This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2EDK - 00474). We would also like to express our gratitude to the Choose Life Foundation for providing continuous support to the GMP Lab &#34;Dimitris Lois&#34; of the Institute of Cell Therapy.

This study was approved by the Ethics Committee of the University of Patras and the Institutional Review Board of the University General Hospital of Patras. Prior to obtaining biological specimens, informed consent was obtained from healthy individuals. The eligibility assessment of participants was conducted according to institutional procedures and in compliance with JACIE standards for leukapheresis. If this protocol is to be implemented in a clinical ACT setting, all procedures must adhere to Good Manufacturing Practice (GMP) guidelines and be performed in GMP-compliant clean rooms. Special safety precautions are necessary when working with viral vectors, and these procedures should be conducted within a dedicated biosafety cabinet. In this study, the described procedures were conducted within a validated H35 HEPA Hypoxystation (see Table of Materials) to ensure a controlled and monitored environment. Decontamination of all hazardous liquids should be carried out using 10% bleach solution.
1. PBMC isolation and T cell activation (Day 0)
<ol>
	<li>Collect cells from the donor apheresis bag (leukopaks, see Table of Materials) into 50 mL tubes, depending on the initial volume.</li>
	<li>Perform two washes of the cell bag by infusing 15 mL of complete hematopoietic media (+5% HSA, commercial serum-free hematopoietic media, see Table of Materials).</li>
	<li>Conduct polysaccharide-based density gradient centrifugation at a 1:2 (reagent:sample) dilution. Centrifuge at 800 x g for 30 min at room temperature with breaks off (A/D, 9/0).</li>
	<li>Using a micropipette, collect PBMCs into a clean 50 mL tube.</li>
	<li>Wash the cells twice with 1x PBS, filling up to 50 mL for each wash (400 x g, 10 min, room temperature). Resuspend the cells in 5 mL of complete media.</li>
	<li>Count the cells using trypan blue using either a standard hemocytometer or an automated cell counter (see Table of Materials).</li>
	<li>Dilute the cells with complete media to achieve a concentration of 2&#160;x 106 cells/mL.</li>
	<li>Activate 20 x 106 PBMCs by adding 10 &#181;L of T cell stimulation reagent (see Table of Materials) per 2 x 106 starting cells. 
	NOTE: This protocol is designed for the activation of 20 x 106 PBMCs but can be adjusted for larger cell numbers as needed.</li>
	<li>Add rhIL-2 (20 U/mL) (see Table of Materials), mix gently, and transfer the mixture into an appropriate vessel (e.g., 6-well plate).</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 for 72 h.</li>
</ol>
2. Lentiviral T cell transduction (Day 3)
<ol>
	<li>Transfer the cell culture into a suitable vessel, such as a 50 mL conical tube. Mix well and centrifuge at 300 x g for 10 min at room temperature (RT) to remove the activation reagent. Carefully discard the supernatant completely&#160;and resuspend the pellet in 1 mL of complete medium. 
	NOTE: At this stage, it is possible to thaw the virus on ice.</li>
	<li>Count the cells using trypan blue.</li>
	<li>Resuspend the cells in complete&#160;medium to achieve a concentration that allows for plating 0.5 x 106 cells in a total volume of 400 &#181;L per well in a 24-well plate. Add rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) (see Table of Materials). 
	NOTE: Ensure to account for the volume of viruses to be added. Refer to step 2.4.</li>
	<li>In a separate tube, add Vectofusin-1 (see Table of Materials) to Opti-MEM medium at a concentration of 10 &#181;g/mL, with a volume equal to the viral volume to be used. 
	NOTE: Consider the total cell culture volume when calculating the concentration of Vectofusin-1. See step 2.3. Alternatively, Vectofusin-1 GMP can be used as a GMP-grade reagent.</li>
	<li>Combine the concentrated virus at a Multiplicity of Infection (MOI) of 40 with the Vectofusin-1/Opti-MEM mixture (1:1 ratio) and mix thoroughly. Add this mixture to the cell culture and adjust the total volume to 400 &#181;L per well using complete medium, if necessary.</li>
	<li>Place the lid on the plate, seal it with paraffin film, and centrifuge at 1000 x g for 2 h at 32 &#176;C. Subsequently, incubate the plate at 37 &#176;C, 5% CO2 overnight.</li>
</ol>
3. Virus removal and T cell expansion (Day 4)
<ol>
	<li>Collect the cells and pool them into a suitable vessel, such as a 50 mL tube.</li>
	<li>Centrifuge the cells at 400 x g at room temperature (RT) for 5 min. Carefully remove the supernatant completely and resuspend the pellet in 1 mL of complete media.</li>
	<li>Count the cells using trypan blue. Resuspend the cells in complete media at a concentration of 1 x 106 cells/mL. Add rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) (see Table of Materials).</li>
