690 corneal gene therapy: past experiences, current trends and future perspectives

690 Corneal Gene Therapy Past Experiences, Current Trends and Future Perspectives manufacturing process and its associated release testing can be ac complished in a reasonable timeframe to be clinical[.]

manufacturing process and its associated release testing can be accomplished in a reasonable timeframe to be clinically relevant We will present data on the tumor harvest / purification process as well as the xenograft vaccine quality control testing plan 688 Aiming at Alcoholism with Ribozyme Genes: Silencing the mRNA for an Alcohol Detoxifying Enzyme Lorena Lobes-Gonzalez, I Carlos Munoz-Brauning, I Amalia Sapag.' 'Gene Pharmacotherapy Laboratory, Department ofPharmacological and Toxicological Chemistry, Faculty ofChemical and Pharmaceutical Sciences, Universidad de Chile , Santiago, Chile Protection against alcoholism is provided by a natural aversion to alcohol in certain individuals or by pharmacological intervention in alcoholics Ethanol is detoxified by oxidation to acetaldehyde and then to acetate by alcohol dehydrogenase and aldehyde dehydrogenase-2 (ALDI-I2) Impairment of ALOI·J2 results in accumulation ofacetaldehyde leading to unpleasant physical effects and aversion to ethanol This condition is observed in individuals carrying an inactivating mutation in the ALDH2 gene and in alcoholics treated with disulfiram, a nonspecifictoxic drug which inactivatesALDH2 by covalent modification Similar protection might be achieved by diminishing enzyme activity with highly specific ribozymes designed to silence the mRNA for ALDI-I2 Hairpin and hammerhead ribozymes targeted to a single region of rat ALOI-12 mRNA (nucleotides 1551-1571)were tested in rat hepatomacells in culture Antisense (blockade) effects were assessed with control ribozymes incapable of cleaving RNA H4-II-E-C3 cells were lipofcctcd with plasmids carrying ribozymc genes driven by a CMV promoter and the ALDH2 activity in cell extracts was measured spectrophotometrically.The hairpin ribozyrne provided a 42 % reduction in the ALDH2 activity, 24 % due to cleavage and 18 % due to antisense action The combined effects of this hairpin ribozyme amount to -70 % reduction oftheALDH2 activity when protein half life and lipofection efficiency are taken into account The same hairpin ribozyme delivered in a plasmid construct which endows it with a longer RNA tail on its 3' side suppressed 36 % ofthe ALDH2 activity, 13% by e1eavage and 23 % by blockade, indicating that overall ribozyme action and cleavage efficiency are sensitive to nanking sequences In contrast, the hammerhead ribozyme provided a modest reduction of 19 % in ALOI-12 activity due entirely to antisense effects In conclusion, a hairpin ribozyme directed to nts 1553-1569 of the ratALDH2 mRNAis a good candidatefor in vivo experiments geared towards developing genetic drugs for alcoholism Supported by FONDECYT 1040555, UChile PG 06504, [CM P99-03[F and [CM P05-001E 689 Targeting Activator Protein-1 To Investigate Protective Phytocompounds Against UVB Radiation-Induced Photoaging in Mouse Vanisree Staniforth, Wen-Ching Huang, Ning-Sun Yang 'Agricultural Biotechnology Research Center; Agricultural Biotechnology Research Center; Taipei Taiwan UV radiation is the main environmental factor that causes premature aging of the skin UVB radiation induces ligand-independent activation of cell surface growth receptors and cytokine receptors, which in tum results in the activation of down stream signaling pathways These pathwaysconvergeto stimulatetranscriptionfactor activator protein-I (AP-I) UVB radiation induced AP-I regulates the expression of matrix-mctalloprotcinascs (MMPs), which have been shown to play important role in degradation of extra-cellular matrix and acceleration of skin aging We have initiated a study to identify the phytocompounds that can abrogate the aging effects S264 of UVB radiation For this purpose, mouse skin transfected with transgenicpromotercontainingAP-I bindingsites driving luciferase gene expression was irradiated with UVB and treated with various phytocompounds Weobserved that UVB irradiation ofmouse skin resultedin significantinductionofAP-I activationand expression of MMPs Our results indicated that phytocompounds such as caffeic acid, luteolin, ferulie acid and geraniol significantly inhibited UVB radiation induced activation of AP-[ transcription factor CafTeic acid and ferulic acid showed significant inhibition ofUVB-induced MMP-9 expression Whereas, luteolin and geraniol significantly inhibitedUVB-inducedMMP-2expression.Our resultssuggestthat the above-mentionedphytocompoundsmay have potentialto confer protection against photoaging Further investigation is in progress to study the effects of the above-identified phytocompounds on morphologicalchanges associated with photoaging and to elucidate the detailed mechanistic basis underlying those effects 690 Corneal Gene Therapy: Past Experiences, Current Trends and Future Perspectives Eytan A Klausner,I Dan Peer,' Robert L Chapman, I Richard E Multack,? Shridhar V Andurkar,' 'Pharmaceutical Sciences, Midwestern University Chicago College ofPharmacy Downers Grove, IL; lAnesthesia, CBR Institute for Biomedical Research, Boston, MA; JOphthalmolog)~ Midwestern University Chicago College ofOsteopathic Medicine, Downers Grove, IL Gcne therapy to the cornea can potentially correct inherited and acquired diseases of the cornea, which arc second only to cataract as the leading cause of vision loss We have searched the literature in order to characterize various aspects ofcorneal gene therapy preclinical studies; vectors, delivery systems, modes of'administration, the animal species used,and diseases that were treated Experiments in corneal gene therapy were applied to acquired, but not inherited, corneal diseases DNA vaccination, RNA interference and gene transfer of cytokincs, growth factors and enzymes modulated the cornealmicroenvironment Positiveresultswere obtainedin preclinical studies for prevention and treatment of corneal graft rejection, neovascularization, haze and herpetic stromal keratitis Aqueous solution was the most common corneal gene deliverysystem While in gene therapy, viral vectors are used much more commonly than non-viral vectors or physical techniques, viral vectors were used in only 45.7% of the corneal gene therapy studies (see Table I) In corneal gene therapy, as in gene therapy in general, viral vectors yielded stronger gene expression that was accompanied by more severe adverse effects, in comparison to non-viral vectors The difficulty in obtaining gene expression following topical ocular administration was circumvented by invasive ocular modes of administration.Thereby, in general, injection into the corneal anterior chamber and the stroma Icd to gene expression in the corneal endothelium and stroma, respectively, regardless ofvcctor type or animal species studied Experiments for investigating corneal gene therapy for differentcorneal diseases may require the usc of various animal species and a certain disease might be best studied using a particular animal species For example, almost all (21/23, 91.3%) of the studies involving herpetic stromal keratitis were conducted on mice (see Figure I) Opportunitiesin the fieldof cornealgene therapy lie in expanding the array of corneal diseases investigatedand in the Molecular Therapy Yofume 15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene Tllcr.lpr application ofrecent designs using safer vectors exhibiting reduced imrnunogenicity and longer duration of gene expression mental diseases and for the development of prenatal gene therapy for specific disorders Table t: The gene transfer method used in ill ~'il'O corneall(ene delivery peer-reviewed lIublic ations(t994-2006) P ~ ' , ercentage Vector No of Pubhcahons Pub licatio ns 25 ,27,2, -.; Adcnovirnl vectors Non-adenoviral viral vectors 118.5 I Naked DNA 2.9 Non-viiiil vectorsothcr than '7 ,6 naked DNA I ,7 Physical mcthods 9,8 G FP positive organs by different g cst atlO~a I ECG - T EIO Ell E08 E09 I!aLoLinjection f.ctoderm -derived organs I I I B.!1!in ( -) ~(~) -,( -) ito) Rctina ( ~) ~(-) - - -(-) Comea (:l ~( ~) _ _ _ (')- - - I S kin ( ~J (-)- , ( - ) (:) ~ 1· l\ lescd er m-d erived organs Id ney l ( ~) ~ ( - L , .lH c - - I(: ) Bladder (-) «+» «+» (-) G o.!!ad(le-,t!!;!oyan')_ _ _ «: ~) ~ « : !J) -l« ±») _ _ (-) (-) _ _ _«±»_ _ «+)).-.,(,) Uterns lVa g i i l3 l~a l (:l _ _ _ « ±) ~« + ) -r~ S m ~ l3 t y es i cl e (:l { :)- ,(:) 1: F: ndode r m-d en \'ed orga ns I T hY!I1US , ( ~ ) ; ( ~ )~ ( ~) {:~ iThroid (-) ( - )~ ( - )-L iver « iJ) ~( ~) ~( :) - - - _ k) L 1I!1£ «+»- -« +).