Name: generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins
Uploaded: 2018-06-14T15:00:00
Description: Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

Vi anerkender Brian Tullius for hans venlige hjælp i redigering håndskriftet.

Sund donor buffy frakker blev fremstillet som kildemateriale fra Central Ohio Region amerikanske Røde Kors. Denne forskning var besluttet på at være undtaget forskning af den institutionelle Review Board af landsdækkende children's Hospital.
1. menneskelige NK celle oprensning og udvidelse
<ol><li>Isolere PBMC fra Buffy Coat12.<ol>
<li>Lag 35 mL af buffy coat stikprøve på 15 mL af Ficoll-Paque.</li>
		<li>Der centrifugeres ved 400 x g i 20 minutter uden bremse o...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-mediated+delivery+of+proteins+enables+efficient+protein-based+genome+editing+in+vitro+and+in+vivo.">Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.</a> Nature Biotechnology. 33 (1), 73-80 (2015).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+delivery+of+genome-editing+proteins+using+bioreducible+lipid+nanoparticles.">Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.</a> PNAS. 113 (11), 2868-2873 (2016).</li><li>Liang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+DNA-free+genome+editing+of+bread+wheat+using+CRISPR/Cas9+ribonucleoprotein+complexes.">Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.</a> Nature Communications. 8, 14261 (2017).</li><li>DeWitt, M. A., Corn, J. E., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+via+delivery+of+Cas9+ribonucleoprotein.">Genome editing via delivery of Cas9 ribonucleoprotein.</a> Methods. , 9-15 (2017).</li><li>Rezvani, K., Rouce, R., Liu, E., Shpall, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Natural+Killer+Cells+for+Cancer+Immunotherapy.">Engineering Natural Killer Cells for Cancer Immunotherapy.</a> Molecular Therapy. 25 (8), 1769-1781 (2017).</li><li>Sutlu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+intracellular+antiviral+defense+mechanisms+augments+lentiviral+transduction+of+human+natural+killer+cells:+implications+for+gene+therapy.">Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.</a> Human Gene Therapy. 23 (10), 1090-1100 (2012).</li><li>Viel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+inhibits+the+activation+and+functions+of+NK+cells+by+repressing+the+mTOR+pathway.">TGF-beta inhibits the activation and functions of NK cells by repressing the mTOR pathway.</a> Science Signaling. 9 (415), (2016).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> JoVE. (48), (2011).</li><li>Lee, D. A., Verneris, M. R., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition,+preparation,+and+functional+assessment+of+human+NK+cells+for+adoptive+immunotherapy.">Acquisition, preparation, and functional assessment of human NK cells for adoptive immunotherapy.</a> Methods in Molecular Biology. , 61-77 (2010).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5 (3), 407-415 (2015).</li><li>Eyquem, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+a+CAR+to+the+TRAC+locus+with+CRISPR/Cas9+enhances+tumour+rejection.">Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.</a> Nature. 543 (7643), 113-117 (2017).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+highly+efficient+mammalian+cell+engineering+via+Cas9+protein+transfection.">Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.</a> Journal of Biotechnology. 208, 44-53 (2015).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Chmielecki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Profiling+of+a+Large+Set+of+Diverse+Pediatric+Cancers+Identifies+Known+and+Novel+Mutations+across+Tumor+Spectra.">Genomic Profiling of a Large Set of Diverse Pediatric Cancers Identifies Known and Novel Mutations across Tumor Spectra.</a> Cancer Research. 77 (2), 509-519 (2017).</li><li>Schultz, L. M., Majzner, R., Davis, K. 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DNA-afhængige ændring af NK celler har udfordrende4,9. Vi, derfor indført direkte en syntetisk præfabrikerede ribonucleoprotein (RNPs) kompleks og Cas9 protein som oprenset protein i primær og udvidet NK celler8. Denne metode er tilladt os at fjerne udjævningen, hale, og andre processer, transcriptional og translationel startet af RNA polymerase II, som kan forårsage NK celle apoptose forbundet med DNA-afhængige transduktion metode...

