418 Application of Therapeutic Artificial miRNAs in the CNS Non Allele Specific Silencing of Mutant and Wildtype Huntingtin Demonstrates Therapeutic Efficacy in Huntington''''s Disease Mice Molecular The[.]
OLIGONUCLEOTIDE THERAPIES broader and more rapid use of the OPEN ZFN selection methodology and provide additional support for the potential use of ZFNs as an important approach for gene therapy 416 Treatment of Canine Leukocyte Adhesion Deficiency Using a SIN Lentiviral Vector and Human CD18 Promoter Expressing Canine CD18 Michael J Hunter,1 Laura M Tuschong,1 Cedar J Fowler,2 Everette J R Nelson,1 Tanya H Burkholder,3 Thomas R Bauer, Jr.,1 Dennis D Hickstein.1 Experimental Transplantation and Immunology, National Cancer Institute, Bethesda, MD; 2HHMI/NIH Research Scholars Program, Howard Hughes Medical Institute, Chevy Chase, MD; Department of Veterinary Resources, National Institutes of Health, Bethesda, MD Hematopoietic stem cell gene therapy would be enhanced by the development of vectors harboring tissue-and developmental stagespecific cellular promoters to express the therapeutic transgene in the target cell population This approach would mitigate the potential adverse effects of inappropriate tissue expression and might be expected to reduce insertional activation of nearby oncogenes that lead to oligoclonal hematopoiesis and leukemia To develop and test a modified promoter cassette with the features described above for our target disease canine leukocyte adhesion deficiency (CLAD), we cloned portions of the human CD18 promoter (1 Kb, 776 bp, and 306 bp) into a SIN lentiviral vector upstream of the canine CD18 cDNA, and used this vector to transduce CLAD CD34+ cells In CLAD, defects in the leukocyte integrin CD18 result in the inability to express CD11/CD18 heterodimers on the leukocyte surface leading to lifethreatening bacterial infections Transduction of CLAD CD34+ cells in vitro with the SIN lentiviral vector with the kb human CD18 promoter resulted in the highest percentage of CD18+ cells; nearly 15% of the CLAD CD34+ cells were CD18+ when assessed days after an overnight transduction We used the SIN lenti vector with the Kb human CD18 promoter to treat two dogs with CLAD Autologous, CLAD CD34+ cells were transduced overnight and infused following a single, non-myeloablative dose of 200cGy total body irradiation (TBI) The percentage of CD18+ leukocyte compartments weeks following infusion of the two dogs were comparable: dog A1, CD18+/ PMN 0.3%, CD18+/CD3+ cells 0.4%, CD18+/B-cells 0.7%, CD18+/ CD14+ cells 0.9%; dog A2, CD18+/PMN 0.5%, CD18+/CD3+ cells 0.8%, CD18+/B-cells 1.2%, CD18+/CD14+ cells 1.4% Both treated dogs are now months of age and have had correction of the CLAD phenotype In contrast, untreated CLAD dogs succumb to overwhelming infection within a few months of life Reversal of the CLAD phenotype with low numbers of CD18+ neutrophils results from selective migration of CD18+ neutrophils from the blood into the tissues Although both CLAD dogs in this study have had a clinical response, additional regulatory elements of the human CD18 promoter/enhancer will be required to ensure long-term reversal of the phenotype in this and other genetic leukocyte diseases These studies represent requisite translational studies in the development of new vector designs for the treatment of children with the human counterpart of CLAD, namely LAD 417 Efficient MGMTP140K-Mediated Selection of Long Term Repopulating Cells in a Nonhuman Primate Model Brian C Beard,1 Grant D Trobridge,1,2 Megan L Welsh,1 HansPeter Kiem.1,2 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of Washington, Seattle, WA In vivo selection of genetically modified hematopoietic repopulating cells has many potential therapeutic applications For some applications which require relatively high levels of gene marking, such as hemoglobinopathies, in vivo selection may be required to increase initially low levels of gene-modified cells Here we demonstrate efficient post-transplantation selection of long-term hematopoietic repopulating cells using methylguanine methyltransferase (MGMTP140K) in a primate model In vivo selection was studied in macaques (M nemestrina) that received CD34-enriched cells transduced with VSVG-pseudotyped HIVderived lentivirus vectors expressing MGMTP140K and a reporter gene green fluorescent protein (GFP) or MGMTP140K only Two macaques were conditioned with a myeloablative dose of total body irradiation and a third macaque was conditioned with a nonmyeloablative dose of busulfan (4 mg/kg/day for days) After stable engraftment monkeys were treated with O6-benzylguanine (O6BG) and BCNU Following myeloablative transplantation the monkeys transplanted with cells gene-modified with a vector expressing MGMTP140K and GFP, in vivo selection was determined by flow cytometry In one monkey (following cycles of O6BG/ BCNU) the granulocytes rose from ∼20% to 70% and in the lymphocytes from ∼20% to 50% In the second monkey (following a single cycle of O6BG/BCNU) the granulocytes rose from ∼25% to 42% and in the lymphocytes from ∼12% to 22% Following nonmyeloablative transplantation and a single cycle of O6BG/BCNU the monkey transplanted with cells gene-modified with the vector expressing only MGMTP140K increased overall gene marking, determined by real time (RT)-PCR, in total white blood cells rose from a provirus copy number of 0.04 (∼4% gene marking) to 0.16 (∼16% gene marking) Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed Importantly, multilineage selection of hematopoietic cells was achieved and clonality studies are underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach In summary, MGMT selection is efficient and well tolerated in macaques and these large animal studies should be highly predictive for clinical applications and will help to further improve HSC gene therapy Oligonucleotide Therapies 418 Application of Therapeutic Artificial miRNAs in the CNS: Non-Allele-Specific Silencing of Mutant and Wildtype Huntingtin Demonstrates Therapeutic Efficacy in Huntington’s Disease Mice Ryan L Boudreau,1 Jodi L McBride,1 Ines Martins,1 Shihao Shen,1 Yi Xing,1 Barrie J Carter,2 Beverly L Davidson.1 University of Iowa, Iowa City, IA; 2Targeted Genetics, Seattle, WA RNA interference (RNAi) provides a promising therapeutic approach to treat several human diseases However, the safety of RNAi-based therapies remains a concern Previously, we compared the efficacy and safety of short-hairpin RNA (shRNA) and artificial microRNA (miRNA) expression vectors in vitro and in vivo We found that shRNAs are more potent but induce toxicity in cell cultures and in mouse brain, whereas artificial miRNAs are expressed at lower levels and display better safety profiles We have since tested the Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S163 OLIGONUCLEOTIDE THERAPIES artificial miRNA strategy in a pre-clinical therapeutic trial aimed at assessing the viability of a non-allele-specific silencing strategy for Huntington’s disease (HD) HD is a dominant, fatal neurodegenerative disease caused by mutant huntingtin (htt) protein which primarily affects the striatum Currently, there are no effective treatments for HD, thus warranting the investigation of novel therapeutic strategies Recently, we and others demonstrated that silencing mutant htt using RNAi provides therapeutic benefit in HD mice We have since found that silencing wildtype htt in adult mouse striatum does not induce overt toxicity However, given the role of htt in various cellular processes, it remains unknown whether nonallele-specific silencing of both wildtype and mutant htt is a viable therapeutic strategy for HD Here, we tested whether co-silencing wildtype and mutant htt provides therapeutic benefit and is tolerable in HD mice Mice were injected intrastriatally with viral vectors expressing htt-specific artificial miRNAs Following treatment, HD mice showed significant reductions in both wildtype and mutant htt, and demonstrated improved motor coordination and survival As a preliminary assessment of the safety of a non-allele-specific silencing strategy for HD, we performed transcriptional profiling to evaluate the effects of reducing wildtype htt in adult mouse striatum We identified gene expression changes that are concordant with previously described roles for htt in various cellular processes In addition, several abnormally expressed transcripts associated with early-stage HD were also differentially expressed in our studies, but intriguingly, those involved in neuronal function changed in opposing directions Whether this reversal of HD-related transcriptional deficits will occur and prove beneficial in a therapeutic setting remains to be determined Together, these encouraging and surprising findings support further testing of non-allele-specific RNAi therapeutics for HD This work was funded by the Lori C Sasser Fellowship, National Institute of Health and Hereditary Disease Foundation 419 Lethal Toxicity Caused by Striatal Delivery of shRNAs in Mouse Models of DYT1 Dystonia: Implications for Therapeutic Design Janine N Martin,1 Nicolle Wolken,6 Timothy Brown,4 William T Dauer,3 Michelle E Ehrlich,2 Pedro Gonzalez-Alegre.5 Genetics, U of Iowa, Iowa City, IA; 2Neurology, Mt Sinai School of Medicine, New York City, NY; 3Neurology and Pharmacology, Columbia U, New York City, NY; 4Biochemistry and Molecular Biology, Thomas Jefferson U, Philadelphia, PA; 5Neurology, U of Iowa, Iowa City, IA; 6Neurology, U of Iowa, Iowa City, IA DYT1 is the most common inherited dystonia, a disabling neurologically based movement disorder This incurable disease is caused by the deletion of a glutamic acid residue in the protein torsinA (torA(∆E)) A common, dominantly inherited mutation likely resulting from a dominant negative effect of torA(∆E) over torA(WT) indicates allele-specific silencing of torA(∆E) could be a potential therapeutic strategy for DYT1 We have previously tested shRNAs that achieve this goal in cultured neuronal cells without triggering inflammatory responses and we completed the following