genetically modifying car t cells using a crispr-cas9 system

Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

To disrupt granulocyte-macrophage colony-stimulating factor, utilize a guide RNA, as described in the manuscript. Incubate transfection reagents at room temperature for 30 minutes. After incubation, add them to the 293T cells that have reached 70% to 90% confluency, and culture the transfected cells at 37 degrees Celsius, 5% CO2 to produce lentivirus.
At 24 and 48 hours, harvest, and concentrate virus-containing supernatant by ultracentrifugation in 50-milliliter ultracentrifuge tubes, and freeze at minus 80 degrees Celsius for future use. Then, on day one, gently resuspend the T cells to break up rosettes.
In a BSL-2 plus approved laboratory, to generate CAR T cells, add CAR19 lentivirus and GM-CSF-targeting CRISPR lentivirus to the stimulated T cells. On day three and day five, for successfully transduced lenti-CRISPR-edited T cells carrying puromycin resistance, treat cells with puromycin dihydrochloride at a concentration of 1 microgram of puromycin per milliliter.

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1. CART19 cell production
<ol>
	<li>T-cell isolation, stimulation, and ex-vivo culture
	<ol>
		<li>Carry out all cell culture work in a cell culture hood utilizing appropriate personal protective equipment. Harvest peripheral blood mononuclear cells (PBMCs) from de-identified normal donor blood cones collected during apheresis as these are known to be a viable source of PBMCs.</li>
		<li>To isolate PBMCs, add 15 mL of a density gradient medium to a 50 mL density gradient separation tube. Dilute the donor blood with an equal volume of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) to avoid cell trapping.
		<ol>
			<li>Add the diluted donor blood from the cone into the separation tube, careful not to disturb the interface between the blood and the density gradient medium. Centrifuge at 1,200 x g for 10 min at RT.</li>
			<li>Decant the supernatant into a new 50 mL conical, wash with PBS + 2% FBS by bringing up to 40 mL, centrifuge at 300 x g for 8 min at RT, aspirate supernatant, resuspend in 40 mL of buffer, and count cells.</li>
		</ol>
		</li>
		<li>Isolate T cells from PBMCs via a negative selection magnetic bead kit using a fully automated cell separator according to the manufacturer&#39;s protocol.</li>
		<li>To culture the isolated T-cells, prepare T-cell medium (TCM) that consists of 10% human AB serum (v/v), 1% penicillin-streptomycin-glutamine (v/v), and serum-free hematopoietic cell medium. Sterilize the medium by filtering through a 0.45 &#181;m sterile vacuum filter and then with a 0.22 &#181;m sterile vacuum filter.</li>
		<li>On Day 0 (the day of T-cell stimulation), wash CD3/CD28 beads prior to stimulation of T-cells. To wash, place the required volume of beads (use enough CD3/CD28 beads for a ratio of 3:1 beads:T cells; note the concentration of beads can be variable) in a sterile 1.5 mL microcentrifuge tube and resuspend in 1 mL of TCM.</li>
		<li>Place in contact with a magnet.</li>
		<li>After 1 min, aspirate the TCM and resuspend in 1 mL of fresh TCM to wash the beads.</li>
		<li>Repeat this procedure for a total of three washes.</li>
		<li>After the third wash, resuspend the beads in 1 mL of TCM.</li>
		<li>Count the T-cells.</li>
		<li>Transfer beads to the T cells at a ratio of 3:1 beads:cells.</li>
		<li>Dilute cells to a final concentration of 1 x 106 cells/mL.</li>
		<li>Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>T cell transfection and transduction
	<ol>
		<li>Carry out lentiviral work using BSL-2+ precautions, including cell culture hoods, personal protective equipment, and disinfection of used materials with bleach before disposal.</li>
		<li>Acquire a chimeric antigen receptor-T19 (CART19) construct in a lentiviral vector. 
		NOTE: The CART19 construct utilized here was designed and then synthesized de novo using a commercially available protein synthesis vendor. The CAR construct was subsequently cloned into a third-generation lentivirus under control of an EF-1&#945; promoter. The single chain variable region fragment is derived from the FMC63 clone and recognizes human CD19. The CAR19 construct possesses a second generation 4-1BB costimulatory domain and CD3&#950; stimulation (FMC63-41BBz).</li>
		<li>Perform lentiviral production as previously described.
		<ol>
			<li>In brief, to produce lentivirus, utilize 293T-cells that have reached 70-90% confluency.</li>
			<li>Allow incubation for 30 min at room temperature of transfection reagents including 15 &#181;g of the lentiviral plasmid of interest, 18 &#181;g of a gag/pol/tat/rev packaging vector, 7 &#181;g of a VSV-G envelope vector, 111 &#181;L of the pre-complexing reagent, 129 &#181;L of the transfection reagent, and 9.0 mL of the transfection medium before adding to the 293T-cells. Then culture the transfected cells at 37 &#176;C, 5% CO2.</li>
