824 AAV mediated gene therapy for murine infantile neuronal ceroid lipofuscinosis (INCL, batten disease)

824 AAV Mediated Gene Therapy for Murine Infantile Neuronal Ceroid Lipofuscinosis (INCL, Batten Disease) Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� �[.]

GENE THERAPY FOR THE NERVOUS SYSTEM I GENE THERAPY FOR THE NERVOUS SYSTEM I 821 Neprilysin Gene Transfer Reduces Amyloid Pathology in Transgenic Mouse Models of Alzheimer’s Disease Robert A Marr,1 Edward Rockenstien,2 Atish Mukherjee,3 Mark S Kindy,3 Louis B Hersh,3 Fred H Gage,1 Inder M Verma,1 Eliezer Masliah.2 Laboratory of Genetics, The Salk Institute, La Jolla, CA, United States; 2Department of Neurosciences, The University of California San Diego, La Jolla, CA, United States; 3Department of Biochemistry, The University of Kentucky, Lexington, KY, United States The degenerative process of Alzheimer’s disease is linked to the accumulation of amyloid-β (Aβ) peptides in the brain Neprilysin has recently been implicated as a major extracellular Aβ degrading enzyme which mediates catabolism of Aβ in mice We set out to test if gene delivery of neprilysin to the effected areas of the brain could be therapeutic in mouse models of amyloidosis A lentiviral vector expressing human neprilysin (Lenti-Nep) was constructed and shown to efficiently express biologically active protein We show that unilateral intra-cerebral injection of Lenti-Nep reduced amyloidβ deposits by half relative to the untreated side Furthermore, LentiNep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice as shown by MAP2 immunoreactivity These data further support a role for neprilysin in regulating cerebral amyloid deposition and demonstrates a new potential gene-therapeutic approach to Alzheimer’s disease therapy 822 Silencing Huntingtin with siRNA Scott Q Harper,1 Patrick D Staber,1 Sarah K Fineberg,1 Henry L Paulson,1,2 Beverly L Davidson.1,3 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin protein This gain of function mutation results in striatal and cortical neuron degeneration leading to severe neurological impairment and death An effective treatment will require a reduction of mutant huntingtin protein Recent advances by this laboratory and others have demonstrated the feasibility of treating dominant genetic disorders using siRNA (small inhibitory RNA) Using in vitro synthesized siRNA and plasmids expressing hairpin siRNAs from the U6 promoter, we tested 10 different siRNAs corresponding to various regions of the human huntingtin transcript (siHDs) U6 plasmids or siRNAs were cotransfected with target plasmids expressing either the full-length normal human huntingtin cDNA or a truncated transcript encoding exons 1-3 and containing an expanded polyglutamine tract (hHunt-82Q) RNA and protein were harvested from transfected cells 48 hrs later, and siRNA mediated silencing was assayed by phosphorimager quantification of northern and western blots We observed a dose-dependent reduction in huntingtin expression with some non-specific effects at the highest ratios of siRNA:target tested To date, our most effective U6driven siHD reduce hHunt-82Q mRNA and protein levels in cell culture by 86% and 55% respectively We are currently developing viral vectors to deliver our most effective siHDs to brains of HD mouse models Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy 823 Characterizing Pol II and Pol III Driven siRNA: Application to Spinal Cerebellar Ataxia Type I Haibin Xia,1 Qinwen Mao,1 Nathan Kiewiet,1 Beverly L Davidson.1,3 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA siRNA has been widely used for gene silencing in vitro, and more recently in vivo In most instances, siRNA expression has been driven from ubiquitously expressed Pol III promoters such as the U6 or H1 promoters In prior work, we found that a modified CMV (Pol II) promoter, combined with a minimal polyA cassette, could successfully drive siRNA expression for efficient diminution in target sequence expression In this study, we compared the levels of siRNA expressed from the two promoters, and the cellular location of the expressed siRNA We also tested the effects of hairpin length, distance between promoter and hairpin, and between hairpin and polyA, on siRNA expression levels and gene silencing In Pol II and Pol III promoter comparison studies, we found that target diminution was modestly increased when the siRNA was expressed from a Pol III promoter, even though the levels of total RNA expressed were much higher Fractionation studies revealed that Pol II expressed siRNA was found predominately in the cytoplasm, while most Pol III expressed siRNA was in the nucleus In studies on the Pol II promoter system, we identified bp as the maximal amount of flanking sequence tolerated 5’ and 3’ of the hairpin, and found that 19 or 21 bp hairpins were optimal for gene silencing These characteristics were used to build siRNA against ataxin-1 Spinal cerebellar ataxia type I is a classical CAG repeat disease, due to a polyglutamine expansion in ataxin-1; therapies would require silencing gene expression Ataxin-1 