studies to determine the efficacy of those hairpins in vivo Two different mouse models of the disease, DYT1 knockin (KI) mice (129/SvJ strain) and transgenic (TG) mice overexpressing human torA(∆E) under the DARPP32 promoter (DARPP32-TorA(∆E)) (C57BL/6 background), along with control littermates were used The study design included bilateral striatal injections of rAAV2,1.CMV.GFP vectors encoding either U6shTorA(∆E) (therapeutic vector), U6shMis (control mismatched shRNA) or no shRNA at 2-6 months of age and behavioral testing (open field, rotarod and staircase reaching tests) at baseline and post injection before sacrificing the mice for biochemical and histological analysis Unexpectedly, a cohort of DYT1 KI and control mice (n: 10 per genotype/vector) displayed mortality rates of 70% and 53% for those receiving the therapeutic and control hairpins respectively, S164 occurring 3-4 weeks post injection, while the GFP only control vector did not induce toxicity In parallel experiments completed in DARPP32-TorA(∆E) TG mice and control littermates, the mortality rate was 41% and 28% in those receiving the therapeutic vector and control shMis respectively, with no toxicity in the GFP only group Mortality in the TG model was reduced when compared to the KI model and occurred 6-8 weeks post injection, perhaps reflecting the different genetic background This delay allowed us to complete behavioral analysis While baseline behavioral analysis did not display any difference, TG and control mice receiving the therapeutic vector and control shRNAs exhibited significant hyperactivity in open field behavior and did significantly worse on rotarod testing weeks post injection when compared to those injected with GFP only vectors, indicating striatal neuronal dysfunction In conclusion, our studies demonstrate that expression of U6-shRNA in the mammalian brain can lead to fatal toxicity, even when cell culture studies did not predict toxicity Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of therapeutic RNAi trials Future studies will explore the mechanism of toxicity and modifying the therapeutic vector to abolish toxicity 420 Rational Design of MicroRNA-Based RNA Interference Leads to More Potent Targeting of Hepatitis B Virus and Long Term Viral Inhibition In Vivo Kathy Keck,1 Ryan Spengler,1 Maria Scheel,1 Ye Ding,2 Anton P McCaffrey.1 Internal Medicine, University of Iowa, Iowa City, IA; 2Wadsoorth Center, New York Department of Health, Albany, NY Hepatitis B virus (HBV) is a small DNA virus that chronically infects 400 million people Previously, we conducted proof-ofprinciple experiments showing that RNA interference (RNAi) could degrade HBV RNAs in mice (McCaffrey et al 2003 Nature Biotechnology 21, 639) Recently Grimm et al expressed the short hairpin RNA (shRNA), HBVU6#2, described in our previous study, using self-complementary adeno-associated virus serotype (scAAV8) in HBV transgenic mice (Grimm et al 2006 Nature 441, 537) While this resulted in substantial HBV knockdown in mice, it also resulted in acute toxicity and death The authors concluded that the high levels of shRNA expression required to observe HBV knockdown oversaturated the RNAi machinery preventing endogenous microRNAs (miRNAs) expression We have utilized recent mechanistic insights to rationally design more potent HBV RNAi triggers We expressed our HBV RNAi triggers in the context of the endogenous miRNA, miR30 All our rationally designed HBV RNAi triggers showed significant silencing and eight were significantly more potent than HBVU6#2 A two step model was used to model the hybridization of the guide strand with the target RNA A regression analysis identified thermodynamic features that were highly correlated with RNAi activity Interestingly, miRNAbased RNAi triggers compete with endogenous miRNAs Pairs of miRNA-based RNAi triggers were expressed in HBV transgenic mice using scAAV8 HBV serum viral genomes were reduced by two logs over 50 days HBV surface antigen expression was similarly reduced Inhibition slowly diminished until remaining stable at ∼90% knockdown for over 120 days No fatality was observed Thus, we have demonstrated long term stable viral inhibition using scAAV8 delivery of miRNA-based RNAi These general approaches can be applied to inhibiting other viruses as well as treating dominant genetic disorders or cancer Supported by NIH 1R01AI068885-01A1 Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... whether co -silencing wildtype and mutant htt provides therapeutic benefit and is tolerable in HD mice Mice were injected intrastriatally with viral vectors expressing htt -specific artificial miRNAs. .. THERAPIES artificial miRNA strategy in a pre-clinical therapeutic trial aimed at assessing the viability of a non- allele- specific silencing strategy for Huntington’s disease (HD) HD is a dominant,... Together, these encouraging and surprising findings support further testing of non- allele- specific RNAi therapeutics for HD This work was funded by the Lori C Sasser Fellowship, National Institute