To achieve scale in the cell and gene therapy field, we must overcome manufacturing barriers. In traditional ex vivo CAR T manufacturing, T cells are harvested from the patient, engineered with viral vectors, expanded, and reinfused to the patient, who has also had to endure lymphodepletion. This is laborious, expensive, and burdensome for the patient. In contrast, generating CAR T cells directly in the patient with a simple infusion would enable off-the-shelf production of CAR-T therapies and drastically improve patient access, while avoiding the straining lymphodepletion procedure.
How do we make this happen? In my view, single-stranded DNA (ssDNA) can make in vivo CAR T therapeutics a commercial reality.
Viral vectors are the current, conventional workhorses for autologous and allogeneic CAR T manufacturing because they can deliver large genetic payloads and integrate into the genome to provide long-term expression. However, genetic integration comes at the cost of insertional mutagenesis risks and has unpredictable long-term effects. Cancer patients, already immunocompromised, thus face a complex mix of additional risks of genotoxicity, inflammation, and immune responses from a viral vector-based in vivo CAR-T treatment.
mRNA has also been explored as a potential in vivo approach for CAR T therapeutics. mRNA, delivered with lipid nanoparticles (LNPs), is a non-viral and transient gene vector alternative with many advantages. But CAR-T cells engineered with the quickly fading mRNA will not persist for the weeks or months needed to eliminate tumors. To maintain the required CAR-T cell activity, frequent redosing, on the scale of days, possibly even hours, will be needed, which creates new risks and health burdens for the patient.
Why ssDNA and LNPs?
Using DNA rather than RNA for non-viral delivery is not a new idea. Plasmid DNA has been tested in a range of contexts, from vaccines to gene therapy, but has faced hurdles related to innate immune activation, particularly in immune cells such as T cells, and inefficient nuclear entry. What distinguishes ssDNA is that it overcomes several of these key barriers.
ssDNA can be used both for transient expression and for permanent genetic CAR integration, allowing to engineer fit-for-purpose therapies. Interest in ssDNA as a non-viral vector has been peripheral traditionally, and it has been frequently written off due to production and expression challenges.
For in vivo CAR-T, where we aim to use a simple infusion to reprogram T cells directly inside the body, the genetic vector delivery system must be safe, durable, scalable, and specific. Lipidnanoparticles (LNP) offer several advantages that meet the requirements. It is a clinically validated delivery system, with billions of vaccine doses demonstrating overall favorable safety profile and the manufacturing processes are well established, making LNPs scalable and compatible with large-scale drug production. The composition of LNPs can be tuned to optimize biodistribution, stability, and cellular uptake, offering flexibility for different therapeutic applications. Furthermore, because mRNA and ssDNA share similar biophysical properties, existing mRNA LNP formulations provide a useful foundation for developing LNP systems suitable for ssDNA delivery.
The road ahead
Our research data show that ssDNA delivered via LNPs achieves robust expression in primary immune cells without triggering the innate immune alarms seen with double-stranded plasmids. Early data also suggests that ssDNA vectors can sustain therapeutically relevant expression over extended periods. They are also fine-tunable with multiple technologies to meet diverse therapeutic needs. Although the field is still young, rapid progress in the field is beginning to establish ssDNA as an alternative to today’s delivery vectors.
We stand at the cusp of a new era in immune cell therapies. Viruses started it, and mRNA opened our imagination for design, delivery, and democratization of in vivo immune cell therapies. Single-stranded DNA coding for CAR (or other therapeutic) genes delivered with LNPs offers a compelling, non-viral, redosable, duration-controllable, off-the-shelf solution to cancer treatment. A simple injection and the patient’s own T cells can be transformed into cancer-fighting machines. If the CAR-T activity wanes, the oncologist simply redoses. No debilitating lymphodepletion required, no trip to a specialized GMP facility needed, no months of waiting for a treatment, and no burden of lifelong genetic alteration.
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