Controlled Transcription of Exogenous mRNA in Platelets Using Protocells

Abstract

Transcribing exogenous RNA in eukaryotic cells requires delivering DNA to their nuclei and changing their genome. Nuclear delivery is often inefficient, limiting the potential scope of gene therapy and synthetic biology. These challenges may be overcome by techniques that allow for extranucleate transcription within eukaryotic cells. Protocells have been developed that enable transcription inside of liposomes; however, it has not yet been demonstrated whether this technology can be extended for use within eukaryotic cells. Here we show RNA-synthesizing nanoliposomes allow transcription of exogenous RNA inside anucleate cells. To accomplish this, components of transcription were encapsulated into liposomes and delivered to platelets. These liposomes were capable of light-induced transcription in platelets, providing proof-of-concept that protocell technology can be adapted for use within mammalian cells.

Platelets loaded with liposome‐encapsulated thrombin have increased coagulability

“…Washed platelets were loaded with liposomes as previously described, with minor modifications . Briefly, 4.3 μ m of human alpha thrombin or DFP active site blocked human alpha thrombin (Haematologic Technologies Inc. Essex Junction, VT, USA) in Tris‐buffered saline (TBS) was added to a 5‐mg lipid film, consisting of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine:cholesterol:1,2‐dioleoyl‐sn‐glycero‐3‐ phosphoethanolamine (DOPC:chol:DOPE; 23:23:4; Avanti Polar Lipids, Alabaster, AL, USA, cholesterol from Sigma, Oakville, Canada), and extruded five times through a 200‐nm filter using a LIPEX extruder (Northern Lipids Inc., Burnaby, Canada).…”

“…Purified liposomes were added to isolated platelets (1:100 v/v) and incubated at room temperature for 1 h with gentle agitation, in order for uptake to occur. The uptake of liposomes into platelets was confirmed as previously described . To remove excess liposomes, platelets and liposomes were centrifuged at 250 × g for 5 min and the supernatant was removed.…”

“…These studies demonstrated that enhancing platelet coagulability could improve overall hemostasis in vivo ; however, clinical use of this technology would currently require bone marrow transplantation, which is not appropriate for cases of acute hemorrhage. Previously, we have modified platelet function by loading platelets with transcriptional machinery, allowing anucleate platelets to transcribe exogenous RNA . This was achieved by encapsulating several biomolecules into liposomes, including a functional enzyme.…”

Communicating artificial cells

“…Alternatively, controlled RNA synthesis can be achieved inside of a delivered artificial cell. Although not used for vaccine development, artificial cells carrying T7 RNA polymerase and caged nucleotides have been shown to synthesize RNA in a light controlled manner inside of anucleated cells, such as platelets [72].…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Optimization of photo‐induced transcription of mRNA in liposomes is accomplished by incorporating PEG within the liposome, which removes DNA and unencapsulated T7RNAP through a column of anionic exchange. Hence, extranuclear transcription engineering in mammalian cells to express exogenous RNA, without genome modification, enlightens the applicability of such technology in gene therapy, RNAi‐based therapeutics, or generally molecular biology (Chan, Novakowski, Law, Klein‐Bosgoed, & Kastrup, 2015).…”