			<li>Harvest, centrifuge (900 x g for 10 min), filter (0.45 &#181;M nylon filter), and concentrate supernatant at 24 and 48 h by ultracentrifugation at either 13,028 x g for 18 h or 112,700 x g for 2 h.</li>
			<li>Freeze at -80 &#176;C for future use.</li>
		</ol>
		</li>
		<li>On Day 1, gently resuspend T-cells to break up the rosettes of T-cells that had been stimulated at 1 x 106/mL on Day 0.</li>
		<li>Under appropriate BSL-2+ precautions for all lentiviral work, add fresh or frozen harvested virus to the stimulated T-cells at a multiplicity of infection (MOI) of 3.0.</li>
	</ol>
	</li>
	<li>CAR T-cell expansion
	<ol>
		<li>During the phase of expansion, continue to incubate the transduced T-cells at 37 &#176;C, 5% CO2. Count CAR T-cells on days 3 and 5, and add fresh, pre-warmed TCM to the culture to maintain a CAR T-cell concentration of 1 x 106/mL.</li>
		<li>Remove beads from the transduced T-cells 6 days after stimulation (Day 6) using a magnet. Harvest, resuspend, and place T cells in 50 mL conical tubes in a magnet for 1 min. Then collect the supernatant (contains the CAR T-cells), and discard the beads.</li>
		<li>Place the collected CAR T-cells back in culture at a concentration of 1 x 106 cells/mL to resume expansion.</li>
		<li>On Day 6, assess surface expression of the CAR by flow cytometry. 
		NOTE: Several methods can be used to detect the surface expression of CAR, such as staining with a goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody or by staining with a CD19 specific peptide, conjugated to a fluorochrome. Here, take an aliquot (about 100,000 T-cells) from the culture and wash with flow buffer prepared with Dulbecco&#39;s phosphate-buffered saline, 2% fetal bovine serum, and 1% sodium azide. Stain the cells with the anti-CAR antibody and wash twice. Stain the cells with live/dead stain and CD3 monoclonal antibody (OKT3). Wash the cells and resuspend in flow buffer. Acquire on a cytometer to determine transduction efficiency.</li>
	</ol>
	</li>
	<li>CAR T-cell cryopreservation
	<ol>
		<li>To harvest and cryopreserve CAR T-cells 8 days after stimulation (Day 8 of T cell expansion), harvest the cells from culture.</li>
		<li>Spin down for 5 min at 300 x g.</li>
		<li>Resuspend in freezing medium at 10 million cells per mL per vial in freezing medium consisting of 10% dimethyl sulfoxide and 90% fetal bovine serum.</li>
		<li>Freeze in a freezing container to achieve a rate of cooling of -1 &#176;C/min in a -80 &#176;C freezer and then transfer to liquid nitrogen after 48 h.</li>
		<li>Prior to their use for in vitro or in vivo experiments, thaw CAR T-cells in warm TCM.</li>
		<li>Wash the cells to dilute and remove the dimethyl sulfoxide and resuspend to a concentration of 2 x 106 cells/mL in warm TCM. Rest overnight at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Granulocyte macrophage colony-stimulating factor (GM-CSF k/o) CART19 production
<ol>
	<li>To disrupt GM-CSF, utilize a guide RNA (gRNA) targeting exon 3 of human GM-CSF (CSF2) selected via screening gRNAs previously reported to have high efficiency for the CSF2 gene that encodes for the human cytokine GM-CSF. 
	NOTE: A commercially synthesized research grade Cas9 third generation lentiviral construct containing this gRNA (under a U6 promoter) was used. The construct contains a puromycin resistance gene. The sequence of the gRNA is GACCTGCCTACAGACCCGCC.</li>
	<li>To produce lentivirus, utilize 293T cells that have reached 70-90% confluency.</li>
	<li>Allow 15 &#181;g of the lentiviral plasmid of interest, 18 &#181;g of a gag/pol/tat/rev packaging vector, 7 &#181;g of a VSV-G envelope vector, 111 &#181;L of the pre-complexing reagent, 129 &#181;L of the transfection reagent, and 9.0 mL of the transfection medium to incubate for 30 min at room temperature.</li>
	<li>Add transfection reagents to the 293T-cells. The culture at 37 &#176;C, 5% carbon dioxide (CO2).</li>
	<li>Harvest, centrifuge (900 x g for 10 min), filter (0.45 &#181;M nylon filter), and concentrate supernatant at 24 and 48 h by ultracentrifugation at either 13,028 x g for 18 h or 112,700 x g for 2 h and freeze at -80 &#176;C for future use.</li>
	<li>On Day 1, gently resuspend the T cells to break up rosettes.</li>
	<li>In a BSL-2+ approved laboratory, add a frozen or freshly harvested virus to the stimulated T-cells to generate CAR T-cells. Transduce T-cells with both the CAR19 lentivirus and GM-CSF targeting CRISPR lentivirus. Add CAR19 lentivirus at a MOI of 3. Since titration of the CRISPR lentivirus was not feasible, use virus particles generated from a 15 ug plasmid preparation to transduce 10 x 106 T cells.</li>
	<li>See the remaining steps of T-cell stimulation, expansion, and cryopreservation as discussed in step 1.</li>
