Genome Editing and Advanced Manufacturing of Hematopoietic Stem Cell-Based Therapeutics
Pankaj Kumar Mandal, PhD
Office of Tissues and Advanced Therapies
Division of Cellular and Gene Therapies
Tumor Vaccines and Biotechnology Branch
Pankaj K. Mandal, PhD is a Senior Staff Fellow in the Tumor Vaccines and Biotechnology Branch (TVBB) of OTAT. His research interests include hematopoietic stem cell (HSC) biology and genetically engineered hematopoietic stem cell (HSC)-- based therapeutics.
Dr. Mandal graduated from Haryana Agricultural University, Hisar, India in 2002 with a BS in veterinary sciences and animal husbandry (2002), completed an MS in veterinary immunology at Indian Veterinary Research Institute, Izatnagar, India (2004), and received his PhD in veterinary medicine from Ludwig Maximilian University, Munich (2009).
He conducted his postdoctoral research training at Harvard Medical School and Boston Children’s Hospital, (2010-2014), focusing on developing a modified mRNA-based cellular reprogramming protocol for deriving induced pluripotent stem (iPS) cells, generating hematopoietic stem cell (HSC)-specific reporter strains of mice, and evaluating the efficacy of CRISPR/Cas9 genome editing in human cells. From 2014-2018, he served as an instructor in pediatrics at Harvard Medical School and was a Senior Scientist in the Discovery Biology group at Omega Therapeutics (2018-2019). In his current role as Senior Staff Fellow at DCGT, Dr. Mandal leads a research group focused on understanding the advanced manufacturing of CRISPR-edited HSC-based therapeutics.
Recent advances in gene therapy, genetic engineering, and stem cell therapeutics, have helped to promote a paradigm shift from conventional palliative treatments to regeneration and cures. Cell- and gene-based therapies and devices could provide long-term cure through replacement or regeneration of diseased tissue. These advances have brought newer cell-based treatments, such as CAR-T cells and HSC gene therapy, into clinical practice. By generating functionally competent cell types through differentiation, CRISPR/Cas9 engineered HSC-based cellular therapeutics hold great promise for treatment of hematological disorders, such as sickle cell anemia, beta-thalassemia, and severe combined immunodeficiency. Despite significant advances in the development of HSC-based therapies over the past decade, the lack of optimized protocols for HSC expansion ex vivo and manufacturing of quality HSC products has stalled their widespread use.
Our research program is focused on understanding advanced manufacturing of genome-edited HSC-based therapeutics. We propose to identify and define optimal conditions for cost-effective, large scale manufacturing of genome-edited, HSC-based therapeutics. Such work will help us to establish a set of Critical Quality Attributes (CQA), evaluate submissions in this product class, and establish FDA regulatory guidelines for HSC-based therapeutics.
HSCs are the regenerative unit of hematopoietic tissue. Through self-renewal and differentiation, HSCs sustain steady-state hematopoiesis by generating various blood cell types throughout life. Therefore, they have the potential to provide curative treatments for various blood disorders.
The conditions for very robust ex vivo gene editing in human HSPCs using CRISPR/Cas9 have been well-established; however, the latest findings indicate that very low percentage of gene-edited HSPCs are able to engraft following transplantation. This reflects our limited understanding of the large-scale manufacturing of HSCs under advanced manufacturing conditions. Sub-optimal and poorly defined culture conditions, gene editing, and gene therapy reagents can adversely affect the ability of ex vivo manipulated HSPC to engraft and sustain long-lasting, balanced hematopoiesis in transplant recipients. This lack of cost-effective, industrial-scale, and reproducible manufacturing of quality HSC-based therapeutics with defined Quality Attributes (QA) is the major roadblock to the safe, effective, and widespread use of this therapy.
Cost-effective large-scale manufacturing of quality HSC-based therapeutics requires an in-depth knowledge of cell behavior and response to advanced manufacturing conditions. Taking a Quality by Design (QbD) approach, our goal is to define the critical parameters that affect HSC activity during advanced manufacturing. Using cells from an HSC-specific reporter mouse, human HSCs/HSPCs, CRISPR/Cas9 technology (for genome editing and functional genomics), and a combination of small molecules, growth factors, and cytokines, we propose to optimize the ex vivo culture conditions for large- scale HSC expansion and develop an advanced manufacturing protocol for CRISPR/Cas9-engineered HSC-based therapeutics.
- Bruna S. Paulsen*, Pankaj K. Mandal*, Richard L. Frock, Baris Boyraz, Rachita Yadav, Srigokul Upadhyayula, Paula Gutierrez-Martinez, Wataru Ebina, Anders Fasth, Tomas Kirchhausen, Michael E. Talkowski, Suneet Agarwal, Frederick W. Alt, and Derrick J. Rossi (2017) Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR/Cas9 genome editing. Nat. Biomed. Eng. 1(11):878-888.
- Pankaj K. Mandal*, Leonardo M. R. Ferreira*, Ryan Collins, Torsten B. Meissner, Christian L. Boutwell, Max Friesen, Vladimir Vrbanac, Brian S. Garrison, Alexei Stortchevoi, David Bryder, Kiran Musunuru, Harrison Brand, Andrew M. Tager, Todd M. Allen, Michael E. Talkowski, Derrick J. Rossi, and Chad A. Cowan (2014). Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9 Cell Stem Cell 15(5): 643–652.
- Roi Gazit*, Pankaj K. Mandal*, Wataru Ebina, Ayal Ben-Zvi, Cesar Nombela-Arrieta, Leslie E. Silberstein, Derrick J. Rossi (2014). Fgd5 identifies hematopoietic stem cells in the murine bone marrow and is not required for definitive hematopoiesis J. Ex. Med. 211(7):1315-31