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Will Non-Viral CAR-T Cells be used to treat more diseases in future?
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Will Non-Viral CAR-T Cells be used to treat more diseases in future?
Chimeric Antigen Receptor T-cell (CAR-T) therapy is a powerful technology that has revolutionized the field of immunotherapy.
Its impressive efficacy in refractory and relapsed hematological malignancies has opened new possibilities for treating solid tumors as well.
However, the widespread application of cell therapies has been hindered by the limitations of commonly used viral vectors for T-cell transduction.
In the era of mRNA vaccines and precise gene editing with CRISPR/Cas9, new non-viral T-cell engineering approaches are emerging as more versatile, flexible, and sustainable alternatives for the next generation of CAR-T cell manufacturing.
Limitations of Viral Vector CAR-T Cells
So far, most CAR-T cell therapies approved or under investigation in clinical trials have utilized viral vectors, especially retroviral and lentiviral vectors.
Viral vectors are standardized systems with a history of efficient gene transfer and long-term use, demonstrating safety in adoptive T-cell therapy.
However, the capacity of viral vectors to carry large genes is limited by the size of the viral capsid, which is approximately 100 nm in diameter and typically cannot accommodate genes larger than 8-9 kb. As CAR-T technology evolves, there is a need to introduce additional activating components or CARs with different specificities to achieve enhanced efficacy, safety, and applicability.
Moreover, clinical-grade viral vector production often takes two to three weeks and requires skilled personnel to be conducted in Good Manufacturing Practice (GMP) facilities with Biosafety Level 2 (BSL2) containment. This high cost, complexity, and the need for personalized therapies ultimately affect the price of CAR-T products, potentially reaching hundreds of thousands of dollars per patient, making them inaccessible to the general population.
Finally, viral vectors come with inherent immunogenic risks, which arise from humoral and cellular immune responses to vector-encoded epitopes, potentially limiting the efficacy and persistence of transduced cells.
Non-Viral Approaches to T-Cell Engineering
The Nobel laureate geneticist Barbara McClintock, in her research on corn kernel color variations in the 1940s, initially discovered the existence of mobile sequences within the genome, known as transposable elements (TEs) or transposons at the time. TEs are divided into two classes, retrotransposons, and DNA transposons.
Retrotransposons move using an RNA intermediate through a copy-and-paste mechanism, representing the most common class of transposons in the human genome. DNA transposons move through a DNA intermediate and are used in gene transfer applications.
Most DNA transposon families have an element encoding a transposase enzyme gene flanked by inverted terminal repeat sequences (ITRs).
The transposase recognizes and binds to elements within the ITRs, catalyzing the excision of the transposon from its original location and its integration into another location in the genome.
Currently, widely used transposon systems include Sleeping Beauty (SB) and piggyBac (PB). Non-viral transposon vectors offer versatility, low immunogenicity, and ease of production but often have lower transfection efficiency compared to viral vectors.
Non-viral transposition of mRNA is typically achieved through methods like electroporation or nanoparticle delivery. Once inside the cell and without the need to reach the nucleus, mRNA is translated into the encoded protein and is usually lost after 2-4 cell divisions. This approach circumvents the risk of genotoxicity associated with integrated vectors, making it a safe and viable strategy in CAR-T cell applications.
Sleeping Beauty Transposon
Emerging from a long evolutionary “slumber,” the SB, found in fish genomes, became the first transposon to exhibit activity in vertebrate cells, paving the way for new perspectives in gene therapy. Based on the classical Tc1/mariner DNA II-class TE, these “jumping” units can move from one genomic location to another through a cut-and-paste mechanism. SB vectors consist of two functional components: the transposon DNA carrying the gene of interest flanked by ITRs and the SB transposase, which recognizes ITR sequences and mobilizes the transgene from the donor DNA to a recipient site within the genome.