	<li>Transfer the cells to an appropriate vessel, such as a T25 flask, at a concentration of 0.3-0.5 x 106 cells/cm2 and incubate them at 37 &#176;C, 5% CO2, for 2 days. 
	&#8203;NOTE: If a larger cell population is required, cells can be subcultured (1:2) in the presence of rhIL7 and rhIL-15 every 2 days, following the same incubation conditions mentioned above, until the desired cell numbers are achieved.</li>
</ol>
4. Cryopreservation (Day 14)
NOTE: Ice will be required to transfer cryovials to the storage location during this step. The timing of cryopreservation depends on the desired cell numbers. For this protocol, cryopreservation is performed on Day 14 after achieving a 25-fold expansion.
<ol>
	<li>Once the desired cell number is reached, harvest the cells and transfer them to 50 mL tubes (adjust the number of tubes based on the total volume).</li>
	<li>Centrifuge the tubes at 400 x g at room temperature (RT) for 5 min. Carefully remove the supernatant completely and resuspend the pellet in 10 mL of media. If necessary, pool the cells from multiple tubes.</li>
	<li>Count the cells using trypan blue. Keep an aliquot of 1 x 106 cells for each quality control (QC) testing (refer to Section 5) to assess Vector Copy Number and Transduction Efficiency.</li>
	<li>Centrifuge the tubes at 400 x g for 10 min at RT. At this point, label the cryovials accordingly.</li>
	<li>Completely remove the supernatant and resuspend the pellet in a cryopreservation solution consisting of 50% HSA and 40% HBSS at a concentration of 1 x 107 cells per mL per cryovial. Transfer the cell suspension into the appropriate number of cryovials and add 10% dimethyl sulfoxide (DMSO) to each cryovial.</li>
	<li>Place the cryovials on ice and promptly transfer them to a controlled-rate freezer for cryopreservation. After cryopreservation, transfer the cryovials to a monitored liquid nitrogen tank for long-term storage.</li>
</ol>
5. Quality control (Day 14)
NOTE: For release testing, the product underwent several tests, including sterility tests for microbial growth, and endotoxin level assessment. These tests were conducted in certified laboratories at the University General Hospital of Patras and the University of Patras. Mycoplasma presence was determined in-house using a biochemical detection assay and measured on a luminometer following the manufacturer&#39;s instructions (see Table of Materials). Cell phenotype (Figure 1) as well as cell number and viability were assessed in-house by flow cytometry (see section 5.1) and an automated cell counter&#160;(see Table of Materials), respectively. Release criteria were established based on published data and recommendations10,11for GMP-produced cell products (Table 1).
<ol>
	<li>Perform transduction efficiency-flow cytometry staining (Day 14) 
	NOTE: An aliquot of 1 x 106 cells will be stained for viability (7-AAD) and CD3 marker2 to evaluate the transduction efficiency (see Table of Materials). If the gene of interest, such as in the case of CAR T cells, needs to be detected, the specific antibody should be in a different color from that of the CD3 marker and 7-AAD to avoid compensation. Appropriate control samples should be taken into account.
	<ol>
		<li>Place the sample in a FACS tube and add 500 &#181;L of FACS buffer.</li>
		<li>Centrifuge the tube at 400 x g at RT for 5 min.</li>
		<li>Completely discard the supernatant with a pipette and resuspend the pellet in a 1:5 diluted solution of CD3:FACS buffer (see Table of Materials).</li>
		<li>Vortex the sample and incubate for 30 min on ice in the dark.</li>
		<li>Add 500 &#181;L of FACS buffer. Centrifuge at 400 x g for 5 min (RT).</li>
		<li>Completely discard the supernatant and resuspend the pellet in a 1:20 diluted solution of 7-AAD:FACS buffer (see Table of Materials).</li>
		<li>Vortex the sample and incubate for 10 min on ice in the dark.</li>
		<li>Add 200 &#181;L of FACS buffer and analyze the sample using a flow cytometer (see Table of Materials).</li>
	</ol>
	</li>
</ol>

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<li>Sandrin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral+vectors+pseudotyped+with+a+modified+RD114+envelope+glycoprotein+show+increased+stability+in+sera+and+augmented+transduction+of+primary+lymphocytes+and+CD34%2B+cells+derived+from+human+and+nonhuman+primates%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates.</a> Blood. 100 (3), 823-832 (2002).</li>
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N.S. developed the protocol and wrote the manuscript which was reviewed and supervised by A.S.; K.S., G.A. performed additional experiments; D.K., M.L. provided valuable support in the experiments and along with V.Z., N.T. in the review process. N.T. is a member of ProtATonce Ltd. All other authors declare no competing interests.