Cancer immunterapi har været fremført i de seneste år. Genetisk modificerede kimære antigen receptor (bil) T celler er et fremragende eksempel på manipuleret immunceller implementeret i cancer immunterapi. Disse celler blev for nylig godkendt af FDA til behandling mod CD19 + B celle maligniteter, men succes har hidtil været begrænset til sygdomme forsynet med et par markedssegment antigener og rettet mod sådanne begrænset antigene repertoirer er udsat for svigt af immunsystemet undslippe. Derudover har bil T celler været fokuseret på brugen af autologe T celler på grund af risikoen for graft - versus - host sygdom forårsaget af allogene T celler. I modsætning, NK-celler er i stand til at dræbe tumor mål i en antigen-uafhængig måde og forårsager ikke GvHD, hvilket gør dem en god kandidat for cancer immunterapi6,7,8,9.
CRISPR/Cas9 teknologi er blevet brugt for nylig i engineering immunceller, men genetisk omprogrammering NK celler med plasmider har altid været udfordrende. Dette har været på grund af vanskeligheder i transgene levering i et DNA afhængige måde som lentiviral og retroviral transduktion forårsager væsentlige procedure-associerede NK celle apoptose og begrænset produktion af gensplejsede NK celler4 , 9.
Mange medfødte immun celler udtrykker høje niveauer af receptorer for patogen-associeret molekylære mønstre såsom retinoid syre-inducerbar gen jeg (RIG-jeg), som aktiverer øget anerkendelse af fremmed DNA. Undertrykkelse af disse veje har aktiveret højere transduktion effektivitet i NK celler, når du bruger DNA-baserede metoder til genetisk modifikation10.
Her beskriver vi metoden for ved hjælp af en DNA-fri genom-redigering af primære og udvidet menneskelige NK celler udnytte Cas9 ribonucleoprotein komplekser (Cas9/RNPs). Cas9/RNPs består af tre komponenter, rekombinante Cas9 protein kompleksbundet med syntetisk single-guide RNA består af en kompleksbundet crRNA og tracerRNA. Disse Cas9/RNPs er i stand til at holde genomisk mål med højere effektivitet i forhold til udenlandske DNA-afhængige tilgange på grund af deres levering som funktionelle komplekser. Derudover kan hurtig clearance af Cas9/RNPs fra cellerne reducere off target effekter såsom induktion af apoptose. De kan således bruges til at generere knock-outs, eller knock-ins når kombineret med DNA for homologe rekombination6,7. Vi viste at elektroporation af Cas9/RNPs er en nem og relativt effektiv metode, der overvinder de foregående begrænsninger af genetisk modifikation i NK-celler.
TGFβ er en stor immunosuppressive cytokin, som hæmmer aktivering og funktioner af NK celler. Det er blevet foreslået, at målrette TGFβ vej kan øge immun cellefunktioner. Vi målrettet regionen kodning TGBR2 ectodomain, som binder TGFβ11. De repræsentative resultater viser et signifikant fald i niveauet af mRNA udtryk for dette gen og yderligere viser, at de modificerede NK celler bliver resistente over for TGFβ. Derudover bevarer de modificerede celler levedygtighed og proliferativ potentiale, som de er i stand til at blive udvidet post-elektroporation ved hjælp af bestrålede feeder celler. Følgende metode er derfor en lovende metode at genmanipulere NK celler for nogen yderligere kliniske eller forskning formål.

