219. Gene Delivery Strategies To Treat α1-Antitrypsin Deficiency

GENETIC AND METABOLIC DISEASES: PART ONE in the murine model of the human disease mucopolysaccharidosis (MPS) type VII using the SB system to correct β-glucuronidase deficiency. β-glucuronidase is ideal for this evaluation, having a definitive histochemical stain and sensitive quantification assay. The SB transposon plasmid pT/CAGGS-GUSB carrying the human β-glucuronidase cDNA behind the CAGGS composite enhancer-promoter was administered to young adult MPS VII mice either alone (Group 1), or co-injected with transposase plasmid pSB10 at a transposon:transposase molar ratio of 1:1 (Group 2), or 10:1 (Group 3). Forty-eight hours after injection, β-glucuronidase enzymatic activity in plasma and liver of treated MPS mice was markedly elevated (in some subjects to >100-fold above that of wild-type untreated or sham-treated mice). Eight weeks postinjection, β-glucuronidase activity was detected in livers from all three treatment groups. In animals that did not receive transposase (Group 1), β-glucuronidase activity levels were approximately 8fold higher than those in mice co-injected with the transposase plasmid (Groups 2 and 3). Toluidine blue staining showed a dramatic reduction in the number and size of pathologic lysosomes of both hepatocytes and Kupffer cells in all treatment groups. There was a dose-effect, with levels of β-glucuronidase catalytic activity inversely related to the levels of reversal of pathology. In mice with the highest β-glucuronidase activity, levels loss of storage vacuoles appeared complete and indistinguishable from those of wild-type mice. For some mice, storage vacuoles appeared to have been eliminated suggesting that episomal gene expression has the potential as a long-term treatment in liver. To evaluate whether transposition occurred in the livers, PCRbased excision assay was performed to detect events that produce a sequence of predicted length with a characteristic footprint. DNA was isolated from livers of mice sacrificed one week after injection. The excision product was obtained only from mice that received transposase. This assay is a high throughput way to evaluate conditions for transposition in select tissues of whole animals only days after plasmid delivery. Supported by NIH (PO1-HD32652 and R21-OD55571), and the Arnold and Mabel Beckman Foundation.

for enhancing transduction in vivo. We evaluated both selective lobe delivery, and the effects of increasing the dwell time of vector in the liver. Methods: AAV serotype -2 null vector (encodes a nonfunctional expression cassette) was infused into adult Lewis male rats. In order to infuse AAV vector into the liver through the hepatic artery in rats we developed a microsurgery protocol. As a general hepatic artery infusion procedure (Method-1), we temporarily clamped the common hepatic artery (CHA) with a micro-clip and then inserted ultra-thin catheter into the proper hepatic artery through a cut-down of the gastroduodenal artery. 5x1012vg/kg of vector was infused through the catheter over a period of about 1 min. After infusion, we removed the clip on the CHA and removed the catheter. In order to increase the dwell time of vector in the liver, we clamped the venous outflow of the liver just before the AAV infusion through the hepatic artery (Method-2). Five minutes after the infusion, we removed the clamps and confirmed that hepatic blood flow was restored. In a third method (Method-3), we infused the vector into specific liver lobes through the hepatic artery. We clamped the right branch of the hepatic artery and portal vein following the procedure of Method-1, infused the vector, and waited for 5 min. This clamping method resulted in selective blood flow and vector infusion through the left and middle liver lobes. After removing the clamp blood flow through all of the lobes returned to normal. As a liver injury marker, we measured serum alanine amino transferase (ALT) at Days 2, 7, 21. Quantification of AAV genomes in the liver lobes was determined by Southern blotting. Results: None of the rats showed serum ALT elevation indicating that these interventions could be performed without causing liver injury. There was an approximately 5-fold increase in the stable double-stranded vector genomes when the liver circulation was stopped for 5 minutes at the time of vector infusion (Method-2 vs Method-1). This suggested that higher rates of vector transduction will be achievable by increasing vector dwell times in liver. Using Method-3, we found 6-fold-higher levels of transduction in the perfused lobes compared to whole liver administered vector via the hepatic artery while almost no vector genomes were detectable in the non-perfused lobes. This suggests that most of the vector is removed by the liver very rapidly after infusion. Determining the optimal physical parameters for vector delivery will be important for future clinical trials.

219. Gene Delivery Strategies To Treat α1Antitrypsin Deficiency Mark A. Zern,1 Yuyou Duan,1 Jian Wu,1 David S. Strayer.2 1 Medicine, University of California, Davis, Sacramento, CA; 2 Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA.

