259 Rapid Identification of Serum Stable Aptamers From Existing Populations Via Poly 2’ SELEX Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Ther[.]
PHYSICAL/NON-PHYSICAL METHODS OF DELIVERY primal determinant of the efficiency in hydrodynamic delivery This is the first report which demonstrates the actual real-time hemodynamics upon a hydrodynamic injection after gene transfer revealed approximately 15% positively stained cells with anti-hAAT antibody in targeted lobes, and less than 1% in non-injected lobes The positive cells decreased to approximately 8% by the end of weeks Initially, serum biochemistry showed a 2-4 fold increase in liver enzymes and returned to normal ranges after a few days Histological analysis showed transient enlargement of the central vein and the sinusoid recovered within 24 hrs PCR analysis showed that gene transfer was liver specific and no gene expression took place in other organs No negative impacts of the procedure on physiological parameters, including blood pressure, electrocardiogram, and animal growth were observed in the entire period of the study These results suggest that effective and safe hydrodynamic gene transfer can be performed in human-size animals, and the clinical applicability of the procedure This work was supported in part by NIH grants RO1EB002946 and RO1HL075542, and by Japanese Society for the Promotion of Sciences Grant-in-Aid for Scientific Research 22890064 and 23790595 258 Histone-Targeted Polyplexes Use Native Vesicular Trafficking Mechanisms to Reach the Nucleus Nikki L Ross,1 Erik Munsell,1 Millicent O Sullivan.1 Chemical & Biomolecular Engineering, University of Delaware, Newark, DE 257 Parameters for Optimal Hydrodynamic Gene Delivery to Pig Liver Using an Image-Guided and Lobe-Specific Procedure Kenya Kamimura,2 Hiroyuki Abe,2 Takeshi Yokoo,2 Tsutomu Kanefuji,2 Takeshi Suda,2 Guisheng Zhang,1 Yutaka Aoyagi,2 Dexi Liu.1 Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA; 2Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan Image-guided, liver lobe specific hydrodynamic gene delivery was employed to evaluate the effect of various parameters on gene delivery efficiency and the long-term effects on human-size pigs (female, 40-60 kg, n=40) Among the parameters examined, hydrodynamic injection from the intermediate site of a hepatic vein with an injection flow rate of 40 ml/sec, quickly reaching the peak intravascular pressure of 150 mmHg, resulted in the highest level of luciferase reporter gene expression (pCMV-Luc) at 3,143 pg/mg of proteins An optimal volume of DNA solution for sequential injection to the multiple targeted lobes was established at 2.5x lobe volume resulting in luciferase expression at 3,699 pg/mg of proteins in injected lobes The pCAG-hAAT plasmid was hydrodynamically injected with these parameters for optimal hydrodynamic gene delivery to two 40 kg pigs to examine the long-term effect of the procedure, including the persistence of the transgene expression, tissue damage, and plasmid distribution in the organs Immunohistochemical analysis weeks S98 Active cellular division is a hallmark of tissue repair as well as multiple other pathologies, and therefore methods to modulate gene expression profiles in proliferating cells would have enormous therapeutic advantages However, while many types of cells will efficiently internalize nucleic acid assemblies, poorly controlled subcellular trafficking and disassembly represent key hurdles preventing clinical realization We hypothesized that peptide sequences derived from nature’s DNA packaging scaffolds – histone proteins – might enable recapitulation of native nuclear localization and controlled release mechanisms Accordingly, we engineered gene delivery polyplexes by packaging plasmid DNAs (pDNAs) with histone tail sequences containing nuclear localization sequences (NLSs) and key modifications known to activate transcription on chromatin We have demonstrated that these histone-mimetic polyplexes exhibit robust gene transfer activity in cultured fibroblasts relevant to tissue repair Furthermore, we have used cell synchronization, high-resolution confocal microscopy, and fluorescence correlation spectroscopy to identify key steps in polyplex trafficking to the nucleus, including retrograde transport mediated by histone processing enzymes and apparent vesicular trafficking into the nucleus Finally, we have shown that these polyplexes interact with key nuclear proteins to activate rapid unpackaging and transcription in the nucleus Thus, our studies add new understanding of cellular gene transfer mechanisms, with direct relevance to wound repair and potential applicability to a wide range of other therapeutic applications involving gene modulation in dividing cells 259 Rapid Identification of Serum-Stable Aptamers From Existing Populations Via Poly-2’ SELEX Khalid K Alam,1 Jonathan L Chang,2 Donald H Burke.1,2 Department of Biochemistry, University of Missouri, Columbia, MO; 2Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO Direct delivery of RNA aptamer therapeutics through the bloodstream remains an attractive route of drug administration and a viable alternative to vector based delivery of genes that express those RNAs While diverse polymer backbone modifications are available to stabilize RNAs against serum nuclease activity and Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy NEUROLOGICAL DISEASES improve pharmacokinetics, these modifications are often performed post-selection and require an iterative process of trial and error to ensure efficacy In principle, combinatorial screening can accelerate identification of optimal modifications, but such screening can be laborious and many modified nucleotides are incompatible with the diverse starting libraries required of in