699. Self-Inactivating Retroviral Vector-Mediated Gene Therapy for MPS I Mice

699. Self-Inactivating Retroviral Vector-Mediated Gene Therapy for MPS I Mice

Inborn Errors of Lysosomal Metabolism in HSPC. Evaluation of the efficacy of this strategy in the murine model of GLD is on going. 698. Long Term Exp...

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Inborn Errors of Lysosomal Metabolism in HSPC. Evaluation of the efficacy of this strategy in the murine model of GLD is on going.

698. Long Term Expression and Safety of Administration of AAVrh.10CUhCLN2, a Candidate Treatment for Late Infantile Neuronal Lipofuscinosis, to the Brain of Non-Human Primates

Dolan Sondhi,1 Neil R. Hackett,1 Katrina del Fierro,1 Andrew M. Edelstein,1 Ronald G. Crystal.1 1 Weill Cornell Medical College, NY, NY.

Late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal lysosomal storage disorder of childhood, originates from mutations in the CLN2 gene that cause a deficiency of tripeptidyl peptidase (TPP-I) in neurons, resulting in progressive neurodegeneration. Previous studies in our laboratory have demonstrated adeno-associated virus (AAV) serotype 2 to be a safe and partially effective vehicle for gene transfer of hCLN2 in the rat, non-human primate and human CNS. Based on studies with the rhesus monkey-derived rh.10 AAV serotype showing enhanced efficacy in LINCL knockout mice compared to AAV2 serotype, the present study tests the hypothesis that direct CNS injection of AAVrh.10CUhCLN2 to the CNS of non-human primates at doses scalable to humans has both a high efficacy for gene transfer and an acceptable long term safety profile. A total dose of 1.8x1012 genome copies of AAVrh.10CUhCLN2 was administered to the CNS of African Green monkeys (n=8) at 12 locations. Target locations were determined using CAT scan and MRI imaging, and were chosen to include 8 caudal sites and 4 rostral sites including both white and gray matter. One group (n=4) was sacrificed at 7 days following injection, while the other group (n=4) was sacrificed at 90 days to determine short and long-term effects of treatment, respectively. As controls, one monkey was injected with equivalent volume of PBS and sacrificed 7 days post-surgery and PBS injected (n= 4) and sham injected (n= 3) monkeys from a previous study were also used. The vector-injected groups did not differ from the controls in any parameter of general assessment or comprehensive blood profile (complete blood count, chemistry panel) at the time points assessed which included pre-administration, on the day of administration, and day 3, 7, 15, 30, 60, or 90 days post-administration. Blinded videotape analysis of behavior pre-surgery and days 7, 15, 30, 60, and 90 post-administration showed no discernible neurological differences. Histopathological examination of the CNS demonstrated that injection of AAVrh.10CUhCLN2 produced localized slight to mild and transient white matter edema (spongiosis) and gliosis in the region of the injection sites in the 90 day monkeys and two out of four 7 day monkeys, a localized finding also observed with the AAV2 serotype vector, a finding likely due to mechanical trauma from the vector infusion. In 1cm3 cubes of the brain, monkeys evaluated at 7 days showed TPP-I activity levels comparable to the control group. However, in the 4 animals sacrificed at 90 days post vector administration, TPP-I activity in the brain was >2 standard deviations over the mean level for the PBS injected animal in 32 ± 8% (range 15-53%) of the cubes with a total TPP-I activity of 160 ± 18% (range 136 - 214%) compared to the PBS control. Together these findings demonstrate long-term gene expression and safety of TPP-I following AAVrh.10CUhCLN2 administration and support the use of this vector as a new vehicle for therapy of diffuse disorders of the CNS such as LINCL.

Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

699. Self-Inactivating Retroviral Vector-Mediated Gene Therapy for MPS I Mice

Xiucui Ma, Ramin S. Herati, Axel Schambach, Katherine P. Ponder. 1 Internal Medicine, Washington University School of Medicine, Saint Louis, MO; 2Internal Medicine, Washington University School of Medicine, Saint Louis, MO; 3Experimental Hematology, Hannover Medical School, Hannover, Germany; 4Internal Medicine, Washington University School of Medicine, Saint Louis, MO.

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease caused by deficient α-L-iduronidase (IDUA) activity, which results in the accumulation of the glycosaminoglycans heparan and dermatan sulfate. Neonatal intravenous injection of a long terminal repeat (LTR)-intact gamma retroviral vector (γ-RV) is remarkably effective for treating MPS I in mice and dogs, but the risk of insertional mutagenesis is a major concern, and immune responses have occurred after transfer to adults. Self-inactivating (SIN) γ-RV lacks sequences from the 3’ end of the vector, which results in a provirus without retroviral enhancer elements and reduces the chance that it would activate the expression of a nearly oncogene. An additional advantage to SIN γ-RV vectors is that expression is dependent upon the internal promoter, which should allow organ-specific expression to be achieved. This should reduce the chance that expression will occur in antigen-presenting cells, and thereby reduce or eliminate an immune response. Indeed, we recently generated an LTR-intact γ-RV designated Reverse hAAT-IDUA, in which the expression cassette was inverted relative to the LTR and expression was directed by the human α1-antitrypsin promoter (hAAT). After in vivo transfer to adults, this resulted in liver-specific expression and stable IDUA activity in serum at 13 ± 8 units (U)/ml and correction of most of the clinical manifestations of disease. However, this vector had an intact LTR, which could induce tumors in animals. We have now cloned the canine IDUA cDNA into the SIN γ-RV designated pSERS11oPRE to generate hAAT-cIDUA-oPRE-SIN. This contains only 22 bp from the 5’ end and 14 bp from the 3’ end of the U3 region of an MLV LTR. In addition, it contains an internal hAAT promoter and an optimized WPRE (oPRE) that deletes the X protein of the hepatitis virus that has been implicated in carcinogenesis. No expression was observed after transduction of NIH 3T3 fibroblast cells in vitro with an amphotropic vector, although HepG2 hepatoma cells had IDUA activity that was 4 U/mg, suggesting that a high degree of liver specificity was obtained as expected. MPS I mice were injected IV with 1010 transducing units/kg of hAAT-cIDUA-oPRE-SIN. This resulted in 84.62 ± 45.01 U/ml (N=10) of IDUA activity at 1 month, which should be sufficient to result in a marked clinical improvement. This is the first demonstration that a SIN γ-RV can result in prolonged and high levels of serum IDUA activity after IV injection in MPS I mice. Mice will be evaluated for the specificity of gene expression in organs in vivo and for a therapeutic effect. If the clinical effect is as good as expected, this backbone will be used to generate a vector with the human IDUA cDNA that can be used in clinical trials.