	<li>For lentiCRISPR-edited T-cells carrying puromycin resistance, treat cells with puromycin dihydrochloride at a concentration of 1 &#181;g of puromycin per 1 mL on Day 3 and Day 5.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD3 Monoclonal Antibody (OKT3), PE, eBioscience</td>
			<td>Invitrogen</td>
			<td>12-0037-42</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 Monoclonal Antibody (UCHT1), APC, eBioscience</td>
			<td>Invitrogen</td>
			<td>17-0038-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Choice Taq Blue Mastermix</td>
			<td>Denville Scientific</td>
			<td>C775Y51</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td>
			<td>Gibco</td>
			<td>40203D</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX System B4-R2-V2</td>
			<td>Beckman Coulter</td>
			<td>C10343</td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Millipore Sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MPC-S (Magnetic Particle Concentrator)</td>
			<td>Applied Biosystems</td>
			<td>A13346</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human T Cell Isolation Kit&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>17951</td>
			<td>Negative selection magnetic beads; 17951RF includes tips and buffer</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Millipore Sigma</td>
			<td>F8067</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD107a&#160;</td>
			<td>BD Pharmingen</td>
			<td>555800</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation Medium (Medium A)</td>
			<td>Invitrogen</td>
			<td>GAS001S100</td>
			<td></td>
		</tr>
		<tr>
			<td>GenCRISPR gRNA Construct: Name: CSF2</td>
			<td rowspan="5">GenScript</td>
			<td rowspan="5">N/A</td>
			<td rowspan="5">Custom order</td>
		</tr>
		<tr>
			<td>CRISPR guide RNA 1; Species: Human, Vector:</td>
		</tr>
		<tr>
			<td>pLentiCRISPR v2; Resistance: Ampicillin; Copy number:</td>
		</tr>
		<tr>
			<td>High; Plasmid preparation: Standard delivery: 4 &#956;g (Free</td>
		</tr>
		<tr>
			<td>of charge)</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A-21235</td>
			<td></td>
		</tr>
		<tr>
			<td>https://tide.nki.nl.</td>
			<td>Desktop Genetics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human AB Serum; Male Donors; type AB; US</td>
			<td>Corning</td>
			<td>35-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN gamma Monoclonal Antibody (4S.B3), APC-eFluor 780, eBioscience</td>
			<td>Invitrogen</td>
			<td>47-7319-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>L3000075</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>STEMCELL Technologies</td>
			<td>7851</td>
			<td></td>
		</tr>
		<tr>
			<td>Monensin Solution, 1000X</td>
			<td>BioLegend</td>
			<td>420701</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD28 Clone CD28.2</td>
			<td>BD Pharmingen</td>
			<td>559770</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD49d Clone 9F10</td>
			<td>BD Pharmingen</td>
			<td>561892</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human MIP-1&#946; PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>560687</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>NALM6, clone G5&#160;</td>
			<td>ATCC</td>
			<td>CRL-3273</td>
			<td>Acute lymphoblastic leukemia cell line</td>
		</tr>
		<tr>
			<td>Nuclease Free Water</td>
			<td>Promega</td>
			<td>P119C</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.22uM, sterile</td>
			<td>Genesee Scientific</td>
			<td>25-227</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.45uM, sterile</td>
			<td>Genesee Scientific</td>
			<td>25-228</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced-Serum Medium (1X), Liquid</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 Mouse Anti-Human IL-2</td>
			<td>BD Horizon</td>
			<td>562384</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine (100X), Liquid</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Permeabilization Medium (Medium B)</td>
			<td>Invitrogen</td>
			<td>GAS002S100</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Genomic DNA Mini Kit</td>
			<td>Invitrogen</td>
			<td>K182001</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin Dihydrochloride</td>
			<td>MP Biomedicals, Inc.</td>
			<td>210055210</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Anti-Human GM-CSF BV421</td>
			<td>BD Horizon</td>
			<td>562930</td>
			<td></td>
		</tr>
		<tr>
			<td>RoboSep-S</td>
			<td>STEMCELL Technologies</td>
			<td>21000</td>
			<td>Fully Automated Cell Separator</td>
		</tr>
		<tr>
			<td>SepMate-50 (IVD)</td>
			<td>STEMCELL Technologies</td>
			<td>85450</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide, 5% (w/v)</td>
			<td>Ricca Chemical</td>
			<td>7144.8-16</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Sterner, R. M. et al., <a href="https://app.jove.com/v/59629">Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells.</a>&#160;J. Vis. Exp. (2019)
The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.