Lentiviral vectors as gene delivery vehicles are important tools in the field of cell and gene therapy because of their ability to transduce both dividing and non-dividing cells12. However, large-scale production of lentiviral vectors using GMP-compatible methods is still challenging due to various parameters, such as transfection methods and purification steps, which can result in variability in lentivirus batches. Academic centers often obtain viral stocks from viral production biotechnology companies, which provide documentation on the functionality and safety of the virus to meet release criteria13,14.
A successful transduction protocol relies not only on the quality of the viral stock but also on the fitness of the target cells. Efficient stimulation of PBMCs is crucial as a failure in the activation step may lead to low transduction efficiency. Additionally, the choice of promoter and envelope protein can impact the efficiency of the transduction protocol15,16. Different transduction enhancers possess distinct properties that facilitate the successful incorporation of viral particles into target cells17,18,19,20. Vectofusin-1, a non-toxic histidine-rich amphipathic peptide, serves as a transduction enhancer that streamlines the transduction process by eliminating the need for pre-coating on cell culture surfaces18. Furthermore, methods like spinoculation have been shown to increase the susceptibility of target cells to viruses21. Cytokines play a major role as they provide costimulatory signals for full T cell activation and have pleiotropic effects on the proliferation, survival, and function of T cells. IL-7 and IL-15 supplementation creates a favorable environment for CAR T cell expansion due to higher proliferation capacity and lower apoptotic rate22,23,24. In this protocol, a high MOI (MOI = 40) was observed for achieving transduction of T cells (&#62;50%), consistent with other transduction protocols25,26.
The quality of the cell product manufactured under GMP conditions largely depends on the manufacturing facilities, equipment, and availability and quality of GMP-grade ancillary materials. The current product was produced in a GMP facility equipped with a building monitoring system (BMS) that controls and logs environmental conditions throughout all the clean rooms (Grades D, C, B, and A). For release testing, this product underwent sterility tests for microbial growth, mycoplasma detection, and endotoxin level assessment along with functional characterization tests, either performed in certified laboratories or in-house,&#160;meeting all the set criteria (Table 1).
Currently, most T cell therapies are manufactured in automated closed systems such as the CliniMACS Prodigy (Miltenyi Biotec, Germany) or Cocoon (Lonza, Switzerland), among many others under development, which offer ease of handling and consistency between lots11,27,28. However, this limits the production of gene therapies to specialized centers and one product per run. The current protocol has been developed as an open process to minimize costs associated with the procurement of specialized equipment while meeting the set release criteria. In conclusion, a simplified, cost-effective, and GMP-compliant protocol is outlined for generating a gene-modified T cell product with an adequate transduction efficiency (&#62;50%), applicable to CAR T cell production in an academic setting.

The emergence of Adoptive Cell Therapies (ACT) and Chimeric Antigen Receptor (CAR)-T cells have brought about a revolutionary shift in modern clinical practice, establishing a new paradigm of personalized medicine. Particularly, CAR-T cells targeting CD19 have demonstrated exceptional clinical responses and represent the most advanced T cell therapy for B cell malignancies1,2,3,4,5. However, the current commercialized gene-modified T cell therapies heavily rely on viral vectors for gene transfer. The implementation of viral vectors in ACT, especially under Good Manufacturing Practice (GMP) conditions within an academic setting, presents significant challenges due to scalability limitations, specialized equipment requirements, the need for highly skilled personnel, and the use of GMP-compliant reagents for plasmid-mediated viral particle production6,7,8.
Consequently, the manufacturing process for gene-modified T cell therapies is intricate and typically commences with the isolation of Peripheral Blood Mononuclear Cells (PBMCs) from a leukapheresis product. T cells are often enriched from the PBMC pool and activated using anti-CD3/CD28 micro-complexes7,9. Subsequently, T cells are genetically modified with either viral or non-viral vectors, which can be produced in situ or outsourced. To attain the required cell numbers for infusion, gene-modified cells are expanded before final formulation and/or cryopreservation. Finally, the cell product must satisfy various release criteria based on multiple quality control assays9.
This protocol presents a comprehensive methodology utilizing simple techniques for the ex vivo manipulation of healthy PBMCs to manufacture a gene-modified T cell product under GMP-compliant conditions. The protocol incorporates the use of a lentiviral vector, which can be outsourced to a viral production company providing all necessary quantitative/functional, purity, and safety tests (e.g., viral titer, detection of host cell DNA replication-competent lentivirus). For this study, the vector employed is a VSV-g (Vesicular Stomatitis Virus G Protein)-based second-generation lentiviral vector with Green Fluorescent Protein (GFP), as the gene of interest, in constitutive expression.

