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 takker for fremragende støtte for deres laboratorium, teammedlemmer og arbejde med Cluster of Excellence CellNetworks på Heidelberg Universitet, samt ved Chica og Heinz Schaller (CHS) fundament. Vi sætter pris på, at molekylær AAV evolution via DNA-familien blander er blevet et meget aktivt område siden vores første udgivelse for tre år siden, og derfor undskylder over for alle forfattere af relevante publikationer, hvis arbejde kan ikke blive citeret her på grund af pladsmangel.

1. Fremstilling af plasmid sæt koder AAV-capsid Genes <ol><li> For at lette rutinemæssig fremstilling af tilstrækkelige mængder af de forskellige AAV capsid (cap) gener til efterfølgende DNA-shuffling, oprindeligt subklone disse gener i en fælles plasmidskelettet. Det er vigtigt at inkludere identiske flankerende sekvenser på&gt; 20 nukleotider til senere anvendelse som primer bindingssteder for nested PCR og til kloning (fig. 2). </li><li> Under anvendelse af passende primere (se tabe...

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	<li>Grimm, D., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+virus+evolution+to+vector+revolution:+use+of+naturally+occurring+serotypes+of+adeno-associated+virus+(AAV)+as+novel+vectors+for+human+gene+therapy.">From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy.</a> Curr. Gene Ther. 3, 281-304 (2003).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+methods+for+gene+transfer+vectors+based+on+adeno-associated+virus+serotypes.">Production methods for gene transfer vectors based on adeno-associated virus serotypes.</a> Methods. 28, 146-157 (2002).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+gene+therapy+vector+evolution+via+multispecies+interbreeding+and+retargeting+of+adeno-associated+viruses.">In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.</a> J. Virol. 82, 5887-5911 (2008).</li><li>Muller, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+peptide+libraries+displayed+on+adeno-associated+virus+to+select+for+targeted+gene+therapy+vectors.">Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.</a> Nat. Biotechnol. 21, 1040-1046 (2003).</li><li>Perabo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+viral+vectors+with+modified+tropism:+the+adeno-associated+virus+display.">In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.</a> Mol. Ther. 8, 151-157 (2003).</li><li>Zolotukhin, S., Potter, M., Hauswirth, W. W., Guy, J., Muzyczka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"humanized"+green+fluorescent+protein+cDNA+adapted+for+high-level+expression+in+mammalian+cells.">A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.</a> J. Virol. 70, 4646-4654 (1996).</li><li>Wobus, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+the+adeno-associated+virus+type+2+(AAV-2)+capsid:+epitope+mapping+and+identification+of+capsid+domains+involved+in+AAV-2-cell+interaction+and+neutralization+of+AAV-2+infection.">Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection.</a> J. Virol. 74, 9281-9293 (2000).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatality+in+mice+due+to+oversaturation+of+cellular+microRNA/short+hairpin+RNA+pathways.">Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.</a> Nature. 441, 537-541 (2006).</li><li>Nakai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unrestricted+hepatocyte+transduction+with+adeno-associated+virus+serotype+8+vectors+in+mice.">Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice.</a> J. Virol. 79, 214-224 (2005).</li><li>Koerber, J. T., Jang, J. H., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+shuffling+of+adeno-associated+virus+yields+functionally+diverse+viral+progeny.">DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.</a> Mol. Ther. 16, 1703-1709 (2008).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+selection+of+shuffled+AAV+genomes:+a+new+strategy+for+producing+targeted+biological+nanoparticles.">Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.</a> Mol. Ther. 16, 1252-1260 (2008).</li><li>Ward, P., Walsh, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+AAV+Cap+sequences+alter+gene+transduction.">Chimeric AAV Cap sequences alter gene transduction.</a> Virology. 386, 237-248 (2009).</li><li>Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myocardium+tropic+adeno-associated+virus+(AAV)+evolved+by+DNA+shuffling+and+in+vivo+selection.">A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 3946-3951 (2009).</li><li>Perabo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+engineering+of+a+gene+therapy+vector:+directed+evolution+of+adeno-associated+virus.">Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus.</a> J. Gene. Med. 8, 155-162 (2006).</li><li>Maheshri, N., Koerber, J. T., Kaspar, B. K., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+adeno-associated+virus+yields+enhanced+gene+delivery+vectors.">Directed evolution of adeno-associated virus yields enhanced gene delivery vectors.</a> Nat. Biotechnol. 24, 198-204 (2006).</li><li>Wu, Z., Asokan, A., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotypes:+vector+toolkit+for+human+gene+therapy.">Adeno-associated virus serotypes: vector toolkit for human gene therapy.</a> Mol. Ther. 14, 316-327 (2006).</li><li>Kwon, I., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+gene+delivery+vectors:+molecular+engineering+and+evolution+of+adeno-associated+viral+vectors+for+enhanced+gene+transfer.">Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer.</a> Pharm. 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Her har vi skitseret væsentlige eksperimentelle trin og retningslinjer for AAV kapsid engineering via DNA-familien blander og for evolution i celler eller dyr. I det væsentlige er disse protokoller standardiserede versioner af de procedurer, vi først rapporteret i AAV feltet i 2008 3. Mens en byge af opfølgende undersøgelser af andre har rapporteret talrige ændringer fx 10-13, vores nuværende versioner repræsenterer grundlæggende strategier der giver reproducerbare resultater og s...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> DNase I </td><td> Invitrogen </td><td> 18068-015 </td></tr><tr><td> Polyethylenimin (PEI) </td><td> Sigma-Aldrich </td><td> 408727 </td></tr><tr><td> Restriktionsenzymer </td><td> NEB </td><td> Various </td></tr><tr><td> T4 DNA-ligase </td><td> NEB </td><td> M0202T </td></tr><tr><td> Gelekstraktionskittet </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 (en del af kit) </td></tr><tr><td> EDTA (25 mM) </td><td> Invitrogen </td><td> 18068-015 (del af sæt) </td></tr><tr><td> Tris </td><td> Roth </td><td> 4855,2 </td></tr><tr><td> Ampicillin natriumsalt </td><td> Roth </td><td> K029.2 </td></tr><tr><td> dNTP&#39;er (10 mM, 100 pi) </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> Plasmid Mega prep kit </td><td> Qiagen </td><td> 12181 </td></tr><tr><td> Ultracentrifuge </td><td> Beckman-Coulter </td><td> Optima L90K </td></tr><tr><td> Quick-Seal centrifugerør </td><td> Beckman-Coulter </td><td> 342414 </td></tr><tr><td> Elektroporation enhed </td><td> Bio-Rad </td><td> GenePulserXcell </td></tr><tr><td> Termocycler </td><td> Eppendorf </td><td> Vapo Protect </td></tr><tr><td> Varme blok </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 (+)-plasmidet </td><td> Stratagene </td><td> 212207 </td></tr><tr><td> cap5F (Pac-site i gule, CAP5-specifikke sekvenser med fed skrift): GACTCTTAATTAACAGGT ATGTCTTTTGTTGATCACCCTCC </td><td> IDTDNA </td><td> Brugerdefineret primer </td></tr><tr><td> cap5R (Asc I stedet for grøn, CAP5-specifikke sekvenser med fed skrift): GTGAGGGCGCGCC TTAAAGGGGTCGGGTAAGGTATC </td><td> IDTDNA </td><td> Brugerdefineret primer </td></tr></tbody></table>

