2670.View ArticlePubMedGoogle Scholar
- Nathwani AC, Davidoff AM, Hanawa H, Hu Y, Hoffer FA, Nikanorov A, Slaughter C, Ng CY, Zhou J, Lozier JN, Mandrell TD, Vanin EF, Nienhuis AW: Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques. Blood. 2002, 100 (5): 1662-1669. 10.1182/blood-2002-02-0589.View ArticlePubMedGoogle Scholar
- Wang L, Calcedo R, Nichols TC, Bellinger DA, Dillow A, Verma IM, Wilson JM: Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy. Blood. 2005, 105 (8): 3079-3086. 10.1182/blood-2004-10-3867.View ArticlePubMedGoogle Scholar
- Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JJE, Ragni M, Manno CS, Sommer JM, Jiang H, Pierce GF, Ertl HC, High KA: CD8+ T-cell responses to adeno-associated virus capsid in humans. nature medicine. 2007, 13 (4): 419-422. 10.1038/nm1549.View ArticlePubMedGoogle Scholar
- Sabatino DE, Mackenzie TC, Peranteau W, Edmonson S, Campagnoli C, Liu YL, Flake AW, High KA: Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther. 2007, 15 (9): 1677-1685. 10.1038/sj.mt.6300219.View ArticlePubMedGoogle Scholar
- Mingozzi F, High KA: Immune responses to AAV in clinical trials. Curr Gene Ther. 2007, 7 (5): 316-324. 10.2174/156652307782151425.View ArticlePubMedGoogle Scholar
- Ponder KP, Wang B, Wang P, Ma X, Herati R, Wang B, Cullen K, O'Donnell P, Ellinwood NM, Traas A, Primeau TM, Haskins ME: Mucopolysaccharidosis I cats mount a cytotoxic T lymphocyte response after neonatal gene therapy that can be blocked with CTLA4-Ig. Mol Ther. 2006, 14 (1): 5-13. 10.1016/j.ymthe.2006.03.015.View ArticlePubMedGoogle Scholar
- Petry H, Brooks A, Orme A, Wang P, Liu P, Xie J, Kretschmer P, Qian HS, Hermiston TW, Harkins RN: Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice. Gene Ther. 2008, 15 (1): 54-60. 10.1038/sj.gt.3303037.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.