P.B. Hackett and R. S. McIvor have a financial interest in Discovery Genomics, Inc.

218. Delivery Parameters Affecting LiverDirected AAV Transduction Kazuo Ohashi,1 Hiroyuki Nakai,1 Linda B. Couto,2 Yoshiyuki Nakajima,3 Mark A. Kay.1 1 Department of Pediatrics, Program in Human Gene Therapy, Stanford University Medical Center, Stanford, CA, United States; 2 Avigen Inc., Alameda, CA, United States; 3First Department of Surgery, Nara Medical University, Kashiahra, Nara, Japan. Purpose: AAV-2 vectors have therapeutic potential for the treatment of hepatodeficiency disorders. However, even when the vector is delivered at very high doses into the portal vein or hepatic artery, a relatively small number of hepatocytes are stably transduced. Thus, we studied alternative delivery routes as a means S86

Background: Human α1-antitrypsin (α1AT) deficiency is caused by mutations in the coding sequences for α1AT, leading to pulmonary fibrosis and cirrhosis of the liver. The absence of blood α1AT is responsible for the pulmonary component of the disease, which reflects poorly tissue damage by neutrophil elastase and other enzymes that are normally neutralized by α1AT. Hepatic cirrhosis occurs because mutant α1AT protein cannot be adequately exported from the hepatocytes that synthesize it, and its intracellular accumulation damages these cells. Any strategy to treat α1AT deficiency must therefore both protect the liver by eliminating the mutant protein and supply adequate blood levels of wild type (wt) α1AT to prevent pulmonary fibrosis. Methods: We therefore designed a hammerhead ribozyme (AT589) to target the mutant α1AT transcript. Because this ribozyme would also recognize wt α1AT transcript, we modified the coding sequence for wt α1AT so that this modified human α1AT mRNA would not be sensitive to the ribozyme but would retain the wt amino acid sequence. Since recombinant SV40-derived vectors (rSV40s) deliver their genes to the liver permanently and with great efficiency, and do Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

GENETIC AND METABOLIC DISEASES: PART ONE not elicit immune responses, the two transgenes were cloned into rSV40 backbone vectors. The modified human α1AT cDNA, driven by an SV40 promoter, was used to produce SV(AT). SV(AT589.t) carried the ribozyme, under the control of the methionine tRNA (pol III) promoter. Transgenic mice expressing mutant human α1AT were used as targets for the ribozyme-carrying vector. Normal mice were given SV(AT), carrying the modified wt human α1AT. Each mouse received a total of 4 daily injections of the respective vectors via indwelling portal vein catheters. Control mice received saline only. Serum levels of human α1AT, which is immunologically distinct from mouse α1AT, were followed over time by ELISA. Results: Sera from normal mice receiving SV(AT) were tested from 1 to 50 weeks post-injection. Blood values of human α1AT fluctuated, but remained between 1 and 3 μg/ml throughout the assay period. (By this ELISA, normal mouse serum has no detectable human α1AT.) The effect of the ribozyme targeting human α1AT mRNA was tested by measuring levels of mutant human α1AT in the blood of transgenic mice 3 to 6 weeks post-transduction. Administration of SV(AT589.t) lowered blood levels of mutant α1AT to 53 ± 5% of pretreatment values (P = 0.027, Wilcoxon signed rank test). Blood levels of human α1AT in control mice were unchanged (P = 0.17). Conclusions: As a target for gene therapy, α1AT deficiency presents special challenges, requiring both long term delivery of a wild type protein and simultaneous removal of a harmful mutant protein. We describe a strategy applying two different rSV40 vectors separately to this task, and demonstrate that each is effective in vivo. Further optimization is necessary, to increase blood wt α1AT levels, to decrease mutant α1AT levels and to combine the two in vivo. Such a combination strategy may be a model for genetic therapy of α1AT deficiency and other similar diseases.

with detection by a charge coupled device camera. A subgroup was euthanized and tissues analyzed for luciferase. Anti-rAAV2 and anti-luciferase ELISA and lymphocyte proliferation assays (LPA) were performed to assess whether rAAV administration induced immune responses. Adult mice that had received in utero administration of rAAV-EF1α-luc and naive controls were administered rAAV-CMV-luc, a serotype 2 rAAV with the CMV promoter. Stable gene expression was observed in all animals persisting for over two years in mice following rAAV injection in utero. Postnatally, ELISA of plasma samples showed no antibody response to AAV2 capsid proteins in contrast to control adult animals injected with the same vector. No antibody responses developed to luciferase when AAV2, AAV5, or AAV1 vectors were injected in utero. Subsequent adult IP injection into in utero AAV2-injected mice resulted in stable postnatal boosting of expression without inducing humoral responses against luciferase. Humoral immunity did develop to rAAV. Similar studies with AAV1 and AAV5 are in progress, as well as analysis of cytokine profiles. Taken together, these findings demonstrate functional tolerance to the luciferase protein is established by in utero delivery and expression of rAAV. Our results are consistent with those of others in which administration of high doses of antigens in the perinatal period preferentially stimulate tolerogenic TH2 responses. Assessing the role of the dose of vector and route of delivery will be important in the development of strategies for tolerance induction.

