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

Nikolaos Savvopoulos; Klearchos Stampolitis; Georgios Alexandropoulos; Dionysia Kefala; Memnon Lysandrou; Vassiliki Zacharioudaki; Nikos Tsolakos; Alexandros Spyridonidis

doi:10.3791/65097

Una procedura conforme alle GMP per la generazione di cellule T geneticamente modificate

JoVE Journal
Bioengineering
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JoVE Journal Bioengineering

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

Una procedura conforme alle GMP per la generazione di cellule T geneticamente modificate

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06:47 min

October 6, 2023

DOI: 10.3791/65097-v

Nikolaos Savvopoulos¹, Klearchos Stampolitis¹, Georgios Alexandropoulos¹, Dionysia Kefala¹, Memnon Lysandrou¹, Vassiliki Zacharioudaki¹, Nikos Tsolakos², Alexandros Spyridonidis¹

¹Institute of Cell Therapy, Bone Marrow Transplantation Lab, Department of Medicine,University of Patras, ²Protatonce Ltd, Demokritos Science Park

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Overview

This protocol describes a simplified and GMP compliant process of transducing human primary T-cells with a gene of interest. This method is designed to be cost-effective and avoids the use of expensive closed system manufacturing platforms.

Key Study Components

Area of Science

Gene therapy
Cellular transduction
Immunotherapy

Background

Transduction of T-cells is crucial for developing gene-modified cell therapies.
CAR T-cells represent a significant advancement in treating hematologic malignancies.
Current methods often rely on costly manufacturing systems.
GMP compliance is essential for clinical applications.

Purpose of Study

To provide a cost-effective method for T-cell transduction.
To ensure compliance with GMP standards.
To facilitate the generation of gene-modified therapies.

Methods Used

Isolation of PBMC from donor blood via leukapheresis.
Polysaccharide-based density gradient centrifugation.
Maintaining a reagent to sample ratio of 1:2.
Washing cells with PBS by centrifugation.

Main Results

The protocol allows for efficient isolation of PBMCs.
Demonstrates compliance with GMP standards.
Provides a foundation for developing CAR T-cell therapies.
Cost-effective compared to existing methods.

Conclusions

This method can enhance the accessibility of gene-modified therapies.
It addresses the need for cost-effective solutions in cell therapy.
Future applications could significantly impact hematologic cancer treatment.

Frequently Asked Questions

What is the significance of GMP compliance?

GMP compliance ensures that the manufacturing process meets quality standards necessary for clinical applications.

How does this method compare to traditional transduction methods?

This method is more cost-effective and does not require expensive closed system platforms.

What types of therapies can this protocol support?

It can support the generation of various gene-modified cell therapies, including CAR T-cells.

What is the role of PBMC in this protocol?

PBMCs are the target cells that are transduced with the gene of interest.

Can this method be used in academic centers?

Yes, it is designed to be applicable in academic settings without the need for expensive equipment.

What is the expected outcome of using this protocol?

The expected outcome is the successful transduction of T-cells, enabling the development of effective cell therapies.

Questo protocollo delinea il processo di trasduzione delle cellule T umane primarie con un gene di interesse, garantendo la compatibilità con gli standard delle buone pratiche di fabbricazione (GMP).

Questo protocollo descrive un processo semplificato e conforme al PNL di trasduzione di cellule T primarie umane con il gene di interesse. Questa particolare tecnica è stata progettata per essere conveniente e conforme al PNL senza l'uso di costose piattaforme di produzione a sistema chiuso attualmente disponibili in molti centri accademici. Questo metodo potrebbe potenzialmente essere applicato alla generazione di terapie cellulari geneticamente modificate, tra cui, ma non solo, le cellule CAR-T, che hanno rappresentato un cambiamento di paradigma per affrontare le neoplasie ematologiche.

Per isolare la PBMC dalle cellule raccolte dopo la leucoaferesi del sangue del donatore, eseguire una centrifugazione a gradiente di densità a base di polisaccaridi del campione, mantenendo un rapporto reagente/campione di 1:2, centrifugando la miscela a 800 G per 30 minuti a temperatura ambiente con i freni disattivati. Quindi, utilizzando una micropipetta, raccogliere le PBMC in una provetta pulita da 50 millilitri. Lavare le celle due volte con PBS mediante centrifugazione.

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