<table>
	<tbody>
		<tr>
			<td>RosetteSep&#8482; Human NK Cell Enrichment Cocktail</td>
			<td>STEMCELL Technologies</td>
			<td>15065</td>
			<td>The RosetteSep&#8482; Human NK Cell Enrichment Cocktail is designed to isolate NK cells from whole blood by negative selection.</td>
		</tr>
		<tr>
			<td>Ficoll-Paque&#174; PLUS</td>
			<td>GE Healthcare - Life Sciences</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 tracrRNA</td>
			<td>Integrated DNA Technologies</td>
			<td>1072532</td>
			<td>TracrRNA that contains proprietary chemical modifications conferring increased nuclease resistance. Hybridizes to crRNA to activate the Cas9 enzyme</td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 crRNA</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Synthetically produced as Alt-R&#174; CRISPR-Cas9 crRNA, based on the sequence of designed gRNA targeting the gene of intrest</td>
		</tr>
		<tr>
			<td>Alt-R&#174; Genome Editing Detection Kit</td>
			<td>Integrated DNA Technologies</td>
			<td>1075931</td>
			<td>Each kit contains T7EI endonuclease, T7EI reaction buffer, and T7EI assay controls.</td>
		</tr>
		<tr>
			<td>Platinum&#174; Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen</td>
			<td>11304-011</td>
			<td></td>
		</tr>
		<tr>
			<td>4D-Nucleofector&#8482; System</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IL-2 Protein</td>
			<td>Novartis</td>
			<td>65483-0116-07</td>
			<td></td>
		</tr>
		<tr>
			<td>P3 Primary Cell 4D-Nucleofector&#8482; X Kit</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td>Contains pmaxGFP&#8482; Vector, Nucleofector&#8482; Solution, Supplement, 16-well Nucleocuvette&#8482; Strips</td>
		</tr>
		<tr>
			<td>Non-targeting: Custom siRNA, Standard 0.05 2mol ON-TARGETplus</td>
			<td>Dharmacon</td>
			<td>CTM-360019</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; S.p. Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies</td>
			<td>1074181</td>
			<td>Cas9 Nuclease</td>
		</tr>
		<tr>
			<td>DNeasy&#174; Blood &#38; Tissue Handbook</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>ThermoFisher</td>
			<td>C3099</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;</td>
			<td>Biolegend</td>
			<td>580706</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 Control Kit, Human</td>
			<td>Integrated DNA Technologies</td>
			<td>1072554</td>
			<td>Includes tracrRNA, HPRT positive control crRNA, negative control crRNA#1, HPRT Primer Mix, and Nuclease-Free Duplex Buffer.</td>
		</tr>
		<tr>
			<td>IDTE pH 7.5 (1X TE Solution)</td>
			<td>Integrated DNA Technologies</td>
			<td>11-01-02-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; Cas9 Electroporation Enhancer</td>
			<td>Integrated DNA Technologies</td>
			<td>1075915</td>
			<td>Cas9 Electroporation Enhancer</td>
		</tr>
	</tbody>
</table>

generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins

CRISPR/Cas9 teknologi fremskynder genom engineering i mange celletyper, men hidtil gen levering og stabil gen ændring har været udfordrende i primære NK celler. For eksempel resulterede transgen levering ved hjælp af lentiviral eller retroviral transduktion i en begrænset udbytte af genetisk manipuleret NK celler på grund af væsentlige procedure-associerede NK celle apoptose. Vi beskriver her en DNA-fri metode for genom-redigering af menneskelige primære og udvidet NK celler ved hjælp af Cas9 ribonucleoprotein komplekser (Cas9/RNPs). Denne metode er tilladt effektive knockout af TGFBR2 og HPRT1 gener i NK-celler. RT-PCR data viste en betydelig nedgang i gen expression niveau, og cytotoksicitet analyse af et repræsentativt celle produkt foreslog at RNP-modificeret NK-celler blev mindre følsomme over for TGFβ. Genetisk modificerede celler kunne være udvidet post-elektroporation ved stimulation med bestrålede mbIL21-udtrykker feeder celler.

Elektroporation effektivitet
For at optimere elektroporation af Cas9/RNPs, vi har testet 16 forskellige programmer med transduktion af normal god landbrugspraksis ikke rettet mod siRNA og DNA plasmid til NK-celler. Flow flowcytometri analyse viste, at EN-138 havde den højeste procentdel af celle levedygtighed og transduktion effektivitet (35% live normal god landbrugspraksis positive celler) for både partikler (<...

Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

Vi præsenterer her, en protokol for at genetisk ændre primære eller udvidede menneskets naturlige dræberceller (NK) celler ved hjælp af Cas9 Ribonucleoproteins (Cas9/RNPs). Ved hjælp af denne protokol, genereret vi NK menneskeceller mangelfuld for omdanne vækst faktor – b receptor 2 (TGFBR2) og hypoxanthine phosphoribosyltransferase 1 (HPRT1).

Generation af Knock-out primære og udvidet menneskelige NK celler ved hjælp af Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Research

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Lille RNA transfektion i primære humane Th17 Celler af Next Generation Elektroporation

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En Flow flowcytometri-baserede cytotoksicitet analyse til vurdering af menneskers NK celleaktivitet

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...