vitro selection due to low yields during transcription or cDNA synthesis To bridge this gap, we developed a novel selection scheme, termed “Poly-2’ SELEX,” in which pre-enriched RNA aptamer libraries are subjected to additional rounds of selection with 2’- modified pyrimidines that confer serum stability Herein we report on the identification of serum-stable RNA aptamers against HIV reverse transcriptase (HIV RT) using this novel variation of SELEX technology Pre-enriched libraries originally selected with standard RNA were subjected to three additional rounds of selection in parallel trajectories in which the 2’-hydroxyl of pyrimidines were replaced with 2’-NH2, 2’-F, or 2’-O-Methyl functional groups during library transcription Each trajectory yielded new populations that bound and inhibited the selection target Next generation sequencing and comparative sequence analysis of these populations revealed enrichment of aptamers within particular modification trajectories that either accommodate or adapt to these various 2’- modifications Primer extension assays confirmed that these newly identified aptamers retain strong inhibition of HIV RT and they exhibited significantly longer half-lives in serum compared to standard RNA When coupled with high-throughput sequencing and bioinformatic analysis, Poly2’ SELEX has the capacity to identify existing aptamers suitable for direct delivery while avoiding potentially costly and difficult selections de novo or time-consuming ad hoc optimization Neurological Diseases 260 Long-Term Neurologic Correction in the Pompe Disease Mice By Intrathecal Gene Therapy Juliette Hordeaux,1,2,3 Laurence Dubreil,1,2 Cynthia Robveille,1,2 Quentin Pascal,1,2 Johan Deniaud,1,2 Mireille Ledevin,1,2 Candice Babarit,1,2 Frederic Jamme,4 Corinne Huchet,3,5 Catherine Caillaud,6,7 Marie-Anne Colle.1,2 PAnTher UMR703, INRA French National Institute for Agricultural Research, Nantes, France; 2LUNAM, Oniris NantesAtlantic National College of Veterinary Medicine, Food Science and Engineering, Nantes, France; 3LUNAM, University of Nantes, Nantes, France; 4SOLEIL French National Synchrotron Facility, Gif-sur-Yvette, France; 5U915, INSERM French National Institute for Health and Medical Research, Nantes, France; U845, INSERM French National Institute for Health and Medical Research, Paris, France; 7Necker Enfant Malade Institute, University of Paris Descartes, Paris, France Pompe disease is a lysosomal storage disorder caused by acidα-glucosidase (GAA) deficiency, leading to glycogen storage The disease manifests as a fatal cardiomyopathy in infantile form Enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history The emergent neurologic phenotype and the poor correction of skeletal muscles in survivors are currently attributed to central nervous system (CNS) glycogen storage, uncorrected by ERT Hence, we hypothesized that intrathecal delivery of AAV-gaa in Pompe disease mice would correct the neurologic and neuromuscular manifestations of the disease GAA-KO mice were injected with ssAAVrh10-gaa into the cisterna magna at one month Their neurologic and motor skills including auditory function were periodically monitored from three to twelve months by hindlimb clasping reflex, brainstem auditory evoked potentials, wire-hang test, and accelerating rotarod Grip strength measurement and in situ muscular contractile activity were recorded at end points - and 12 months Glycogen content, GAA activity, and disease-related pathology were assessed in the CNS and muscles We also analyzed the treatment related chemical changes in motor neurons and white matter tracts of spinal cord using Fourier transform infrared (FT-IR) microspectroscopy with a synchrotron light source We demonstrate a significant functional neurologic correction in treated animals from months onward, a neuromuscular improvement from months onward, and a partial restoration of the muscular strength at end points The entire CNS, including the regions most affected by the disease such as the brainstem, spinal cord, and spinal ganglia show enzymatic, biochemical and histological correction Muscle glycogen storage is not cleared by the treatment, thus suggesting that the restoration of muscle functionality is directly related to the CNS correction This unprecedented widespread CNS cure and its impact on the global neuromuscular function offer new perspectives for the management of patients 261 AAVrh10-ATP7A Administration to the Cerebrospinal Fluid, in Combination with Subcutaneous Copper, Normalizes Neurological Outcomes in a Mouse Model of Menkes Disease Marie Reine Haddad,1 Eun-Young Choi,1 Stephen G Kaler.1 Section on Translational Neuroscience, Molecular Medicine Program, NICHD, NIH, Bethesda, MD Menkes disease is a X-linked recessive neurodegenerative disorder of copper metabolism produced by diverse mutations in a copper transporting ATPase, ATP7A The disorder presents within weeks of birth and and is often fatal by age to years Subcutaneous injections of copper alone can normalize outcomes if commenced within 10 days of age, but only in the context of a favorable mutation that enables Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S99 ... subjected to additional rounds of selection with 2’- modified pyrimidines that confer serum stability Herein we report on the identification of serum- stable RNA aptamers against HIV reverse transcriptase... trajectory yielded new populations that bound and inhibited the selection target Next generation sequencing and comparative sequence analysis of these populations revealed enrichment of aptamers within... variation of SELEX technology Pre-enriched libraries originally selected with standard RNA were subjected to three additional rounds of selection in parallel trajectories in which the 2’-hydroxyl of