Name: using crispr/cas9 to knock out gm-csf in car-t cells
Uploaded: 2019-07-22T12:30:00
Description: Watch this Scientific Journal Video about Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells at JoVE.com

This work was supported through grants from K12CA090628 (SSK), the National Comprehensive Cancer Network (SSK), the Mayo Clinic Center for Individualized Medicine (SSK), the Predolin Foundation (SSK), the Mayo Clinic Office of Translation to Practice (SSK), and the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician-Scientist Scholarship (RMS).

This protocol follows the guidelines of Mayo Clinic&#39;s Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC).
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 viab...

<ol>
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In this report, we describe a methodology to utilize CRISPR/Cas9 technology to induce secondary modifications in CAR-T cells. Specifically, this&#160;is demonstrated using lentiviral transduction with a viral vector that contains gRNA targeting the gene of interest and Cas9 to generate GM-CSFk/o CART19 cells. We had previously shown that GM-CSF neutralization ameliorates CRS and NT in a xenograft model.12&#160;As previously described, GM-CSFk/o CAR-T cells allow for the inhib...

Chimeric antigen receptor T (CAR-T) cell therapy exhibits great promise in the treatment of cancer.1,2&#160;Two CAR-T cell therapies targeting CD19 (CART19) were recently approved in the United Stated and in Europe for the use in B cell malignancies after demonstrating striking results in multicenter clinical trials.3,4,5&#160;Barriers to more widespread use of CAR-T cells are limited activity in solid tumors and associated toxicities including cytokine release syndrome (CRS) and neurotoxicity (NT).3,5,6,7,8,9&#160;To enhance the therapeutic index of CAR-T cell therapy, genome engineering tools such as zinc finger nucleases, TALENs, and CRISPR are employed to further modify CAR-T cells in an attempt to generate less toxic or more effective CAR-T cells.10,11
In this article, we describe a method to generate CRISPR/Cas9 edited CAR-T cells. The specific goal of this method is to genetically modify CAR-T cells during CAR-T cell manufacturing via CRISPR/Cas9 to generate less toxic or more effective CAR-T cells. The rationale for developing this methodology is built on lessons learned from clinical experience of CAR-T cell therapy, which indicates an urgent need for novel strategies to increase the therapeutic window of CAR-T cell therapy and to extend the application into other tumors and is supported by the recent advances in synthetic biology allowing multiple modifications of CAR-T cells that have started to enter the clinic. While several genome engineering tools are being developed and applied in different settings, such as zinc finger nucleases, TALENs, and CRISPR, our methodology describes CRISPR/Cas9 modification of CAR-T cells.10,11&#160;CRISPR/Cas9 is an RNA-based bacterial defense mechanism that is designed to eliminate foreign DNA. CRISPR relies on endonucleases to cleave a target sequence identified through a guide RNA (gRNA). CRISPR editing of CAR-T cells offers several advantages over other genome engineering tools. These include precision of the gRNA sequence, simplicity to design a gRNA targeting the gene of interest, high gene editing efficiency, and the ability to target multiple genes since multiple gRNAs can be used at the same time.
Specifically in the methods described here, we used a lentivirus encoding CRISPR guide RNA and Cas9 to disrupt a gene during CAR transduction of T cells. In selecting an appropriate technique to edit CAR-T cells, we suggest the technique described here is an efficient mechanism to generate research grade CAR-T cells, but because the long term effect of permanent integration of Cas9 into the genome is unknown, we propose this methodology to develop proof of concept research grade CAR-T cells but not for producing good manufacturing practice grade CAR-T cells.
In particular, here we describe the generation of granulocyte macrophage colony stimulating factor (GM-CSF) knockout CAR-T cells targeting human CD19. These CAR-T cells were generated by transduction with lentiviral particles encoding a guide RNA specific to GM-CSF (gene name CSF2) and Cas9. We previously found that GM-CSF neutralization ameliorates CRS and NT in a xenograft model.12&#160;GM-CSFk/o CAR-T cells allow for the inhibition of GM-CSF during the manufacturing process, effectively reducing production of GM-CSF while enhancing CAR-T cell anti-tumor activity and survival in vivo compared to wildtype CAR-T cells.12&#160;Thus, here we provide a methodology to generate CRISPR/Cas9 edited CAR-T cells.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">CD3 Monoclonal Antibody (OKT3), PE, eBioscience</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:123px;">12-0037-42</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3 Monoclonal Antibody (UCHT1), APC, eBioscience</td>
			<td>Invitrogen</td>
			<td style="width:123px;">17-0038-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choice Taq Blue Mastermix</td>
			<td>Denville Scientific</td>
			<td style="width:123px;">C775Y51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td>
			<td>Gibco</td>
			<td style="width:123px;">40203D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">CytoFLEX System B4-R2-V2</td>
			<td style="width:217px;">Beckman Coulter</td>
			<td style="width:123px;">C10343</td>
			<td>flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">dimethyl sulfoxide</td>
			<td style="width:217px;">Millipore Sigma</td>
			<td style="width:123px;">D2650-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:217px;">Gibco</td>
