RNA-BASED LOGIC CIRCUITS WITH RNA BINDING PROTEINS, APTAMERS AND SMALL MOLECULES
Engineered synthetic RNA-based genetic circuits are provided that are regulated exclusively at the post-transcriptional level.
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This application is a divisional of U.S. application Ser. No. 15/509,258, filed Mar. 7, 2017, which is national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2015/049045, filed Sep. 8, 2015, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application 62/047,137, entitled “RNA-BASED LOGIC CIRCUITS WITH RNA BINDING PROTEINS, APTAMERS AND SMALL MOLECULES,” filed Sep. 8, 2014 and of U.S. provisional application 62/195,747, entitled “RNA-BASED LOGIC CIRCUITS WITH RNA BINDING PROTEINS, APTAMERS AND SMALL MOLECULES,” filed Jul. 22, 2015, the entire disclosures of each of which are herein incorporated by reference in their entireties.
FEDERALLY SPONSORED RESEARCHThis invention was made with government support under W911NF-11-2-0054 awarded by the Army Research Office (ARO). The government has certain rights in the invention.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEBThis application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 3, 2022, is named M065670359US03-SEQ-GIC and is 30,394 bytes in size.
FIELD OF INVENTIONEngineered synthetic RNA-based genetic circuits are provided that are regulated exclusively at the post-transcriptional level.
BACKGROUND OF INVENTIONMessenger RNA (mRNA) as a platform for gene transfer has numerous advantages over plasmid DNA including the lack of requirement for crossing the nuclear envelope, and importantly, negligible risk of genomic integration (1-2). The recent progress in development of chemical mRNA modifications made it possible to use in vitro synthesized mRNA with high stability and low immunogenicity as a powerful tool for gene therapy (3-6). Self-replicating RNA is also gaining interest for biomedical applications (7-8).
However, synthetic biology has remained DNA-centered and genetic circuit design always relies exclusively or partially on transcriptional regulation. The development of parts and devices has also been focused primarily on promoter and transcription factor libraries (9-10).
SUMMARY OF INVENTIONThe promise of synthetic biology is that the engineered genetic circuits will provide sophistication of output control that can never be achieved with traditional pharmaceuticals. Encoding the regulation exclusively at post-transcriptional level and RNA delivery of desired logic circuits may enable the benefits of synthetic biology tools while offering the safety of non-DNA therapeutics. However, no control mechanisms have been developed to regulate replicon-based expression. While there have been a number of efforts to engineer post-transcriptional devices based on microRNA, aptamers, or aptazymes (11), most are characterized by a very low dynamic range and importantly, the devices are not suitable for construction of scalable circuits.
Devices based on RNA-binding proteins (RBPs), however, can be easily wired together to create synthetic circuits of various complexities or to interconnect cellular and synthetic signaling pathways.
According to one aspect, synthetic RNA circuits are provided. The circuits include a first RNA molecule comprising at least one sequence recognized by at least one first microRNA that is/are specifically expressed in a first cell type, and a sequence encoding a protein that specifically binds to a RNA motif and inhibits protein production; and a second RNA molecule comprising at least one sequence recognized by at least one second microRNA that is/are not expressed in the first cell type or is expressed at a low level relative to a second cell type, at least one RNA motif and a sequence encoding an output molecule.
In some embodiments, in a cell that expresses the at least one first microRNA but does not express the at least one second microRNA, the at least one first microRNA represses translation of or degrades the sequence encoding the protein that specifically binds to a RNA motif and inhibits protein production, thereby allowing expression of the output molecule.
In some embodiments, the output molecule is a protein. In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the first cell type is a cancer cell. In some embodiments, the at least one first microRNA is miR-21. In some embodiments, the at least one second microRNA is selected from the group consisting of miR-141, miR-142 and miR-146.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, synthetic RNA circuits are provided. The circuits include a first RNA molecule comprising at least one sequence recognized by a protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule; a second RNA molecule comprising at least one sequence recognized by a second protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production; and a third RNA molecule comprising at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the second protein that specifically binds to a RNA motif and inhibits protein production. In some embodiments, the circuits further include the siRNA molecule or microRNA molecule that binds to the third RNA molecule. In some embodiments, the siRNA molecule is a synthetic siRNA molecule, or wherein the microRNA molecule is an endogenously expressed microRNA molecule.
