Innate Immunity and Cell Death

March 22, 2023 / by Jim Williams, Ph.D.

The bane of non-viral cell and gene therapy

In my previous discussions on the benefits of NanoplasmidTM vectors, I touched on eliminating protein and antibiotic markers and the advantage of the RNA-OUT marker system, as well as structured DNA repeats and the plasmid replication challenge.

As part of an ongoing Nanoplasmid discussion, this post will touch on the functional issues with canonical plasmid backbones. It has been discovered that these drawbacks manifest themselves after vector transfection into target cells. These include vector backbone mediated transgene silencing leading to vector inactivation, and metabolic perturbation resulting in cellular toxicity. References as noted are listed at the end of this post.

The first functional limitation of canonical plasmid vectors that was characterized in 2004 is bacterial backbone-mediated promoter inactivation¹. Subsequently, various investigators identified bacterial backbone-mediated cellular toxicity that dramatically reduces cell viability after ex vivo transfection of primary cells for cell therapy.

Lu et al in 2012² demonstrated that bacterial regions ≥1 kb resulted in transgene expression silencing in vivo, while shorter spacers ≤500 bp exhibited prolonged transgene expression. This work showed that prolonged duration transgene expression in gene and cell therapy vectors can be maintained with ≤500 bp bacterial backbone vectors.

Cellular toxicity is also dramatically reduced with ≤500 bp bacterial backbone vectors, which improves payload genome integration with transposon vectors³ and increases gene editing frequency using a CRISPR HDR template vector⁴.

Nanoplasmid platform benefits
Nanoplasmid vectors have a ≤500 bp bacterial backbone. Nanoplasmid vectors combine the RNA-OUT antibiotic free marker with a specialized bacterial R6K replication origin in place of the traditional pUC replication origin. Nanoplasmids can be produced in high yield and quality in bacterial fermentation.

Benefit #1: Reduced vector inactivation
Nanoplasmid vectors can exhibit prolonged transgene expression and up to 10-fold higher overall transgene expression than plasmid vectors after in vitro or in vivo delivery⁵, ⁶. Gene silencing is hypothesized to be a consequence of vector inactivation through heterochromatin formation on large >1 kb untranscribed plasmid backbones⁷, ⁸; we hypothesize that higher overall expression level with Nanoplasmid vectors is due to reduced heterochromatinization mediated vector inactivation leading to increased nuclear localization of ‘non-inactivated’ vectors.

Benefit #2: Reduced transfection toxicity
Some large plasmid backbone mediated toxicity is potentially due to heterochromatin mediated ‘danger signal’ innate immunity activation and cellular perturbation by inactivated transfected plasmid DNA. Consistent with this hypothesis, reduced cellular perturbation at the transcriptome level have been reported after in vitro transfection of Nanoplasmid vectors compared to plasmid⁹, ¹⁰ or Lentiviral vectors¹¹.

Superior cell viability and gene integration frequencies, compared to plasmid comparators, has been reported for non-viral CAR T cell manufacturing using Nanoplasmid PiggyBac transposon vectors¹², or Nanoplasmid Homology-Directed Repair template vectors for CRISPR/Cas9 mediated gene editing¹³.

For PiggyBac modified CAR T cells, the improved transposition efficiency and reduced toxicity with Nanoplasmid compared to plasmid resulted in a CAR-T product with shorter manufacturing timelines and increased percentage of desirable Tscm cells with superior efficacy and survival in animal models.

CAR T cells produced with a Nanoplasmid vector in the P-BCMA-101 clinical trial NCT03288493 showed robust expansion curves, increased Overall Response Rate and increased depth of response compared to CAR-T cells manufactured with a plasmid vector¹². This improved cell therapy performance with Nanoplasmid vectors is likely due to the benefits of reduced cellular toxicity combined with reduced vector inactivation resulting in increased gene integration into healthier cells.

The bottom line
Nanoplasmid vector technology and selection methods can offer significant advantages over traditional plasmid DNA. At only 500 bp, the Nanoplasmid bacterial backbone is engineered to reduce plasmid-associated transfection toxicity and payload silencing. Furthermore, dramatically improved transgene expression allows minimization of the DNA delivery demand, further lessening observed toxicity and vector inactivation during cell transfection. This efficiency speeds manufacturing and increases yields in time-sensitive applications.

Nanoplasmids, with the combined advantages of antibiotic-free selection and high transgene expression, are a smaller plasmid vector that performs better for a range of clinical applications than traditional plasmid. The platform is highly beneficial for a wide variety of cell and gene therapy applications.

