Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy.

Jennifer E. Adair,Sara P. Kubek,Timothy Waters,Jonah D. Hocum,Shelly Heimfeld,Kevin G. Haworth,Grant D. Trobridge,Hans-Peter Kiem

doi:10.1038/ncomms13173

Abstract

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However, current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility, limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here, we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow, and reconstitute human haematopoiesis in immunocompromised mice. Importantly, nonhuman primate autologous gene-modified CD34+ cell products are capable of stable, polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy, there is enormous potential for this approach to treat patients on a global scale.

Highlights

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases
There is tremendous potential for haematopoietic stem cell (HSC) and progenitor (CD34 þ ) cell gene therapy for many diseases, but as the field closes in on large global health burdens such as HIV and haemoglobinopathies, lack of a portable technology for standardized manufacture of gene-modified CD34 þ blood cell products becomes a critical barrier to widespread clinical use
Ex vivo manufacturing generally includes (1) immunomagnetic bead-based isolation of target CD34 þ cells, (2) CD34 þ cell supportive culture conditions with (3) defined gene modification reagents and conditions and (4) removal of residual manufacturing reagents for preparation and testing of the final cellular product for infusion. All of these steps are carried out under current Good Manufacturing Practices, but the CD34 þ cell source (that is, bone marrow (BM) or growth factor mobilized leukapheresis (HPC-A)), and the therapeutic genetic modification vary depending on the target patient population

Summary

Introduction

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. Ex vivo manufacturing generally includes (1) immunomagnetic bead-based isolation of target CD34 þ cells, (2) CD34 þ cell supportive culture conditions with (3) defined gene modification reagents and conditions and (4) removal of residual manufacturing reagents for preparation and testing of the final cellular product for infusion All of these steps are carried out under current Good Manufacturing Practices (cGMP), but the CD34 þ cell source (that is, bone marrow (BM) or growth factor mobilized leukapheresis (HPC-A)), and the therapeutic genetic modification vary depending on the target patient population. An HES sedimentation protocol for up to 1.8 l of BM was developed using customized programming for the CliniMACS Prodigy device (Miltenyi Biotec GmbH) This commercially available device permits automated pre-processing, immunomagnetic labelling and separation of target cells, including CD34 þ cells and T cells, from human HPC-A products[16,17], and is capable of large scale, automated Ficollbased RBC depletion from BM18

Objectives

Methods

Results

Conclusion