Name: engineering and evolution of synthetic adeno-associated virus (aav) gene therapy vectors via dna family shuffling
Uploaded: 2012-04-02T12:00:00
Description: Watch this Scientific Journal Video about Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling at JoVE.com

Forfatterne ønsker å takke for utmerket støtte av laboratoriet deres, gruppemedlemmer og arbeidet ved Cluster of Excellence CellNetworks ved Heidelberg Universitet, samt av Chica og Heinz Schaller (CHS) stiftelse. Vi forstår at molekylær AAV evolusjon via DNA familie shuffling har blitt en svært aktiv feltet siden vår første publikasjon tre år siden, og derfor beklager til alle forfattere av relevante publikasjoner som arbeidet kunne ikke bli sitert her på grunn av plassbegrensninger.

1. Utarbeidelse av Plasmid sett koding AAV kapsid Genes <ol><li> For å lette rutine utarbeidelse av tilstrekkelige mengder av de ulike AAV kapsid (CAP) genene for påfølgende DNA shuffling, først subclone disse genene inn i en felles plasmid ryggrad. Det er viktig å inkludere identiske flankerer sekvenser av&gt; 20 nukleotider for senere bruk som primer bindingssteder for nestet PCR og kloning (Fig. 2). </li><li> Bruk av egnede primere (se tabell for eksemplarisk primere for AAV5), PCR fo...

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Her har vi skissert viktige eksperimentelle skritt og retningslinjer for AAV kapsid prosjektering via DNA familie stokke og for evolusjon i celler eller i dyr. I hovedsak er disse protokollene er standardiserte versjoner av prosedyrene vi først rapportert innen AAV feltet i 2008 3. Mens en kave av oppfølgingsstudier av andre har rapportert om en rekke modifikasjoner for eksempel 10-13, våre nåværende versjoner representerer grunnleggende strategier og ga reproduserbare resultater samt...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> DNase jeg </td><td> Invitrogen </td><td> 18068-015 </td></tr><tr><td> Polyethylenimine (PEI) </td><td> Sigma-Aldrich </td><td> 408727 </td></tr><tr><td> Restriksjonsenzymer </td><td> NEB </td><td> Various </td></tr><tr><td> T4 DNA ligase </td><td> NEB </td><td> M0202T </td></tr><tr><td> Gel utvinning kit </td><td> Qiagen </td><td> 28704 </td></tr><tr><td> Phusion II polymerase Kit </td><td> Finnzymes (NEB) </td><td> F-540S </td></tr><tr><td> HotStar Hifi polymerase Kit </td><td> Qiagen </td><td> 202602 </td></tr><tr><td> DMSO </td><td> Finnzymes (NEB) </td><td> F-540S (del av sett) </td></tr><tr><td> EDTA (25 mm) </td><td> Invitrogen </td><td> 18068-015 (del av sett) </td></tr><tr><td> Tris </td><td> Roth </td><td> 4855.2 </td></tr><tr><td> Ampicilin sodium salt </td><td> Roth </td><td> K029.2 </td></tr><tr><td> dNTPs (10 mm, 100 mL) </td><td> Invitrogen </td><td> 18427013 </td></tr><tr><td> Iodixanol (OptiPrep) </td><td> Axis-Shield </td><td> 1114739 </td></tr><tr><td> Phenolred </td><td> Merck </td><td> 107241 </td></tr><tr><td> Plasmidet mega prep kit </td><td> Qiagen </td><td> 12181 </td></tr><tr><td> Ultrasentrifuger </td><td> Beckman-Coulter </td><td> Optima L90K </td></tr><tr><td> Quick-Seal sentrifugerør </td><td> Beckman-Coulter </td><td> 342414 </td></tr><tr><td> Electroporation enhet </td><td> Bio-Rad </td><td> GenePulserXcell </td></tr><tr><td> Termisk cycler </td><td> Eppendorf </td><td> Vapo Protect </td></tr><tr><td> Oppvarming blokk </td><td> BIOER </td><td> MB-102 </td></tr><tr><td> Fluorescens mikroskop </td><td> Olympus </td><td> IX81 </td></tr><tr><td> FACS analysator </td><td> Beckman-Coulter </td><td> Cytomics FC500 MLP </td></tr><tr><td> MegaX DH10B T1R celler </td><td> Invitrogen </td><td> C640003 </td></tr><tr><td> Benzonase </td><td> Merck </td><td> 101695 </td></tr><tr><td> Adenovirus-5 </td><td> ATCC </td><td> VR-5 </td></tr><tr><td> pBlueScript II KS (+) plasmid </td><td> Stratagene </td><td> 212207 </td></tr><tr><td> cap5F (Pac jeg stedet i gule, cap5-spesifikke sekvenser i fet skrift): GACTCTTAATTAACAGGT ATGTCTTTTGTTGATCACCCTCC </td><td> IDTDNA </td><td> Custom primer </td></tr><tr><td> cap5R (Asc jeg nettstedet i grønt, cap5-spesifikke sekvenser i fet skrift): GTGAGGGCGCGCC TTAAAGGGGTCGGGTAAGGTATC </td><td> IDTDNA </td><td> Custom primer </td></tr></tbody></table>