specific siRNAs (F1 through F11) were generated One F11 led to very effective gene silencing as measured by western blot Our data support the utility of Pol II promoters for gene silencing studies, with application to an important class of dominant inherited disorders 824 AAV-Mediated Gene Therapy for Murine Infantile Neuronal Ceroid Lipofuscinosis (INCL, Batten Disease) M Griffey,1 E Bible,2 J Cooper,2 D Wozniak,3 S Rothman,4 C Vogler,5 M Sands.1 Internal Medicine, Washington University Medical School, St Louis, MO, United States; 2Neuropathology, King’s College, London, United Kingdom; 3Psychiatry, Washington University Medical School, St Louis, MO, United States; 4Neurology, Washington University School of Medicine, St Louis, MO, United States; 5Pathology, St Louis University, St Louis, MO, United States The Neruonal Ceroid Liposfuscinoses (NCL, Batten Disease) are a group of inherited pediatric neurodegenerative diseases Children with NCL suffer from blindness, seizures, a decline in motor and mental abilities, and finally premature death Autofluorescent storage material accumulates in many cell types, including neurons This accumulation leads to neuronal loss and widespread brain atrophy The earliest form of NCL has an onset at 1.5 years of age and is known as Infantile Neuronal Ceroid Lipofuscinosis (INCL) The primary defect in INCL is due to mutations in the gene that encodes the lysosomal hydrolase palmitoyl protein thioesterase (PPT1) A murine model of PPT1-deficiency that exhibits many of the same clinical signs as humans with INCL was recently created by Dr S Hofmann’s group (University of Texas Southwestern) To determine the efficacy of a viral-mediated gene therapy approach to treat the PPT1-deficient mouse we created a recombinant adeno-associated viral (AAV) vector containing the human PPT1 cDNA and tested its S317 GENE THERAPY FOR THE NERVOUS SYSTEM I properties in vitro and in vivo Our in vitro data indicate that the rAAV-PPT1 vector produces enzymatically active rPPT1 that is able to cross-correct PPT1(-) cells We have now examined the in vivo characteristics of our rAAV-PPT1 vector In PPT(-) mice given intra-cranial injections (2/hemisphere) of 0.5 μl rAAV-PPT1 (1.7 X 10 IU/ml) at birth, there are peaks of PPT1 activity corresponding to the injection sites The average PPT1 specific activity was 75 nmol/mg/h, representing about 25% of normal levels (+/+ range = 250-350 nmol/mg/h) The secondary elevation of another lysosomal enzyme observed in untreated PPT(-) mice was reduced throughout much of the brain in the rAAV-PPT1 treated animals indicating a therapeutic response Storage material observed on toluidine blue stained sections was reduced in rAAV-PPT1 treated animals In addition, quantitative measurements showed that the levels of autofluorescent material were significantly (p < 0.05) reduced in the rAAV-PPT1 treated animals and this reduction extended at least 2mm from the injection site There was a significant (p < 0.001) improvement in the brain weights of rAAV-PPT1 treated animals The brain weights of normal, PPT1(-), and rAAV-PPT1 treated mice at months of age average 0.403 g, 0.313 g, and 0.384 g, respectively The treated animals also displayed significant (p < 0.05) increases in cortical volume and thickness To determine if the treated animals displayed functional improvements, we examined their motor function using the rotarod behavioral test At five months of age the rAAV-PPT1 treated animals showed significant (p < 0.05) improvement in their ability to remain on the rotarod during all test days and trials Additional testing at months is currently underway Experiments to determine if the lifespan is increased and the seizure frequency is reduced in the rAAV-PPT1 treated mice are also in progress These data indicate that AAV-mediated gene therapy at birth provides both significant biochemical and clinical improvements in the murine model of INCL 825 The Effects of Pre-Immunization to wtAAV on Single and Multiple Administrations of rAAV in the Brain Carmen Peden,1 Corinna Burger,2 Nick Muzyczka,2 Ron Mandel.1 Neuroscience, University of Florida College of Medicine, Gainesville, FL; 2Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL Epidemiological studies report the prevalence of natural immunization to wild type AAV (wtAAV) to approach 80% of the population, with 30% having antibodies capable of preventing AAV infection Likewise, some peripheral administrations of rAAV in naïve animals are reported to generate an immune response, preventing further applications of rAAV mediated gene therapy due to production of neutralizing antibodies (nAb), and/or a cellular immune response generated against the transgene product The bloodbrain-barrier (BBB) provides some immunologic privilege to the brain parenchyma, and the immune response to rAAV administration in the brain of a naïve animal has been minimal However, vector administration may cause sufficient disruption of the BBB to promote an immune response in a previously immunized animal We tested the hypothesis that intracerebral rAAV