Collaboration investigates the delivery of mRNA using DNA origami as a gene therapy approach for enabling CAR-T cell generation in vivo to increase therapeutic efficacy and lower costs
By Benjamin Boettner
(BOSTON) — The “Immuno-engineering to Improve Immunotherapy” (i3) Center at Harvard’s Wyss Institute for Biologically Inspired Engineering selected Elizabeth Carstens, M.D., and Yang (Claire) Zeng, M.D., Ph.D. as the 2022 recipients of its annual grant award. The newly formed research team is exploring DNA origami as a vehicle for the targeted delivery of messenger RNA (mRNA) molecules encoding chimeric antigen receptors (CARs) to T cells in order to generate the cancer-fighting CAR-T cells in the living body. If possible, this novel gene therapy approach could eliminate the cumbersome and expensive process of creating and multiplying CAR-T cells ex vivo for their use in adoptive cell therapies.
The i3 Center was formed as an NIH-funded initiative in 2020 to innovate and accelerate new types of biomaterials-based immunotherapies developed through cross-institutional and cross-disciplinary research led by its members, who combine world-leading expertise in the cancer immunology and bioengineering fields. Each year, the i3 Center supports a new research program that is performed by researchers of its founding collaborating institutions, with the intend to add a new multidisciplinary facet to the Center’s central agenda. Carstens’ and Zeng’s collaboration unites the expertise of Eric Smith’s lab for Synthetic Biology and Cellular Engineering at the i3 Center and Dana-Farber Cancer Institute (DFCI) on improving CAR-T cell therapies (Carstens) with that of William Shih’s group at the Wyss Institute and DFCI on developing new methodology for the creation and use of mRNAs and various DNA nanostructures in novel cancer vaccine and therapy approaches (Zeng).
Addressing key challenges to CAR-T cell therapy
“The current process of generating commercially available CAR-T cell products is extremely costly and tedious as it involves the isolation of appropriate T cells from patients, genetically manipulating them so that they express a tumor cell-binding CAR-T cell receptor, and a time-consuming expansion step to reach therapeutically active cell numbers,” explains Carstens. “By re-engineering this process, using mRNA and DNA origami technology, and moving it into the body, we hope to be able to make it faster, and more efficient, and achieve robust therapeutic effects, all at significantly lower costs.”
The first CAR-T cell (chimeric antigen receptor-T cell) therapies have been approved by the Federal Drug Administration (FDA) to treat certain types of leukemias and lymphomas, which are tumors of the immune system. More recently CAR-T cell therapy has also been expanded to multiple myeloma, and there are ongoing efforts for a number of solid malignancies. To generate CAR-T cell products, which are “living drugs,” first, T cells are collected from patients and genetically engineered outside of the body to express an artificial T cell receptor molecule on their surface which is part normal T cell receptor and part tumor cell-binding “antigen receptor.” Over a period of weeks and sometimes longer, the engineered cells need to be amplified in cell cultures that minimize any loss of their functions. Once CAR-T cells are administered to patients, the added chimeric receptor allows them to specifically recognize cancer cells and unleash a potent T cell response that destroys them. This CAR-T cell manufacturing process is completed in specialized facilities, and depends on the availability of reagents, skilled personnel, and timely transportation, which together greatly contribute to the high costs of CAR-T cell therapies, and putting them out of reach for some cancer patients.
Bringing DNA nanotechnology to immunotherapy
DNA origami have been discussed as potential delivery vehicles for drugs to treat cancer and other diseases. “DNA origami, which have not advanced into clinical settings yet, are biocompatible, can be assembled in a highly programmable way, and offer the opportunity to bind a variety of ‘guest molecules’ to them, such as therapeutic mRNA and different therapy-enhancing molecules,” said Zeng. “Carrying the right combination of guests in the right configuration, they could help convert T cells on-site into potent cancer-fighting agents,” she explained.
T and other cells take up mRNA by the process of “endocytosis” that encloses them into small membrane vesicles called “endosomes” at the cell surface, which are then internalized into the cell’s interior. As a barrier to most mRNA therapies, therapeutic mRNA molecules are usually trapped within endosomes without getting access to the protein-synthesizing machinery that translates them into functional therapeutic proteins. “In addition to binding CAR-encoding mRNA molecules and an antibody that allows T cell targeting to differently shaped DNA origami, we are also testing an ‘endosome-escape coating’ method developed by our external collaborator Nicholas Stephanopoulos, Ph.D. at Arizona State University.” The method consists of a small protein peptide that, when bound to the surface of DNA origami, could allow them and their therapeutic cargo to break out of endosomes and thus unfold their therapeutic effects.
The researchers will first focus on optimizing the right configurations of mRNA, targeting antibody and endosome escape molecules on origami with different shapes – first, by using cultivated T cells as an in vitro model, and later mice with multiple myeloma as a well-defined in vivo model for testing CAR-T cell therapies. “If our approach proves successful in the treatment of multiple myeloma, it could also be a new gateway for the development of CAR-T cell therapies against solid tumors which so far have alluded this type of immunotherapy,” said Carstens. Developing immunotherapies for solid tumors is a key goal pursued at the i3 Center.
Zeng is an Instructor in Medicine at DFCI and Harvard Medical School (HMS). She holds an M.D. and a Ph.D. degree from Peking Union Medical College and Tsinghua University. In her postdoctoral studies, she focused on the biology of mesenchymal stem cells, new delivery strategies for cell therapies, and immunotherapies. Working on William Shih’s team at the Wyss Institute and DFCI, she has developed DoriVac, a novel vaccine platform that uses DNA origami to present tumor-specific and pathogen-specific antigens and adjuvant molecules with nanoscale precision to dendritic cells to generate stronger immune responses against cancer cells and infectious organisms. The DoriVac technology is currently being translated towards clinical applications at the Wyss Institute and is an integral part of one of the i3 Center’s principal research directions.
Carstens received her M.D. degree at UT Southwestern Medical School in Dallas, Texas, and completed her medical residency in internal medicine at the Johns Hopkins Hospital before starting her hematology/oncology fellowship at DFCI and joining Eric Smith’s group. She helped develop novel adeno-associated virus (AAV) vectors for gene delivery at the Rice University, and as a Fellow in the Medical Research Scholars Program that the National Institutes of Health in Bethesda, pursued strategies to potentiate immunotherapy by targeting a molecule of the complement system on cancer cell surfaces. At the DFCI, Carstens is building in vitro and in vivo expertise in the development of novel nucleic acid-based immunotherapies.
The i3 Center is co-led by David Mooney, Ph.D., a Founding Core Faculty member of the Wyss Institute and the Robert P. Pinkas Family Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences, and F. Steven Hodi, Jr, M.D., Director of the Melanoma Center and The Center for Immuno-Oncology at DFCI, and Professor of Medicine at HMS. Carstens’ and Zeng’s project is the third to be supported by the i3 Center and the grant follows earlier grants awarded to cancer immunologists Eric Smith, M.D., Ph.D., and Rizwan Romee, M.D. at DFCI.