220. Tolerance to Transgene-Encoded Protein after In Utero Injection of Adeno-Associated Viral Vectors

Andrew D. Simmons,1 Heini Ilves,1 Michael Kelly,1 Carol Lin,1 Denice Mittelstaedt,1 Minh Nguyen,1 Chris Pacheco,1 Dale Talbot,1 Melanie Tuerk,1 Melinda VanRoey,1 Gabor Veres,1 Tom Dull.1 1 Cell Genesys, Foster City, CA.

Gerald S. Lipshutz,1 Wendy R. Mahler,2 Christopher H. Contag,3 Karin M. Gaensler.2 1 Department of Surgery, University of California, San Francisco, CA; 2Department of Medicine, University of California, San Francisco, CA; 3Pediatrics, Microbiology & Immunology, & Radiology, Stanford University, Palo Alto, CA. Immune responses to therapeutic proteins produced by gene therapy vectors can abrogate the clinical efficacy of these agents. Such inhibitory responses remain a challenging problem, for example in the treatment of hemophilia. Delivery and expression of genes prenatally, prior to the maturation of the immune system, may enable 1) induction of tolerance to therapeutic proteins, and 2) efficient augmentation of gene expression postnatally. We previously showed that in utero delivery of adenoviral vectors containing either the factor VIII (FVIII), or factor IX gene (FIX) resulted in transient correction of the bleeding diathesis in murine hemophilic models without inducing humoral immune responses. Re-administation in adult animals produced high-level re-expression of the factors. Thereafter, immune responses were observed. This lack of tolerance induction was likely in part due to the lack of ongoing expression of the FIX and FVIII genes after the first weeks of life. In the present studies, we have assessed whether delivery of rAAV in utero will produce stable gene expression postnatally and the induction of tolerance to the reporter gene expressed. rAAV containing the luciferase (luc) reporter gene and the elongation factor 1-alpha (EF1α) promoter were constructed. At gestation day 15, pregnant mice were anesthetized and 3x1011 genomes of rAAV-EF1α-luc were injected by intraperitoneal route (IP) into each fetus (n=41). Animals underwent serial examination by in vivo bioluminescent imaging (BLI) of transmitted photons Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

221. Expression of Coagulation Factor VIII in Mice, Rats, and Dogs Following In Vivo Lentiviral Delivery of a Novel Hepatocyte-Restricted Expression Cassette

Hemophilia A, caused by a deficiency in blood coagulation factor VIII (FVIII), is the most common severe hereditary bleeding disorder and a candidate for gene therapy. At present, one of the major limitations to hemophilia A gene therapy is FVIII expression, which is plagued by mRNA and protein instability. To address this problem, we constructed lentiviral vectors expressing the B-domain deleted (BDD) FVIII gene driven by an intron containing, strong constitutive promoter (CAG). Portal vein injection of this lentiviral vector, in the absence of a partial hepatotectomy, resulted in long term FVIII expression in Swiss nu/nu mice (7.6 ng/ml on day 42) and shortterm expression in hemophilia A mice (84.6 ng/ml on day 7). Given that only short term FVIII expression was observed in hemophilia A mice, and that lentiviral vectors can efficiently transduce antigenpresenting cells, we sought to eliminate the humoral immune response by driving FVIII expression from a liver specific promoter. Two novel expression cassettes, designated LE and ET, were developed. In vivo liver histology and splenic FACs analyses using a GFP reporter gene revealed that the ET cassette was robust and hepatocyte restricted. Hemophilia A mice injected with lentiviral vectors driving the expression of an intron containing BDD FVIII from either the LE and ET cassettes showed high levels of FVIII expression (26.9 ng/ml on day 7). Surprisingly, this expression was only observed short term. To address the scalability of lentiviral vectors with the eventual goal of human gene therapy, both low (3.6 x 109 IU) and high (1.7 x 1010 IU) doses were tested in immune deficient rats by portal vein delivery. Stable and high levels of FVIII expression (39.1 ng/ml [low dose] and 333.1 ng/ml [high dose]) were

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