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

Adeno-associerede virale (AAV) vektorer repræsenterer nogle af de mest potente og lovende køretøjer til terapeutisk human genoverførsel grund af en unik kombination af fordelagtige egenskaber 1. Disse omfatter apathogenicity de underliggende vildtype vira og de ​​avancerede metoder til fremstilling af høj-titer, høj renhed og klinisk kvalitet rekombinante vektorer 2. En yderligere særlig fordel ved den AAV-systemet i forhold til andre vira er tilgængeligheden af et væld af naturligt forekommende serotyper, som afviger i væsentlige egenskaber endnu kan alle let konstrueres som vektorer under anvendelse af en almindelig protokol 1,2. Desuden har en række grupper, herunder vores egen nylig udtænkt strategier til at bruge disse naturlige virus som skabeloner til oprettelse af syntetiske vektorer, som enten kombinerer aktiver af flere input serotyper, eller der forbedrer egenskaberne ved et enkelt isolat. De respektive teknologier til at opnå disse mål are enten DNA familien blander 3, dvs fragmentering af forskellige AAV capsid gener efterfulgt af deres re-samling er baseret på partielle homologieme (typisk&gt; 80% for de fleste AAV-serotyper), eller peptid display 4,5, dvs indsættelse af normalt syv aminosyrer i en eksponeret sløjfe af det virale capsid, hvor peptidet ideelt medierer re-målretning til en ønsket celletype. For at opnå maksimal succes, er begge metoder anvendes på en high-throughput fashion, hvor protokollerne er op-skaleret til at give biblioteker på omkring en million forskellige capsid varianter. Hver klon derpå omfatter en unik kombination af talrige parentale virus (DNA shuffling metode) eller indeholder en karakteristisk peptid i den samme virale rygraden (peptid display fremgangsmåde). Den efterfølgende sidste trin er iterativ udvælgelse af et sådant bibliotek på målceller for at berige for de enkelte capsider, der opfylder de fleste eller ideelt alle krav udvælgelsesprocessen. Sidstnævnte fortrinsvis kamines positivt tryk, såsom vækst på en bestemt celletype af interesse, med negativ selektion for eksempel fjernelse af alle capsider reagerer med anti-AAV-antistoffer. Denne kombination øger chancerne for, at syntetiske capsiderne overlevende valget matcher behovene i given applikation på en måde, der sandsynligvis ikke ville have fundet i noget naturligt forekommende AAV isolere. Her fokuserer vi på DNA-familien blander metode som teoretisk og eksperimentelt mere udfordrende af de to teknologier. Vi beskriver og demonstrere alle væsentlige skridt til generering og udvælgelse af blandet AAV biblioteker (fig. 1), og derefter drøfte de faldgruber og kritiske aspekter af protokollerne, at man skal være opmærksom på for at lykkes med molekylær AAV evolution.

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 grundlæggende teknik til molekylær konstruere og udvikle syntetiske Adeno-associerede virus (AAV) genterapivektorer via DNA familie blanding. Desuden leverer vi generelle retningslinjer og repræsentative eksempler på udvælgelse og analyse af individuelle kimære capsider med forbedrede egenskaber på målceller i kultur eller i mus.

Engineering and Evolution af syntetisk Adeno-associeret virus (AAV) Genterapivektorer via DNA Family blander

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

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