			<td style="width:123px;">14190-144&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads MPC-S (Magnetic Particle Concentrator)</td>
			<td>Applied Biosystems</td>
			<td style="width:123px;">A13346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Easy 50 EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">18002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Human T Cell Isolation Kit&#160;</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">17951</td>
			<td>negative selection magnetic beads; 17951RF includes tips and buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Fetal bovine serum</td>
			<td style="width:217px;">Millipore Sigma</td>
			<td style="width:123px;">F8067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Mouse Anti-Human CD107a&#160;</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">555800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Fixation Medium (Medium A)</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:123px;">GAS001S100</td>
			<td></td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:557px;">GenCRISPR gRNA Construct: Name: CSF2 
			CRISPR guide RNA 1; Species: Human, Vector: 
			pLentiCRISPR v2; Resistance: Ampicillin; Copy number: 
			High; Plasmid preparation: Standard delivery: 4 &#956;g (Free 
			of charge)</td>
			<td style="width:217px;">GenScript</td>
			<td style="width:123px;">N/A</td>
			<td>custom order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td style="width:123px;">A-21235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">https://tide.nki.nl.</td>
			<td style="width:217px;">Desktop Genetics</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human AB Serum; Male Donors; type AB; US</td>
			<td>Corning</td>
			<td style="width:123px;">35-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN gamma Monoclonal Antibody (4S.B3), APC-eFluor 780, eBioscience</td>
			<td>Invitrogen</td>
			<td style="width:123px;">47-7319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td style="width:123px;">L3000075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen</td>
			<td style="width:123px;">L34966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lymphoprep</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">07851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monensin Solution, 1000X</td>
			<td>BioLegend</td>
			<td style="width:123px;">420701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human CD28 Clone CD28.2</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">559770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human CD49d Clone 9F10</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">561892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human MIP-1&#946; PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">560687</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Mr. Frosty Freezing Container</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:123px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NALM6, clone G5&#160;</td>
			<td>ATCC</td>
			<td style="width:123px;">CRL-3273</td>
			<td>acute lymphoblastic leukemia cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Promega</td>
			<td style="width:123px;">P119C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.22uM, sterile</td>
			<td>Genesee Scientific</td>
			<td style="width:123px;">25-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.45uM, sterile</td>
			<td>Genesee Scientific</td>
			<td style="width:123px;">25-228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Opti-MEM I Reduced-Serum Medium (1X), Liquid</td>
			<td style="width:217px;">Gibco</td>
			<td style="width:123px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-CF594 Mouse Anti-Human IL-2</td>
			<td>BD Horizon</td>
			<td style="width:123px;">562384</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Glutamine (100X), Liquid</td>
			<td>Gibco</td>
			<td style="width:123px;">10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Permeabilization Medium (Medium B)</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:123px;">GAS002S100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureLink Genomic DNA Mini Kit</td>
			<td>Invitrogen</td>
			<td style="width:123px;">K182001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>MP Biomedicals, Inc.</td>
			<td style="width:123px;">0210055210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td style="width:123px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Anti-Human GM-CSF BV421</td>
			<td>BD Horizon</td>
			<td style="width:123px;">562930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RoboSep-S</td>
			<td style="width:217px;">STEMCELL Technologies</td>
			<td style="width:123px;">21000</td>
			<td>Fully Automated Cell Separator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SepMate-50 (IVD)</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">85450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Sodium Azide, 5% (w/v)</td>
			<td style="width:217px;">Ricca Chemical</td>
			<td style="width:123px;">7144.8-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza</td>
			<td style="width:123px;">04-418Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

using crispr/cas9 to knock out gm-csf in car-t cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Figure 1&#160;shows reduction of GM-CSF in GM-CSFk/o CART19 cells. To verify that the genome of the T cells was altered to knockout GM-CSF, TIDE sequencing was used in the GM-CSFk/o CART19 cells (Figure 1A). CAR-T cell surface staining verifies that the T cells successfully express the CAR surface receptor in vitro by gating on live CD3+ cells (Figure 1B). Intracellular staining...