-.,(:) 11 ~ :~~~~~~~ 131.5 :~~~ I~ ~ ~~~:~~~ ~~~~ sR N A aptamers ,5 , I I Figur e 1: TtM distrib uti on of c:orrntal gene d eliv efY pu b lication s according 10 the anima l species an d ex pe ri mental disease model " I!IIMic e O Rat s • Rabbit s C Sh •• p Monk eys • Dog s fJ 1< Pancreas I nle_stine I «i» «: ; ) ~( -) (-) «±»_ _(:) _H (-) ,( ~) 692 Regulatory Elements for Improved Gene Therapy Vectors Amy C Groth; George Stamatoyannopoulos,' David W Emery.' IDepartment a/Medicine, University 0/ Washington, Seattle, IVA 691 Developmental Stage Determines Distribution of Organ Transduction after Extracoelomic Cavity Injection of Lentiviral Vector Masayuki Endo, Tiago H Coelho, Phillip W Zoltick, Antoneta Radu, Nidal Muvarak, Alan W Flake IThe Children's Center/or Fetal Research, The Children's Hospitala/Philadelphia Philadelphia, PA, The development of fetal gene transfer can be applicable both to fetal gene therapy and developmental biological research: Th~re are several ways to inject genes directly into the fetus for in VIVO fetal gene transfer However, extracoelomic cavity gene transfer (ECGT) has not showed any gene expression in the late gestational stages We hypothesized that early gestational administration into the exracoelomic cavity might result in broader gene transfer to the mesoderm-derived organs due to developmental accessibility To test this hypothesis, we injected lentiviral vector carrying the green fluorescent protein (GFp) reporter gene into the feta~ murine extra-coelomic cavity from the late head fold/early somite stage (E8) to E II Injections were performed under image guidance usWe ing a Visualsonies@ Vev0770 system with a 40 mHz s~anhead observed the injected mice under fluorescence stereomicroscopy at by sequential time points after birth and confirmed G\p e~pression immunohistochemistry We injected total 78 fetal mice lrom E8-E II and total survival rate was 52.6 % In early gestation (E8-EIO), orga~s (Table) significant GFPexpression was observed in ~ul~iple Remarkably, GFp expression was observed m tissues denved from endoderm and mesoderm including genital system at E8, whereas no expression was observed in ectoderm derived tissues After E II , we could not detect GFP expression The observed temporal patterns of gene expression correspond to the expected embryologic accessibility of organ specific cell populations We conclude from this study that early gestational extracoelomic cavity gene transfer has higher efficiency and a broader distribution of transduction to both of mesoderm- and endoderm-derived organs than what has been previously observed later in gestation This model may be useful for investigation of mechanisms of genetic and/or developMolecular Therapy Volume15, Supplement I May 2007 Copyright © Th e AmericanSociety of Gf,.'Jl1; Therapy Enhancers and repressor-blocking insulators are important for strong, stable gene expression from gene therapy vectors However, enhancers have the ability to inappropriately activate cellular genes and cause transformation events It is therefore essential to include clements such as enhancer-blocking insulators that will limit the We regulatory reach of vector enhancers to the relevant t:~nsgenes have developed two functional screens, one for positive regulatory elements (enhancers and repressor-blocking insulators), and one for enhancer-blocking insulators To identify new positive regulatory elements we cloned a library ofsize-selected human genomic DNA fragmen;s into the 3' LTR ofthe GFP-expressing gammaretrovirus, MGPN2 Upon reverse transcription and integration, each cloned fragment is copied to the 5' LTR and the GFP gene is flanked by the candidate element We enriched the library for positive regulatory elements by sorting for the top 2-10% of Grp+ cells, expanding, harvesting virus and transducing new packaging cells Enhancers are likely to be present in this population, and repressor-blockers may be over-represented in all fractions due to their anti-silencing effect After three or five rounds of enrichment, we sequenced the inserts and assayed them for enhancement and stabilization ofgen.e expression One element , 1-1-11, is likely an enhancer Although It did not exhibit enhancer activity in a transient luciferase assay, there was a 4-fold increase in colony formation in a stable integration promoter-trap assay when compared to the neutral spacer, G6pD In a mouse bone marrow colony assay, individual 1-1-11 colonies had a significantly higher mean fluorescence !han the e~pt~ MGPJ;l2 vector (average 115 mfu vs 46 rnfu, p