In some embodiments, the output molecule is a protein. In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, synthetic RNA circuits are provided. The circuits include a first RNA molecule comprising at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule; and a second RNA molecule comprising at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production. In some embodiments, the circuits further include the siRNA molecule or microRNA molecule that binds to the second RNA molecule. In some embodiments, the siRNA molecule is a synthetic siRNA molecule, or wherein the microRNA molecule is an endogenously expressed microRNA molecule.
In some embodiments, the output molecule is a protein. In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, synthetic RNA circuits are provided. The circuits include a first RNA molecule comprising at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding a second protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a first siRNA molecule or microRNA molecule; and a second RNA molecule comprising at least one sequence recognized by the second protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a second siRNA molecule or microRNA molecule. In some embodiments, the circuits further include the siRNA molecule or microRNA molecule that binds to the third RNA molecule. In some embodiments, the siRNA molecule is a synthetic siRNA molecule, or wherein the microRNA molecule is an endogenously expressed microRNA molecule. In some embodiments, the first RNA molecule and/or the second RNA molecule further comprise a sequence encoding one or more output molecules that are not a protein that specifically binds to a RNA motif and inhibits protein production.
In some embodiments, the output molecule is a protein. In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, synthetic RNA circuits are provided. The circuits include an RNA molecule comprising a sequence encoding a destabilization domain fused to an output protein, wherein the destabilization domain facilitates degradation of the output protein in the absence of a small molecule that binds to the destabilization domain.
In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the methods further include administering the small molecule that binds to the destabilization domain to the mammal. In some embodiments, the small molecule that binds to the destabilization domain is administered at different times for expressing the antigen and/or the adjuvant at the different times. In some embodiments, the small molecule that binds to the destabilization domain is administered by oral administration, intramuscular injection of lipid nanoparticles, or by implantation of a polymeric implant for sustained release. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, synthetic RNA circuits are provided. The circuits include a first RNA molecule comprising a sequence encoding a destabilization domain fused to a protein that specifically binds to a RNA motif and inhibits protein production; and a second RNA molecule comprising at least one sequence recognized by the protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule. The destabilization domain facilitates degradation of the protein that specifically binds to a RNA motif and inhibits protein production in the absence of a small molecule that binds to the destabilization domain.
In some embodiments, the output molecule is a protein. In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the output molecule is a fusion of a TetR protein and a second protein; and the RNA molecule(s) further includes an aptamer sequence and a second output molecule. The aptamer sequence is bound by the TetR protein in the absence of tetracycline and the aptamer sequence is positioned relative to the second output molecule so that it suppresses translation of the second output molecule in the absence of tetracycline.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the methods further include administering the small molecule that binds to the destabilization domain to the mammal. In some embodiments, the small molecule that binds to the destabilization domain is administered at different times for expressing the antigen and/or the adjuvant at the different times. In some embodiments, the small molecule that binds to the destabilization domain is administered by oral administration, intramuscular injection of lipid nanoparticles, or by implantation of a polymeric implant for sustained release. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, synthetic RNA circuits are provided. The circuits include an RNA molecule comprising a sequence encoding a TetR protein and a sequence encoding an output protein, and an aptamer sequence that is bound by the TetR protein in the absence of tetracycline. The aptamer sequence is positioned relative to the sequence encoding the output protein so that it suppresses translation of the output protein in the absence of tetracycline. In some embodiments, the aptamer is positioned in the 5′ untranslated region (UTR) of the sequence encoding an output protein.
In some embodiments, the output molecule is a protein. In some embodiments, the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. In some embodiments, the therapeutic protein is a protein for protein replacement therapy, Myr-Akt, or follistatin. In some embodiments, the selection protein is used for selection or purification of a cell in which it is expressed. In some embodiments, the selection protein is a protein that confers drug resistance to a cell. In some embodiments, the fluorescent protein is EGFP, EYFP, or EBFP. In some embodiments, immunomodulator protein is a cytokine. In some embodiments, the cytokine is IL-12, IL-15 or IL-21. In some embodiments, the immunomodulator protein is a immunosuppressant protein. In some embodiments, the cell death protein is hBax.
In some embodiments, the RNA molecules encode more than one output molecule.
