Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing

Marshall, Nikki B.; Oda, Shannon K.; London, Carla A.; Moulton, Hong M.; Iversen, Patrick L.; Kerkvliet, Nancy I.; Mourich, Dan V.

doi:10.1016/j.jim.2007.06.009

Cited by 28 publications

(26 citation statements)

References 49 publications

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“…In addition, a cell penetrating peptide was conjugated to each of the oligomers (PPMOs). The conjugated-peptide readily delivers PMO into primary murine dendritic, macrophage, and T cells and effectively induces splice alterations in vitro with limited impact on cell viability (Marshall et al, 2007). Antisense oligomers designations are given as SD or SA and then the exon targeted (e.g., SA2 targets the splice acceptor site of exon 2).…”

Section: Resultsmentioning

confidence: 99%

“…Surface staining and CFSE dilution were examined by flow cytometry gating on live cells using 7-aminoactinomycin D (7-AAD) (Calbiochem). Intracellular cytokine staining (ICS) was conducted as previously described (Marshall et al, 2007). Data were acquired using Beckman Coulter FC-500 and analyzed using Winlist (Verity) or FlowJo (Tree Star).…”

Section: Flow Cytometrymentioning

confidence: 99%

“…While testing the uptake properties of various argininerich PPMO, we had made the prior observation that delivery was enhanced in T cells following activation stimulus (Marshall et al, 2007). However, those previous data were generated utilizing a fluorescent conjugated PPMO.…”

Section: Activation Dependency Of Ppmo Antisense Activity Of In T Celmentioning

confidence: 99%

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Alternative Splice Forms of CTLA-4 Induced by Antisense Mediated Splice-Switching Influences Autoimmune Diabetes Susceptibility in NOD Mice

Mourich

Oda

Schnell

et al. 2014

Nucleic Acid Therapeutics

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Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.

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Section: Resultsmentioning

confidence: 99%

Section: Flow Cytometrymentioning

confidence: 99%

See 1 more Smart Citation

Alternative Splice Forms of CTLA-4 Induced by Antisense Mediated Splice-Switching Influences Autoimmune Diabetes Susceptibility in NOD Mice

Mourich

Oda

Schnell

et al. 2014

Nucleic Acid Therapeutics

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show abstract

“…However, they possess multiple positive charges at physiological pH as they are rich in basic cationic amino acids such as arginine, lysine, histidine or proline 97,100 and some of them share common features, including important theoretical hydrophobicity and helical moment (reflecting the peptide amphipathicity), the ability to interact with lipid membranes and to adopt a significant secondary structure on binding to lipids 97 .…”

Section: Cell Penetrating Peptides For Targeting Anticancer Dugsmentioning

confidence: 99%

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2013

IJPSR

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“…25 Previously, PMOs have been used to alter gene expression in primary leukocytes, however, for this the cellular membranes needed to be physically or chemically perturbed to facilitate delivery, which can non-specifically decrease cell viability. [26][27][28] We have demonstrated that PMO conjugated with cell-penetrating peptides rich in arginine specifically alter gene expression in cultures of primary murine leukocytes, 29 and in a model of murine skin hypersensitivity, peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) was effective in targeting activated T cells, 30 however, the delivery of the PPMO to human leukocytes is not well characterized. In this study, we assessed whether an arginine-rich (RXR) 4 , cell-penetrating, PPMO targeting FOXP3 would inhibit human T reg function and enhance induction of effector T-cell responses.…”

Section: Introductionmentioning

confidence: 99%

Depleting regulatory T cells with arginine-rich, cell-penetrating, peptide-conjugated morpholino oligomer targeting FOXP3 inhibits regulatory T-cell function

et al. 2011

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CD4 þ CD25 þ regulatory T cells (T reg ) impair anti-tumor and anti-viral immunity. As there are higher T reg levels in cancer patients compared with healthy individuals, there is considerable interest in eliminating them or altering their function as part of cancer or viral immunotherapy strategies. The scurfin transcriptional regulator encoded by the member of the forkhead winged helix protein family (FOXP3) is critical for maintaining the functions of T reg . We hypothesized that targeting FOXP3 expression with a novel arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino (PPMO) based antisense would eliminate T reg and enhance the induction of effector T-cell responses. We observed that the PPMO was taken up by activated T cells in vitro and could downregulate FOXP3 expression, which otherwise increases during antigen-specific T-cell activation. Generation of antigenspecific T cells in response to peptide stimulation was enhanced by pre-treatment of peripheral blood mononuclear cells with the FOXP3-targeted PPMO. In summary, modulation of T reg levels using the FOXP3 PPMO antisense-based genomic strategy has the potential to optimize immunotherapy strategies in cancer and viral immunotherapy.

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Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing

Cited by 28 publications

References 49 publications

Alternative Splice Forms of CTLA-4 Induced by Antisense Mediated Splice-Switching Influences Autoimmune Diabetes Susceptibility in NOD Mice

Alternative Splice Forms of CTLA-4 Induced by Antisense Mediated Splice-Switching Influences Autoimmune Diabetes Susceptibility in NOD Mice

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Depleting regulatory T cells with arginine-rich, cell-penetrating, peptide-conjugated morpholino oligomer targeting FOXP3 inhibits regulatory T-cell function

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