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Multiplexed Circular RNA Delivery for extended expression without integration

Group 4 (1)

Who this is for:

  • Cell therapy groups looking for alternatives to permanent genetic modifications
  • R&D groups looking to over-express a gene of interest for long-periods of time in primary cells, without generating cell lines

Opportunities

  • Extended Expression: mRNA degrades quickly while circular RNA can express for over 5 days
  • No genetic modifications: Circular RNA allows for extended biological impact without impacting the genome
  • No Modified Bases: Mechanoporated circular RNA does not need base modifications for robust expression
  • Multiplexed Engineering: Mechanoporation allows for co-delivery of multiple factors and control over ratio in the cell

Results Obtained

  • Extended Expression of circular RNA constructs vs mRNA in primary cells
  • Enhanced CAR T Mediated Killing in T Cells Mechanoporated with Circular RNA

How We Did It

Portal uses mechanoporation to deliver virtually any cargo to diverse cell types while maintaining cell health. This enables intracellular delivery of impermeable molecules without the complications of electroporation or other delivery methods. Learn more about mechanoporation here! 

Robust Circular RNA Expression in PBMCs and T Cells

Two proof of concept experiments for cRNA in PBMCs and T cells were performed to show robust expression over several days. The first experiment compared GFP expression in PBMCs between cRNA and mRNA (Fig 1). The second experiment boosted T cells with mbIL2 cRNA and compared expression between days one, two, and five (Fig 2).

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Fig 1: GFP cRNA expression is more intense compared to mRNA and lasts longer, up to 10 days following boost in primary human PBMCs

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Fig 2: mbIL2 cRNA expression lasts five plus days following boost of T cells.

T cells Engineered with Simultaneous circRNA Delivery for CD19 CAR and mbIL-12

In a proof of concept study to demonstrate a potential use case for cRNA mechanoporation, we designed cRNA transcripts for a CD19 CAR and two enhancing factors, membrane-bound IL-2 (mbIL-2) and mbIL-12. When boosting CD19 and mbIL-12 together, we see strong codelivery and expression of CD19 CAR with mbIL-12 (Fig 3). Following this combined boost, the cells demonstrated efficient killing >90% at a E:T ratio of 3:1 (Fig 4).

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Fig 3: Flow cytometry was conducted 48h post-boost to assess expression of CD19 CAR and mbIL-12
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Fig 4:  In an experiment where T cells were boosted with mbIL-12 and CD19 CAR cRNA, we observe high correlation of expression of both transcripts and corresponding killing activity in a cytotoxicity assay

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