One significant advantage of this strategy over viral systems is its larger cargo capacity. The size of the insert is inversely related to transposition efficiency, with the optimal size not exceeding 6 kb. However, upgraded versions include two complete transposon units, allowing for a payload of up to 11 kb, thereby expanding the cloning capabilities of SB-based vectors. Additionally, when combined with bacterial artificial chromosomes (BACs), SB can deliver transgenes as large as 100 kb in human embryonic stem cells. In clinical applications, Cooper et al. were the first to use SB-engineered anti-CD19 CAR-T cells in clinical trials, confirming the safety of SB-engineered anti-CD19 CAR-T cells as adjunct therapy in 26 cases of B-ALL and non-Hodgkin lymphoma patients post-autologous or allogeneic hematopoietic stem cell transplantation (HSCT) in two clinical trials (NCT00968760, NCT01497184).
Furthermore, ongoing research in the United States and Europe employs the SB platform for CAR-T studies. The UltraCAR-T platform utilizes non-viral systems via the SB vector to deliver multiple genes. This platform is being used for autologous cells targeting CD33 CAR and mbIL15 (PRGN-3006) in the treatment of r/r acute myeloid leukemia and high-risk myelodysplastic syndrome (MDS). Currently, PRGN-3006 is being evaluated in a dose-escalation/expansion study (NCT03927261) for its safety in adult patients with r/r acute myeloid leukemia and high-risk MDS. Preliminary data show good tolerability of PRGN-3006 infusions and a 50% response rate in patients who received lymphodepleting conditioning, correlating with CAR-T cell expansion and persistence.
Similar to the SB vector, the PB system consists of PB transposase (PBase) in the form of mRNA or DNA and a separate transfection plasmid carrying the gene of interest. PB transposon vectors are characterized by a single open reading frame (ORF) flanked by ITRs on both sides, with characteristic asymmetry in PB ITRs. The transposase recognizes ITRs on both sides of the transposon and integrates the transgene into the genome DNA through a cut-and-paste mechanism. PB exhibits higher transposon mobilization activity in mammalian cells than SB, has a larger cargo capacity (up to 14 kb), and allows for the delivery of multiple transgenes through the design of multiple flip-in cassettes.
Increasing preclinical data support the feasibility and safety of PB-based CAR-T manufacturing platforms, allowing this system to advance into clinical trials.
The CARTELL trial in Australia is a phase I clinical study (ACTRN12617001579381) investigating the efficacy and safety of donor-derived anti-CD19 CAR-T cells obtained through the PB transposon system.
Early results indicate comparable activity to anti-CD19 CAR-T cells generated using high-efficiency viral vectors.
Additionally, two phase I studies are ongoing in Japan (UMIN000030984) and China (NCT04289220
) investigating the feasibility and safety of anti-CD19 CAR-T cells manufactured using the PB system.
In the Japanese study, to date, no patients have displayed dose-limiting toxicities, and one patient has shown sustained B-cell aplasia for nine months.
While transposon-engineered CAR-T cells are still in their early stages of clinical trials, some have demonstrated clinical efficacy. Anti-BCMA CAR-T cells (P-BCMA-101), designed via the PB platform, improved transposition during the manufacturing process, including the use of nanoparticles to reduce the size of the scaffold and bring ITRs closer. The resulting cell product displayed a high composition of memory stem T cells (TSCM). Ninety patients with r/r multiple myeloma received P-BCMA-101 treatment, with early results showing an overall response rate (ORR) of 57% in the dose-escalation cohort and an ORR of 73% when combined with lenalidomide (NCT03288493), with low toxicity.
Over 30 years ago, pioneering research by Malone demonstrated that RNA mixed with lipids could be taken up by human cells and translated into proteins.
Since then, RNA has been used in various aspects of genetic engineering, including restoring the functional expression of mutated genes, knocking down genes for silencing, modifying cell phenotypes, or encoding antigens.
Successful protein expression from RNA depends on its stability and translational efficiency, which are determined by cis-acting elements such as the 5′ cap structure, polyA tail, the composition of the coding sequence, and potential non-coding regions at the 5′ and 3′ ends of the molecule.