• Want to learn more? Contact Jim Williams with your questions
• Visit the Aldevron Nanoplasmids page
• Download our whitepaper, Next Generation Plasmid Technology: Improving Performance Safety and Manufacturing for Today's Therapies
• Watch our video to learn more about Nanoplasmid’s RNA-OUT antibiotic free marker
• Download our Nanoplasmid brochure
• Have a topic to suggest? Let us know

References

1. Chen, Z. Y.; He, C. Y.; Meuse, L.; Kay, M. A. Silencing of Episomal Transgene Expression by Plasmid Bacterial DNA Elements in Vivo. Gene Ther 2004, 11 (10), 856–864. https://doi.org/10.1038/SJ.GT.3302231.
2. Lu, J.; Zhang, F.; Xu, S.; Fire, A. Z.; Kay, M. A. The Extragenic Spacer Length between the 5’ and 3’ Ends of the Transgene Expression Cassette Affects Transgene Silencing from Plasmid-Based Vectors. Mol Ther 2012, 20 (11), 2111–2119. https://doi.org/10.1038/MT.2012.65.
3. Monjezi, R.; Miskey, C.; Gogishvili, T.; Schleef, M.; Schmeer, M.; Einsele, H.; Ivics, Z.; Hudecek, M. Enhanced CAR T-Cell Engineering Using Non-Viral Sleeping Beauty Transposition from Minicircle Vectors. Leukemia 2017, 31 (1), 186–194. https://doi.org/10.1038/LEU.2016.180.
4. Danner, E.; Lebedin, M.; de La Rosa, K.; Kühn, R. A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease. Open Biol 2021, 11 (1). https://doi.org/10.1098/RSOB.200283.
5. Boye, C.; Arpag, S.; Burcus, N.; Lundberg, C.; DeClemente, S.; Heller, R.; Francis, M.; Bulysheva, A. Cardioporation Enhances Myocardial Gene Expression in Rat Heart. Bioelectrochemistry 2021, 142. https://doi.org/10.1016/J.BIOELECHEM.2021.107892.
6. Lu, J.; Williams, J. A.; Luke, J.; Zhang, F.; Chu, K.; Kay, M. A. A 5’ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in Vivo. Hum Gene Ther 2017, 28 (1), 125–134. https://doi.org/10.1089/HUM.2016.140.
7. Riu, E.; Chen, Z. Y.; Xu, H.; He, C. Y.; Kay, M. A. Histone Modifications Are Associated with the Persistence or Silencing of Vector-Mediated Transgene Expression in Vivo. Mol Ther 2007, 15 (7), 1348–1355. https://doi.org/10.1038/SJ.MT.6300177.
8. Suzuki, M.; Kasai, K.; Saeki, Y. Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin. J Virol 2006, 80 (7), 3293–3300. https://doi.org/10.1128/JVI.80.7.3293-3300.2006.
9. Bozza, M.; Green, E. W.; Espinet, E.; de Roia, A.; Klein, C.; Vogel, V.; Offringa, R.; Williams, J. A.; Sprick, M.; Harbottle, R. P. Novel Non-Integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models. Mol Ther Methods Clin Dev 2020, 17, 957–968. https://doi.org/10.1016/J.OMTM.2020.04.017.
10. Roig-Merino, A.; Urban, M.; Bozza, M.; Peterson, J. D.; Bullen, L.; Büchler-Schäff, M.; Stäble, S.; van der Hoeven, F.; Müller-Decker, K.; McKay, T. R.; Milsom, M. D.; Harbottle, R. P. An Episomal DNA Vector Platform for the Persistent Genetic Modification of Pluripotent Stem Cells and Their Differentiated Progeny. Stem Cell Reports 2022, 17 (1), 143–158. https://doi.org/10.1016/J.STEMCR.2021.11.011.
11. Bozza, M.; de Roia, A.; Correia, M. P.; Berger, A.; Tuch, A.; Schmidt, A.; Zörnig, I.; Jäger, D.; Schmidt, P.; Harbottle, R. P. A Nonviral, Nonintegrating DNA Nanovector Platform for the Safe, Rapid, and Persistent Manufacture of Recombinant T Cells. Sci Adv 2021, 7 (16). https://doi.org/10.1126/SCIADV.ABF1333.
12. Ostertag, E. Manufacturing Matters in CAR-T: Small Changes Can Have a Big Impact.
13. Oh, S. A.; Senger, K.; Madireddi, S.; Akhmetzyanova, I.; Ishizuka, I. E.; Tarighat, S.; Lo, J. H.; Shaw, D.; Haley, B.; Rutz, S. High-Efficiency Nonviral CRISPR/Cas9-Mediated Gene Editing of Human T Cells Using Plasmid Donor DNA. J Exp Med 2022, 219 (5). https://doi.org/10.1084/JEM.20211530.