1. CART19 cell production

	T-cell isolation, stimulation, and ex-vivo culture
	
		Carry out all cell culture work in a cell culture hood utilizing ...

Begin with a suspension of T cells and treat them with CAR lentiviruses carrying a gene for the chimeric antigen receptor or CAR and CRISPR lentiviruses.
The CRISPR lentivirus contains genes for the guide RNA and Cas9 nuclease as well as an antibiotic resistance gene.
Both the CAR and CRISPR lentiviruses interact with T cells and release their RNA.
These RNAs are reverse-transcribed and converted into corresponding DNA, which integrates into the T cell DNA.
This enables the synthesis of CAR proteins, guide RNAs, and Cas9 proteins.
The guide RNA forms a complex with Cas9, which specifically recognizes and binds to the target gene on the T cell DNA.
Meanwhile, Cas9 cleaves the DNA, creating a double-stranded break.
This break is repaired via non-homologous end joining, an error-prone repair mechanism, resulting in target gene modification.
This generates genetically modified T cells with chimeric antigen receptors.
Treat the cells with antibiotics to eliminate the unmodified T cells and selectively isolate the genetically modified CAR T cells.

66 次观看. 使用 CRISPR-Cas9 系统对 CAR T 细胞进行基因修饰

视频: 使用 CRISPR-Cas9 系统对 CAR T 细胞进行基因修饰 - 实验

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

同源小鼠 B 细胞淋巴瘤模型对 CD19 轿车 T 细胞的临床前评价

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

科研

JoVE Journal

癌症研究

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain &#946;2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

CRISPR干扰（CRISPRI）在瘦素病原物种中基因沉默的应用

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...

遗传学

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

成績單

Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System