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

Авторы выражают благодарность выдающимся поддержку своей лаборатории, членов команды и работы кластера передового опыта CellNetworks в Гейдельбергском университете, а также Чика и Хайнц Шаллер (CHS) фундамент. Мы понимаем, что молекулярная эволюция AAV с помощью ДНК перетасовки семья стала очень активной области с нашей первой публикации три года назад, и поэтому извинения всем авторам соответствующих публикаций, работа которых не может быть заключено в кавычки здесь из-за нехватки места.

1. Подготовка плазмиды наборы Кодирование AAV капсида Гены <ol><li> Для облегчения рутинной подготовки достаточного количества различных AAV капсида (крышка) гены последующим перетасовка ДНК, первоначально субклон этих генов в общем плазмиды позвоночника. Важно включают идентичные фл...

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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. Res. 25, 489-499 (2008).</li><li>Perabo, L., Huber, A., Marsch, S., Hallek, M., Buning, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+evolution+with+adeno-associated+viral+libraries.">Artificial evolution with adeno-associated viral libraries.</a> Comb. Chem. High. Throughput. Screen. 11, 118-126 (2008).</li><li>McCarty, D. M., Monahan, P. E., 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=Self-complementary+recombinant+adeno-associated+virus+(scAAV)+vectors+promote+efficient+transduction+independently+of+DNA+synthesis.">Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.</a> Gene Ther. 8, 1248-1254 (2001).</li></ol>

Здесь мы выделили существенные экспериментальные шаги и рекомендации по AAV капсида инженерных через ДНК семьи перетасовки и эволюции в клетках или в животных. В сущности, эти протоколы являются стандартными версиями процедуры мы впервые сообщили в поле AAV в 2008 году 3. В то время ка...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td></tr><tr><td> ДНКазы I </td><td> Invitrogen </td><td> 18068-015 </td></tr><tr><td> Полиэтиленимина (PEI) </td><td> Sigma-Aldrich </td><td> 408727 </td></tr><tr><td> Ограничение ферменты </td><td> NEB </td><td> Различный </td></tr><tr><td> T4 ДНК-лигазы </td><td> NEB </td><td> M0202T </td></tr><tr><td> Гель для извлечения набора </td><td> Qiagen </td><td> 28704 </td></tr><tr><td> Phusion II полимеразной Kit </td><td> Finnzymes (НЭБ) </td><td> F-540S </td></tr><tr><td> HotStar Hifi полимеразной Kit </td><td> Qiagen </td><td> 202602 </td></tr><tr><td> ДМСО </td><td> Finnzymes (НЭБ) </td><td> F-540S (часть комплекта) </td></tr><tr><td> ЭДТА (25 мМ) </td><td> Invitrogen </td><td> 18068-015 (часть комплекта) </td></tr><tr><td> Трис </td><td> Рот </td><td> 4855,2 </td></tr><tr><td> Ампициллина натриевая соль </td><td> Рот </td><td> K029.2 </td></tr><tr><td> дНТФ (10 мм, 100 мкл) </td><td> Invitrogen </td><td> 18427013 </td></tr><tr><td> Иодиксанол (OptiPrep) </td><td> Axis-экран </td><td> 1114739 </td></tr><tr><td> Phenolred </td><td> Merck </td><td> 107241 </td></tr><tr><td> Плазмиды мега комплект подготовительной </td><td> Qiagen </td><td> 12181 </td></tr><tr><td> Ультрацентрифуге </td><td> Beckman Coulter- </td><td> Optima L90K </td></tr><tr><td> Быстрая печать пробирки </td><td> Beckman Coulter- </td><td> 342414 </td></tr><tr><td> Электропорация блок </td><td> Bio-Rad </td><td> GenePulserXcell </td></tr><tr><td> Термоциклер </td><td> Эппендорф </td><td> Vapo Protect </td></tr><tr><td> Нагревательный блок </td><td> BIOER </td><td> MB-102 </td></tr><tr><td> Флуоресцентный микроскоп </td><td> Олимп </td><td> IX81 </td></tr><tr><td> FACS анализатор </td><td> Beckman Coulter- </td><td> Cytomics FC500 MLP </td></tr><tr><td> MegaX DH10B T1R клеток </td><td> Invitrogen </td><td> C640003 </td></tr><tr><td> Benzonase </td><td> Merck </td><td> 101695 </td></tr><tr><td> Аденовирус-5 </td><td> ATCC </td><td> VR-5 </td></tr><tr><td> pBlueScript II KS (+) плазмиды </td><td> Stratagene </td><td> 212207 </td></tr><tr><td> cap5F (Pac я сайта желтый, CAP5-специфических последовательностей выделены жирным шрифтом): GACTCTTAATTAACAGGT ATGTCTTTTGTTGATCACCCTCC </td><td> IDTDNA </td><td> Пользовательские грунтовка </td></tr><tr><td> cap5R (по возрастанию я сайт в зеленый, CAP5-специфических последовательностей выделены жирным шрифтом): GTGAGGGCGCGCC TTAAAGGGGTCGGGTAAGGTATC </td><td> IDTDNA </td><td> Пользовательские грунтовка </td></tr></tbody></table>

engineering and evolution of synthetic adeno-associated virus (aav) gene therapy vectors 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 combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular 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

Мы показываем основные техники молекулярной инженерии и развиваться синтетические адено-ассоциированные вирусные (ААВ) вектора генной терапии с помощью ДНК семьи перетасовки. Кроме того, мы предоставляем общие принципы и характерные примеры для отбора и анализа отдельных химерных капсид с улучшенными свойствами на клетки-мишени в культуре или в организме мышей.

Инженерные и эволюции Синтетическая адено-связанный вирус (AAV) генной терапии векторы с помощью ДНК семьи Перетасовка

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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Immunology and Infection

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Генерация рекомбинантного вируса гриппа от ДНК плазмиды

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

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Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Разделение одноцепочечных ДНК двухцепочечной ДНК и РНК из экологического сообщества Вирусный Использование гидроксиапатита хроматографии

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

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Расширение цитотоксических Т-лимфоцитов из пуповинной крови, что целевая цитомегаловирус, вирус Эпштейна-Барр Вирус, и аденовирус

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Тетрациклин-регулируемой клеточная линия продуцирует высоким титром лентивирусных векторов, которые специально направлены дендритных клеток

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Клеточная проточной цитометрии измерить цитотоксическую активность

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Распространение<em&gt; Homalodisca coagulata вирус-01</em&gt; Через<em&gt; Homalodisca vitripennis</em&gt; Культура клеток

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

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Простой и эффективный подход к Construct Mutant осповакцины вирусного вектора

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Оценка эффективности и токсичности РНК Ориентация ВИЧ-1 Производство для использования в генной или терапии наркотиками

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...