Synthetic Messenger RNA as a Tool for Gene Therapy

Rabinovich, Peter M.; Komarovskaya, Marina; Ye, Zhijia; Imai, Chihaya; Campana, Dario; Bahceci, Erkut; Weissman, Sherman M.

doi:10.1089/hum.2006.17.1027

Cited by 63 publications

(48 citation statements)

References 25 publications

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“…2 Only two reports have been published, in which the effect of ARCA on protein expression in DCs was analyzed in comparison with m 7 GpppG. 23,24 By analyzing the effect of the different cap analogs on the half-life and translational efficiency of RNAs in human immature and mature DCs, as well as antigen-specific T cell response on vaccination with RNA in mice to enable read-outs as close as possible to the desired clinical effects, we made several surprising findings.…”

Section: Discussionmentioning

confidence: 99%

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

et al. 2010

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Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5 0 mRNA cap analogs on the kinetics of the encoded protein, we found that m 2 7,2 0 ÀO Gpp S pG (b-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as b-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5 0 mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

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Section: Discussionmentioning

confidence: 99%

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

et al. 2010

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“…This approach has been successfully tested in preclinical trials to develop melanoma vaccines (Kyte et al 2005). Since RNAs do not carry the risk of insertional mutagenesis, another promising application for synthetic mRNAs is gene therapy, for example, introducing a chimeric immune receptor into T-lymphocytes (Rabinovich et al 2006). These observations provide strong motivation to increase protein yields from transfected mRNAs.…”

Section: Mgmentioning

confidence: 99%

Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells

Grudzien-Nogalska

Jemielity²,

Kowalska³

et al. 2007

RNA

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Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the a-, b-, or g-phosphate moieties, m 2 7,29-O Gppp S G, m 2 7,29-O Gpp S pG, and m 2 7,29-O Gp S ppG, respectively. The new compounds were also modified at the 29-O position of the m 7 Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the a-and b-phosphates. Gp 3 G. The greater yield of protein due to combining higher translational efficiency with longer t 1/2 of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.

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“…These cells were grown in medium supplemented with Zeocin (0.2 mg=ml) (Cooper et al, 2005). Polymerase chain reaction DNA templates were made by polymerase chain reaction (PCR) according to the protocol we have described (Rabinovich et al, 2006). Gene amplification was performed with AccuPrime Pfx DNA polymerase (Invitrogen) according to the manufacturer's protocol.…”

Section: Cellsmentioning

confidence: 99%

“…mRNA synthesis with T7 RNA polymerase has been described (Rabinovich et al, 2006). This was performed with an mMESSAGE mMACHINE T7 Ultra kit (Ambion, Austin, TX), using the procedure recommended by the manufacturer.…”

Section: Rna Synthesismentioning

confidence: 99%

“…We developed a fast and effective approach for in vitro synthesis of mRNA that can be transferred into human lymphocytes. RNA electroporation produces a uniform level of expression in lymphocytes and permits simultaneous coexpression of several mRNA transgenes (Rabinovich et al, 2006). In this paper, we report our investigation of various types of lymphocytes in the targeting of hematological and solid tumor cells.…”

Section: Introductionmentioning

confidence: 99%

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Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

et al. 2009

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mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgenemediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3 + CD8 + , CD3 + CD4 + , and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3 + CD8 + lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

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Synthetic Messenger RNA as a Tool for Gene Therapy

Cited by 63 publications

References 25 publications

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells

Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

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