engineering and evolution of synthetic adeno-associated virus (aav) gene therapy vectors via dna family shuffling

Adeno-tilknyttede viral (AAV) vektorene representerer noen av de mest potente og lovende biler for terapeutisk human genoverføring grunn av en unik kombinasjon av gode egenskaper 1. Disse inkluderer apathogenicity av de underliggende hvete virus og de ​​svært avanserte metoder for produksjon av høy-titer, høy renhet og klinisk-grade rekombinante vektorer 2. En ytterligere spesiell nytte av AAV systemet over andre virus er tilgjengeligheten av et vell av naturlig forekommende serotypene som avviker i vesentlige egenskaper ennå kan alle enkelt konstruert som vektorer ved hjelp av en felles protokoll 1,2. Videre har en rekke grupper, inkludert vår egen nylig utviklet strategier for å bruke disse naturlige virus som maler for å lage syntetiske vektorer som enten kombinerer eiendelene til flere input serotyper, eller som forbedrer egenskapene til en enkelt isolere. De respektive teknologier for å oppnå disse målene are enten DNA familie tre shuffling, dvs. fragmentering av ulike AAV kapsid gener etterfulgt av sin re-montering basert på partielle homologies (typisk&gt; 80% for de fleste AAV serotype), eller peptid visning 4,5, dvs. innsetting av vanligvis syv aminosyrer inn en utsatt løkke av viral kapsid der peptid ideelt formidler re-målretting til en ønsket celletype. For maksimal suksess, er begge metodene brukes i en high-throughput fashion der protokollene er opp-skaleres til å gi bibliotekene i rundt en million forskjellige kapsid varianter. Hver klone er da består av en unik kombinasjon av mange foreldre virus (DNA shuffling tilnærming) eller inneholder en særegen peptid innenfor samme viral ryggraden (peptid visning tilnærming). Den påfølgende siste trinnet er iterativ valg av et slikt bibliotek på målceller for å berike for individuelle capsids oppfyller de fleste eller ideelt sett alle krav i utvelgelsesprosessen. Sistnevnte helst kamInes positivt press, for eksempel vekst på en bestemt celletype av interesse, med negativ seleksjon, for eksempel eliminering av alle capsids reagerer med anti-AAV antistoffer. Denne kombinasjonen øker sjansen for at syntetiske capsids overlevende utvalget svarer behovene til den enkelte søknaden på en måte som trolig ikke ville ha blitt funnet i noen naturlig forekommende AAV isolere. Her fokuserer vi på DNA familien shuffling metode som teoretisk og eksperimentelt mer utfordrende av de to teknologiene. Vi beskriver og demonstrerer alle nødvendige skritt for generering og valg av stokkes AAV bibliotekene (Fig. 1), og deretter diskutere fallgruver og kritiske aspekter ved de protokoller som man trenger å være klar over for å lykkes med molekylær AAV evolusjon.

Watch this Scientific Journal Video about Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling at JoVE.com

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

Vi viser den grunnleggende teknikken til å molecularly konstruere og utvikle syntetiske Adeno-tilknyttede viral (AAV) genterapi vektorer via DNA familie shuffling. Videre gir vi generelle retningslinjer og representative eksempler for utvalg og analyse av individuelle chimeric capsids med forbedrede egenskaper på målceller i kultur eller i mus.

Engineering and Evolution of Synthetic Adeno-assosiert virus (AAV) genterapi vektorer via DNA Family shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

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