Plasmid DNA is a critical component of chimeric antigen receptor T-cell (CAR-T) cancer therapies because of its central role in bringing these personalized therapies to life. After T cells are removed from patients, a plasmid DNA vector consisting of transgenes carried by a bacterial backbone transforms the cells into cancer-killing machines.
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But first-generation bacterial vectors are far from perfect. They’re huge—typically 2,000 base pairs in length or larger—and they require the use of antibiotic markers for selection. Those characteristics raise the risk of adverse effects, including transfection toxicity, gene silencing, and antibiotics resistance. Hence the demand for better CAR-T plasmid backbones is on the rise, fueled by regulatory demands from agencies such as the U.S. Food and Drug Administration (FDA), which in new guidelines released this year recommended that new CAR-Ts exclude transgenes that could cause antibiotics resistance.
Improving the plasmid DNA backbone used to make CAR-Ts will result in benefits that extend far beyond improving patient safety and satisfying regulators, however. Plasmid DNA innovations will also improve manufacturing efficiency and yield. And they will support scientific advances in cancer cell therapies that could greatly expand the population of patients who can be treated with them.
Improving gene expression and safety
One major improvement in the design of next-generation plasmid DNA is a reduction in size. When vaccines using plasmid DNA vectors started hitting the market over a decade ago, researchers raised concerns about the long length of their bacterial backbones. One study found that spacer regions over 1,000 base pairs could spur transgene silencing, for example.1
Emerging plasmids with spacer regions that are 500 base pairs in length or even smaller are proving effective in enhancing gene expression. A 2022 study led by Wayne State University in Detroit compared one plasmid with a spacer region of 2,678 base pairs to another that was 454 base pairs in size. The researchers found that in two types of rat cells, gene expression levels were 10-fold higher with the smaller plasmid.2
Given the complexity of creating CAR-Ts from individual patients’ cells, the potential benefits of improved gene expression cannot be overstated. Better gene expression increases cell quality and viability, improving manufacturing yields and easing the process of scaling up production.
Another important innovation in the design of plasmid DNA backbones is the elimination of antibiotic markers. One example of an alternative technology uses non-coding mRNA to spur RNA/RNA interactions. These interactions mediate selection by inhibiting the translation of the marker SacB mRNA, which in turn represses the expression of the enzyme levansucrase. This allows sucrose to be used instead of antibiotics in the selection process,3 in effect eliminating the risk of antibiotics resistance by substituting in a benign substance.
Next-generation DNA plasmids are starting to show promise in clinical trials. In one trial, a CAR-T targeting BCMA that was made with a reduced-size plasmid was tested in patients with relapsed/refractory multiple myeloma. The overall response rate among 90 patients was 57% in those treated with the CAR-T alone and 73% in people who were also treated with rituximab. Toxicity was low.4
Ushering in CAR-T innovations
Reducing the size of plasmid DNA backbones could translate into more efficient manufacturing of CAR-T treatments. A German research team tested the utility of a DNA nanovector in CAR-T development and described how they were able to develop a five-day manufacturing protocol for them—a significant improvement over the three- to four-week process currently required for CAR-T production.5
New plasmid DNA templates are also supporting the development of alternative CAR-T editing methods. For example, some researchers are investigating CRISPR/Cas9-ribonucleoprotein delivery as a method for editing T cells. A 2022 study led by Genentech compared a reduced-size plasmid with a traditional plasmid and double-stranded DNA as CRISPR/Cas9 donor templates. They reported that the small plasmid yielded double the amount of CAR-T cells as did the traditional plasmid and three-fold as many as the double-stranded DNA.6
Other researchers are using small plasmid DNA to in the design of new cell-therapy platforms, such as CAR-NK (natural killer) therapies. Researchers in Ireland, for example, used a small plasmid backbone to make CAR-NK cells from blood taken from a healthy donor. They edited the cells to target acute myeloid leukemia (AML) and expanded them over 25 days to produce several doses of cancer-killing cells.7 The authors concluded that improving the delivery of plasmid DNA was key to making NK cell engineering viable, which, in turn could make it possible to create cell therapies that don’t need to be tailored to individual patients, but rather are allogeneic, or off the shelf.
Whether they’re designed to treat just one patient or many patients, all CAR-T treatments share one challenge: the T cells must be modified using a robust, efficient protocol. That’s why innovations in plasmid DNA are so vital to advancing the CAR-T field. Smaller, safer plasmid DNA backbones are expanding the toolkit for CAR-T developers, helping them reach their goal of bringing life-saving cell therapies to a broader population of cancer patients.
References
1. Williams, J., et al. (2009). Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production. Biotechnol. Adv. Volume 27, Issue 4.
2. Boye, C., et al. (2022). Reduction of plasmid vector backbone length enhances reporter gene expression. Bioelectrochemistry. Volume 144.
3. Williams, J., et al. (2023). Improving cell and gene therapy safety and performance using next-generation Nanoplasmid vectors. Mol. Ther. Volume 32.
4. Moretti, A., et al. (2022). The Past, Present, and Future of Non-Viral CAR T Cells. Front. Immunol. Volume 13.
5. Bozza, M., et al. (2021). A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells. Sci. Adv. Volume 7, Issue 16.
6. Oh, S., et al. (2022). High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA. J. Exp. Med. Volume 219, Issue 5.
7. Gurney, M., et al. (2021). Tc Buster Transposon Engineered CLL-1 CAR-NK Cells Efficiently Target Acute Myeloid Leukemia. Blood. Volume 138, Issue Supplement 1.
Venkata Indurthi has been a member of the Aldevron team since he received his doctorate in pharmaceutical sciences from North Dakota State University, Fargo, ND, in 2016. He has held a variety of positions in increasing responsibility and focus, including Senior Scientist in product and process design, Director of RNA Operations, Director and then Vice President of Research and Development before being named Chief Scientific Officer in 2022. Indurthi received his Bachelor of Science in Biotechnology from SRM University, Chennai, India, in 2010. He has been recognized with several awards and honors from a variety of organizations, served on or lead many panels, and has authored or participated in numerous published articles.