<table><tbody><tr><td>24-well TC-treated plate</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>5 mL FACS tube&amp;nbsp;</td><td>Corning</td><td>352054</td><td></td></tr><tr><td>50 mL Falcon tubes</td><td>Greiner biomedica</td><td>227261</td><td></td></tr><tr><td>6-well TC-treated plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>7-AAD</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>BD FACS CANTO II</td><td>BD Biosciences</td><td>V96300084</td><td></td></tr><tr><td>BSA</td><td>Applichem</td><td>A1391</td><td></td></tr><tr><td>Cell Counter&amp;nbsp;</td><td>Corning Cell Cytosmart</td><td>J21E0081</td><td></td></tr><tr><td>Cryovials</td><td>Greiner biomedica</td><td>122263</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2438</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS)</td><td>Life Technologies</td><td>14190</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>FACS Buffer (1x D-PBS, 0.5% BSA, 2 mM EDTA)</td><td>-</td><td>-</td><td></td></tr><tr><td>FlowJo Software v10.6.2&amp;nbsp;</td><td>TreeStar Inc</td><td>-</td><td></td></tr><tr><td>Gibco CTS Opti-MEM I Medium</td><td>ThermoFisher Scientific</td><td>A4124801</td><td></td></tr><tr><td>GMP rhIL-15</td><td>Miltenyi Biotec</td><td>170-076-114</td><td></td></tr><tr><td>GMP rhIL-2</td><td>Miltenyi Biotec</td><td>170-076-146</td><td></td></tr><tr><td>GMP rhIL-7</td><td>Miltenyi Biotec</td><td>170-076-111</td><td></td></tr><tr><td>Hanks&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14175</td><td></td></tr><tr><td>HEPA Whitley H35 Hypoxystation</td><td>Don Whitley Scientific</td><td>HA0315172H</td><td></td></tr><tr><td>Human Serum Albumin (HSA)</td><td>Baxter</td><td>B05AA01</td><td></td></tr><tr><td>Junior LB 9509 Portable Luminometer</td><td>Berthold Technologies</td><td>6506</td><td></td></tr><tr><td>Lenti-X Provirus Quantitation Kit</td><td>Takara Bio</td><td>631239</td><td></td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>7811</td><td></td></tr><tr><td>MACS GMP T cell TransAct</td><td>Miltenyi Biotec</td><td>170-076-156</td><td></td></tr><tr><td>Mouse Anti-Human&amp;nbsp; CD3 (Clone: UCHT-1)</td><td>BD Biosciences</td><td>557706</td><td></td></tr><tr><td>MycoAlert Plus Detection kit</td><td>Lonza Bioscience</td><td>LT07-703</td><td></td></tr><tr><td>Parafilm ultra-expandable closing film tape 5 cm x 75 m</td><td>D.Dutscher</td><td>90261</td><td></td></tr><tr><td>PeriStem PS-650-DB (apheresis bag)</td><td>Biomed Device</td><td>SC00816</td><td></td></tr><tr><td>Planer Kryo10 Series III</td><td>Planer</td><td></td><td></td></tr><tr><td>T75 flasks</td><td>Greiner biomedica</td><td>658175</td><td></td></tr><tr><td>Trypan blue, 0.4% solution</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>Vectofusin-1 GMP</td><td>Miltenyi Biotec</td><td>170-076-165</td><td></td></tr><tr><td>X-VIVOTM 15 Serum-free Hematopoietic Cell Medium</td><td>Lonza Bioscience</td><td>A1048501</td><td></td></tr></tbody></table>

a gmp-compliant procedure for the generation of gene-modified t cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