RNA is suitable for various cell transfection methods, including electroporation, cationic lipids, and cationic polymers.
There have been many in vitro and preclinical studies, using mRNA to introduce CARs into T cells, tested in model systems of both hematological and solid tumors.
While mRNA-based therapies have shown promise in reducing off-target effects, lowering toxicity, and alleviating integration-related safety concerns, transient protein expression remains a drawback in these applications.
Another method for genetically modifying lymphocytes is transfecting the gene of interest in DNA form along with RNA encoding the transposase. Encoding the transposase as mRNA has several advantages when co-delivered with DNA carrying the target gene. First, the transient expression of the transposase reduces the rate of secondary transposon excisions and reinsertions caused by cut-and-paste events. Second, this approach allows for precise titration of the ratios between SB mRNA and CAR DNA for sustained integration.
The potential safety advantage of transient CAR expression from mRNA could provide lower toxicity in hematological and solid tumor settings.
Clinical studies have been conducted with mRNA-based CAR-T cells targeting CD123 and CD19 in hematological malignancies and mesothelin and c-Met in solid tumors.
Although existing reports from these studies show that mRNA CAR-T cells are generally safe, with few severe adverse events, a common theme is the need for repeated dosing with high concentrations.
The requirement for multiple high-dose infusions of mRNA CAR-T cells is likely related to the lack of persistence of transgene-expressing cells, designed to prolong the duration of activity in these patients, but repeated dosing may introduce additional complications.
The Future of Non-Viral CAR-T Cell Therapy
To address future challenges, in addition to transposon platforms, another non-viral tool in gene engineering is nanocarriers.
The use of nanocarriers is emerging as a potential solution to overcome current obstacles in gene delivery, such as toxicity and low transfection efficiency. In the latest advances in nanotechnology, one of the forefront discoveries by Bozza et al. involves the development of non-integrating DNA nanocarriers capable of generating active CAR-T cells both in vitro and in vivo.
This platform is devoid of viral components, can undergo additional chromosome replication within dividing cells, and maintains sustained transgene expression without integration, all while retaining the advantages of non-viral carriers: non-immunogenicity, simplicity, multifunctionality, and low production costs.
On the other hand, progress is being made in improving CAR-T cell separation, activation, and gene modification using newly developed biomaterials.
One example is the use of synthetic DNA aptamers and complementary reverse technology, allowing the isolation of highly pure and high-yield unlabeled CD8+ T cells from PBMCs in a single separation step.
The main advantage of this approach is the ability to isolate multiple distinct T-cell populations in a single separation step using aptamers with different specificities.
Gene editing and targeted knockout rely on host DNA double-strand break (DSB) repair and homology-directed repair (HDR) processes.
HDR typically occurs at a low frequency in primary cells and is limited to small transgenes, resulting in low transfection efficiency. To deliver DNA inserts more efficiently, CRISPR/Cas9 has recently been combined with transposons to enhance the efficiency of RNA-guided integration using transposase catalysis.
Experiments have been conducted to combine CRISPR/Cas9 with the SB transposon, showing promise for future non-viral clinical applications. Identifying and mitigating the risk of genomic rearrangements and translocations may allow for further development of gene editing in immunotherapy.
In Conclusion: Will Non-Viral CAR-T Cells be used to treat more diseases in future?
Successful CAR-T cell therapies to date have been associated with T cells engineered using viral vectors.
However, relapse, the complexity of the manufacturing process, and the application of these technologies in diseases, including solid tumors, require more sophisticated designs and gene transfer techniques to address these challenges.
To fill these gaps, non-viral technologies are continually advancing, and it is certain that we will see more applications of these techniques in CAR-T cell therapy in the near future.
1. The Past, Present, and Future of Non-Viral CAR T Cells. Front Immunol. 2022; 13: 867013.
Will Non-Viral CAR-T Cells be used to treat more diseases in future?
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