Watch this Scientific Journal Video about Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells at JoVE.com

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Our protocol describes the genetic modification of CAR-T cell during their manufacturing via the CRISPR technology to generate more effective and less toxic product. An advantage of this technique is that CAR-T cell production and genetic manipulation can both occur in one cell with good efficiency. Demonstrating the procedure will be Rosalie Sterner, an MD PhD student, Michelle Cox, a technologist and a graduate student, and Reona Sakemura, a postdoctorate fellow, from my laboratory.After isolating T-cells from harvested peripheral blood mononuclear cells, start culturing them by first preparing T-cell medium and then sterilizing it by filtering it through a 0.45 micrometer sterile vacuum filter and then with a 0.22 micrometer sterile vacuum filter. On the day of T-cell stimulation or day zero, prior to stimulation, wash CD3/CD28 beads by placing the required volume of beads in a sterile 1.5 milliliter microcentrifuge tube and resuspending in one milliliter of TCM. Place the tube in contact with a magnet for one minute.Then aspirate the TCM and resuspend in one milliliter of fresh TCM to wash the beads again and repeat for a total of three washes. And finally, resuspend the beads in one milliliter of TCM. After counting the T-cells, transfer the beads to the T-cells at a ratio of three to one beads to cells.Then dilute the cells to a final concentration of one million cells per milliliter and incubate at 37 degrees Celsius, 5%CO2 for 24 hours. After acquiring a CART19 construct in a lentiviral vector, perform lentiviral production by first incubating lentiviral plasmid, packaging vector, envelope vector, precomplexing reagent, transfection reagent, and transfection medium at room temperature for 30 minutes. Carry out lentiviral work using BSL2+precautions including cell culture hoods, personal protective equipment, and disinfection of used materials with bleach before disposal.After incubation, add these transfection reagents to the 293 T-cells that have reached 70 to 90%confluency and culture the transfected cells at 37 degrees Celsius and 5%CO2. At 24 and 48 hours after transfection, harvest, centrifuge, filter and concentrate virus-containing supernatant by ultracentrifugation and freeze at minus 80 degrees Celsius for future use. On day one, gently resuspend T-cells that have been stimulated at one million cells per milliliter on day zero in order to break up the rosettes of cells.Under appropriate BSL2+precautions, add fresh or frozen harvested virus to the simulated T-cells at a multiplicity of infection of 3.0. Continue to incubate the transduced T-cells at 37 degrees Celsius, 5%CO2. On days three and five, count CAR-T cells and add fresh pre-warmed TCM to the culture to maintain a CAR-T cell concentration of one million cells per milliliter.Six days after simulation, remove beads from the transduced T-cells by first harvesting and resuspending the cells. Transfer the cells in 50 milliliter conical tubes and place the tubes in a magnet for one minute. After collecting the CAR-T cell-containing supernatant and discarding the beads, place the cells back in culture at a concentration of one million cells per milliliter in a culture flask.Use a sample of cells for flow cytometry to assess surface expression of the CARs. Incubate the rest to resume expansion and then harvest and cryo-preserve the cells at day eight as described in the manuscript. To disrupt the 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 293 T-cells that have reached 70 to 90%confluency and culture the transfected cells at 37 degrees Celsius, 5%CO2 to produce a 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 BSL2+approved laboratory, to generate CAR-T cells, add CAR19 lentivirus and GMCSF-targeting CRISPR lentivirus to the simulated T-cells. On day three and day five for successfully transduced lentiCRISPR-edited T-cells carrying puromycin resistance, treat cells with puromycin dihydrochloride at a concentration of one microgram of puromycin per milliliter.Tide sequencing was used to confirm a GM-CSF reduction in GM-CSF knockout CART19 cells with a disruption efficiency of approximately 71%Flow plots of CAR-T cell surface staining gated on live CD3-positive cells show that both wild-type CART19 and GM-CSF knockout CART19 successfully and in a similar fashion express the CAR surface receptor in vitro. On the other hand, intracellular staining of GM-CSF by flow cytometry also gated on live CD3-positive cells demonstrates decreased expression of GM-CSF in the knockout cells compared to wild-type CART19 cells confirming functional success of the knockout. Particular care should be taken during the transduction steps of this procedure as they are the most critical to the development of genetically modified CAR-T cells.The methodology described can potentially be applied to a variety of genes to modify CAR-T cells via CRISPR/CAS9 to help generate a less toxic and more effective product. CRISPR/CAS9 technology provides the strategies to directly target the genome of CAR-T cells to engineer solutions to current clinical shortcomings.

using-crisprcas9-to-knock-out-gm-csf-in-car-t-cells

Research

JoVE Journal

Cancer Research

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

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.

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

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...