Assessment of transduction efficiency 
The transduced cells were evaluated for the expression of the gene of interest (GFP) using flow cytometry. Representative results are depicted in Figure 1. On Day 14, more than 95% of the cells were CD3+, indicating successful T cell activation and expansion. The transduction efficiency within the CD3+ population was measured at 58.7% (TD, Figure 1C) compared to the non-transduced cells (UNTD, Figure 1B) which exhibited a transduction efficiency of 0.51%. Viability analysis showed that more than 95% of the cells were alive after excluding 7-AAD+ cells (Figure 1A). FlowJo Software (see Table of Materials) was used for data analysis.
Assessment of expansion profile 
The transduced cells were expanded in the presence of rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) from Day 4 until the day of cryopreservation (Day 14). Subculturing was performed every 2 days. The cells underwent a 25-fold expansion, as shown in Figure 2, reaching clinically relevant cell numbers based on the initial cell count.
Assessment of quality control 
The product underwent various quality control tests, including sterility testing, mycoplasma detection, and assessment of endotoxin levels. Additionally, functional characterization was performed according to the criteria outlined in Table 1. The product met all the set criteria, indicating its suitability for further use.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65097/65097fig01v2.jpg" /> 
Figure 1: Representative results showing the expression of the GFP transgene on human primary CD3+ T cells. Analysis was performed on singlet alive (7-AADneg) cells (A). The positivity threshold of the transduced (TD) cells (C) was determined based on the non-transduced (UNTD) population (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65097/65097fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65097/65097fig02.jpg" /> 
Figure 2: Expansion profile of transduced T cells. Transduced (TD) T cells were expanded in the presence of rhIL-7 and rhIL-15 for 10 days with subculturing every 2 days (n = 3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65097/65097fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Assessment</td>
			<td>Method</td>
			<td>Criteria</td>
		</tr>
		<tr>
			<td>Sterility</td>
			<td>Development of microorganisms on TSA (Tryptic Soy Agar) or SDA&#160;(Sabouraud&#160;Dextrose&#160;Agar)&#160;plates (outsourced or collaborating certified Microbiology Lab)</td>
			<td>Negative after 7 days</td>
		</tr>
		<tr>
			<td>Mycoplasma</td>
			<td>Mycoalert Plus Detection kit</td>
			<td>Negative</td>
		</tr>
		<tr>
			<td>Endotoxin&#160;</td>
			<td>Gel Clot Limulus Amobecyte Lysate (LAL) test (outsourced or collaborating certified laboratory within University&#8217;s Department of Public Health)</td>
			<td>&#60;0.5 EU/mL</td>
		</tr>
		<tr>
			<td>Cell phenotype</td>
			<td>Flow cytometry (in house)</td>
			<td>CD3+ &#8805;80% 
			GFP+ &#8805;30% (Transduction efficiency)</td>
		</tr>
		<tr>
			<td>Cell number&#160;</td>
			<td>Automated counter (in house)</td>
			<td>&#62;1 x 108 cells</td>
		</tr>
		<tr>
			<td>Viability</td>
			<td>Trypan blue stain (in house)</td>
			<td>&#8805;70% alive cells</td>
		</tr>
		<tr>
			<td>Transgene copies/cell</td>
			<td>Real-time PCR (in house)</td>
			<td>&#8804;50 copies/&#181;g DNA</td>
		</tr>
	</tbody>
</table>
Table 1: Release criteria for the cell product.

Watch this Scientific Journal Video about A GMP-Compliant Procedure for the Generation of Gene-Modified T cells at JoVE.com

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

a-gmp-compliant-procedure-for-the-generation-of-gene-modified-t-cells

Nanyang Technological University

Research

JoVE Journal

Bioengineering

465 Views.  University of Patras. Dit protocol beschrijft een vereenvoudigd en BNP-conform proces voor het transduceren van menselijke primaire T-cellen met genen van belang. Deze specifieke techniek is ontworpen om kosteneffectief te zijn en in overeenstemming te zijn met het BNP zonder het gebruik van dure productieplatforms voor gesloten systemen die momenteel in veel academische centra beschikbaar zijn. Deze methode kan mogelijk worden toegepast op het genereren van gengemodificeerde celtherapieën, inclusief maar niet be...

Video: A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Immunology and Infection

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.

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 ...

Cancer Research

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

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.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

A CRISPR-Cas9 Technique for Gene Editing in T Cells

Source: Agarwal, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62299">Production of Human CRISPR-Engineered CAR-T Cells.</a> J. Vis. Exp. (2021).
This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell&#39;s non-homologous end-joining mechanism, leading to gene knockout.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Designing of sgRNAs and gene ...

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

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