administration and re-administration would not be affected by the presence of circulating antibodies to wtAAV2 Sprague-Dawley rats received a series of three peripheral immunizations using wtAAV2 (109 particles) and an adjuvant, to promote the production of circulating nAb Along with naïve controls, the rats received an injection of rAAV2, encoding human glial cell line-derived neurotrophic factor (hGDNF), rAAV-hGDNF, in the right striatum On day 14, rats in the double injection group received an additional injection in the contralateral striatum of rAAV-hGDNF or vehicle Both single and double administration groups were euthanized either 14 or 28 days S318 after their last surgery for either histologic analysis or quantification of GDNF levels Blood was also drawn at each time point for comparison of antibody titers ELISA quantification revealed the immunized animals produced levels of GDNF below their own baseline levels in the contralateral striatum, while the naïve animals, exhibited a seven-fold increase over baseline GDNF production Moreover, inflammatory markers predominated in the areas of the injections in the pre-immunized cases, and in the immunized double injection groups, were accompanied by actual tissue destruction All animals in the immunized groups developed very high titers of neutralizing antibodies We are currently characterizing the nature of the inflammatory response In summary, we have observed that the presence of a high neutralizing antibody titer, can significantly interfere with successful gene transfer in the brain, and the robust inflammatory response generated is sufficient to cause tissue damage in some cases It may therefore become necessary to employ some immuno-evasive strategy in patients who exhibit pre-existing immunity to wtAAV NM is an inventor on patents related to recombinant AAV technology and; owns equity in a gene therapy company that is commercializing AAV for; gene therapy applications 826 HSV-Mediated VEGF Gene Transfer Prevents Neuropathy in the Diabetic Mouse Munmun Chattopadhyay,1 David Krisky,2 Darren Wolfe,2 James Goss,13 Marina Mata,13 Joseph C Glorioso,2 David J Fink.1,2,3 Neurology, University of Pittsburgh, Pittsburgh, PA, United States; 2Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 3GRECC, Pittsburgh VA Healthcare System, Pittsburgh, PA, United States Peripheral neuropathy is a common complication of diabetes for which there is no available treatment In animal models neurotrophic factors can prevent the progression of nerve damage but these potent peptides cannot be administered to patients in adequate doses by systemic routes We have previously demonstrated that gene transfer achieved by subcutaneous inoculation of a herpes simplex virus (HSV)-based vector expressing neurotrophin-3 or nerve growth factor (NGF) can prevent pyridoxine-induced neuropathy in the rat, and that HSV-mediated gene transfer of NGF can prevent diabetic neuropathy in the mouse In the study we examined whether HSVmediated gene transfer of the vascular endothelial growth factor (VEGF) could be used to prevent progression of neuropathy in diabetic mice A VEGF-expressing vector, T0VEGF, was created in a T0ZHG backbone HSV defective for the viral genes ICP4, ICP27, ICP22, and UL41 by cotransfection with a PacI linearized plasmid (p41:0VEGF) containing VEGF165 under the control of the HSV ICP0 promoter element Expression of VEGF protein was confirmed by Western blot and bioassay, and release of VEGF from transfected primary DRG neurons in culture was determined by ELISA Diabetes was induced in male Swiss-Webster mice (6-8 weeks old) by the injection of streptozotocin (STZ, 100mg/kg twice over 48 hours) Two weeks after STZ, subsets of mice were inoculated subcutaneously in the plantar surface of the right hind foot with of 10 μl containing × 107 pfu of either vector THVEGF or QOZHG (expressing lacZ) Diabetes induced by STZ resulted in a sensory neuropathy manifest by a decrease in the foot sensory nerve amplitude (FSA; control = 22.0 ± 1.8 μV, diabetic = 12.5 ± 1.3 μV) Transduction of dorsal root ganglia in vivo with vector THVEGF by footpad inoculation weeks after STZ administration protected against the decrease in FSA measured weeks after vector (THVEGF = 31 ± 1.6 μV and QOZHG = 14.1 ± 1.7 μV) Peripheral innervation of sweat glands was evaluated by measuring the number of sweat droplets in the hind paw after stimulation by subcutaneous injection Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ... to wtAAV NM is an inventor on patents related to recombinant AAV technology and; owns equity in a gene therapy company that is commercializing AAV for; gene therapy applications 826 HSV -Mediated. .. preventing AAV infection Likewise, some peripheral administrations of rAAV in naïve animals are reported to generate an immune response, preventing further applications of rAAV mediated gene therapy. .. in the rAAV-PPT1 treated mice are also in progress These data indicate that AAV- mediated gene therapy at birth provides both significant biochemical and clinical improvements in the murine model