Functional differences between protamine preparations for the transfection of mRNA

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Abstract

Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20–1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.

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In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies.

Ingmar Hoerr, Reinhard Obst, Hans-Georg Rammensee … (2000)

To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.

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Immunostimulating capacities of stabilized RNA molecules.

Günther Jung, Hans-Georg Rammensee, Jean-Philippe Carralot … (2004)

Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP.

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Novel multi-peptide vaccination in Hla-A2+ hormone sensitive patients with biochemical relapse of prostate cancer.

JENS BEDKE, Klaus Dietz, Dorothee Wernet … (2009)

A phase I/II trial was conducted to assess feasibility and tolerability of tumor associated antigen peptide vaccination in hormone sensitive prostate carcinoma (PC) patients with biochemical recurrence after primary surgical treatment. Nineteen HLA-A2 positive patients with rising PSA without detectable metastatic disease or local recurrence received 11 HLA-A*0201-restricted and two HLA class II synthetic peptides derived from PC tumor antigens subcutaneously for 18 months or until PSA progression. The vaccine was emulgated in montanide ISA51 and combined with imiquimod, GM-CSF, mucin-1-mRNA/protamine complex, local hyperthermia or no adjuvant. PSA was assessed, geometric mean doubling times (DT) calculated and clinical performance monitored. PSA DT of 4 out of 19 patients (21%) increased from 4.9 to 25.8 months during vaccination. Out of these, two patients (11%) exhibited PSA stability for 28 and 31 months which were still continuing at data cut-off. One patient showed no change of PSA DT during vaccination but decline after the therapy. Three patients had an interim PSA decline or DT increase followed by DT decrease compared to baseline PSA DT. Three of the responding patients received imiquimod and one the mucin-1-mRNA/protamine complex as adjuvant; both are Toll-like receptor-7 agonists. Eleven (58%) patients had progressive PSA values. The vaccine was well tolerated, and no grade III or IV toxicity occurred. Multi-peptide vaccination stabilized or slowed down PSA progress in four of 19 cases. The vaccination approach is promising with moderate adverse events. Long-term stability delayed androgen deprivation up to 31 months. TLR-7 co-activation seems to be beneficial.

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Journal

Journal ID (nlm-ta): Drug Deliv

Journal ID (iso-abbrev): Drug Deliv

Title: Drug Delivery

Publisher: Taylor & Francis

ISSN (Print): 1071-7544

ISSN (Electronic): 1521-0464

Publication date (Electronic, pub): 17 August 2020

Publication date (Electronic, collection): 2020

Volume: 27

Issue: 1

Pages: 1231-1235

Affiliations

[a ]Department of Dermatology, University Hospital of Zürich , Zürich, Switzerland

[b ]Faculty of Science, University of Zürich , Zürich, Switzerland

[c ]Faculty of Medicine, University of Zürich , Zürich, Switzerland

[d ]Department of Dermatology and Allergy, University Hospital, LMU Munich , Munich, Germany

[e ]Lausanne University Hospital (CHUV) , Lausanne, Switzerland

[f ]Faculty of Biology and Medicine, University of Lausanne , Lausanne, Switzerland

Author notes

Supplemental data for this article can be accessed here .

CONTACT Steve Pascolo steve.pascolo@ 123456usz.ch University Hospital of Zürich , Department of Dermatology, Gloriastrasse 31, Zürich8091, Switzerland

Article

Publisher ID: 1790692

DOI: 10.1080/10717544.2020.1790692

PMC ID: 7470126

PubMed ID: 32804028

SO-VID: 8bcbbb41-7071-4469-9857-8c338cbd47a0

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Functional differences between protamine preparations for the transfection of mRNA

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Abstract

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RNA drug delivery

Most cited references 20

In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies.

Immunostimulating capacities of stabilized RNA molecules.

Novel multi-peptide vaccination in Hla-A2+ hormone sensitive patients with biochemical relapse of prostate cancer.

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