In some embodiments, the output molecules comprise at least one antigen, and optionally, one or more adjuvants.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae or a fusion of MS2 protein and a protein that inhibits protein production. In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is L7Ae and the RNA motif is one or more Box C/D, K-turn and/or K-loop motifs. In some embodiments, the RNA motif is two K-turn motifs. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the second RNA molecule.
In some embodiments, the protein that specifically binds to a RNA motif and inhibits protein production is a fusion of MS2 protein and a protein that inhibits protein production and the RNA motif is one or more MS2 coat protein binding sites. In some embodiments, the RNA motif is eight MS2 coat protein binding sites. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the second RNA molecule. In some embodiments, the MS2 fusion protein is a fusion of MS2 protein and CNOT7 protein (MS2-CNOT7) or Dm-POP2 protein (MS2-Dm-POP2).
In some embodiments, the RNA molecules comprise modified RNA. In some embodiments, the RNA molecules comprise 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate.
In some embodiments, the RNA molecules are encoded on one or more RNA replicons. In some embodiments, the one or more RNA replicons is/are one or more alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In some embodiments, the RNA molecules are expressed from one or more subgenomic promoters of the one or more replicons, optionally wherein the one or more subgenomic promoters are optimized for length or position in the RNA molecule. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule. In some embodiments, the small molecule is trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the RNA molecules are encoded on one or more plasmids.
In some embodiments, the synthetic RNA circuit further includes a sequence encoding Csy4 protein and a Csy4 recognition site. In some embodiments, the Csy4 protein is a fusion of a destabilization domain and Csy4. In some embodiments, the destabilization domain is regulated by trimethoprim (TMP) or 4-hydroxytamoxifin (4-OHT).
In some embodiments, the synthetic RNA circuit further includes one or more internal ribosomal entry sites (IRESs) for improved polycystronic expression. In some embodiments, the synthetic RNA circuit further includes one or more general translation enhancers (GTEs). In some embodiments, the synthetic RNA circuit is encoded on self-cleaving helper-defective interfering RNA, optionally comprising Csy4, wherein Csy4 is expressed from an internal ribosome entry site (IRES).
According to another aspect, methods of treating cancer in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
According to another aspect, methods of inducing an immune response in a mammal are provided. The methods include administering to a mammal the foregoing synthetic RNA circuits. In some embodiments, the methods further include administering tetracycline to the mammal. In some embodiments, the tetracycline is administered at different times for expressing the antigen and/or the adjuvant at the different times. In some embodiments, the tetracycline is administered by oral administration, intramuscular injection of lipid nanoparticles, or by implantation of a polymeric implant for sustained release. In some embodiments, the synthetic RNA circuit is administered as a first replicon, and further administering a second replicon as ballast to control expression of a protein encoded by the first replicon.
The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Each of the above embodiments and aspects may be linked to any other embodiment or aspect. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, not every component may be labeled in every drawing.
Methods are described herein for safe, programmable control of cell behavior, with minimal risk of harmful genomic integration, through synthetic regulatory circuits encoded exclusively on RNA. Towards the goal of a plug-and-play platform for RNA-encoded regulation several post-transcriptional circuits were created by wiring regulatory devices based on RNA binding proteins. The circuit behavior can also be tuned/controlled via a small molecule dependent aptamer or degradation domain. As demonstrated herein, the circuits function when encoded on self-amplifying RNA replicon, providing means for long-term expression and a potential platform for future therapeutic applications.
Synthetic regulatory circuits encoded on RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. Post-transcriptional circuits were created using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. As demonstrated herein, the circuits function in mammalian cells when encoded on modified mRNA or self-replicating RNA.
In some embodiments, synthetic RNA circuits that are multi-input microRNA-based cell classifiers are provided. Such circuits can include a plurality of RNA molecules. A first RNA molecule includes at least one sequence recognized by at least one microRNA (first microRNA) that is/are specifically expressed in a first cell type, and a sequence encoding a protein that specifically binds to a RNA motif and inhibits protein production. A second RNA molecule includes at least one sequence recognized by at least one different (second) microRNA that is/are not expressed in the first cell type or is expressed at a low level relative to a second cell type, at least one RNA motif and a sequence encoding an output molecule. By sensing the presence and/or absence of the first and second microRNAs, each of which can be a single or a plurality of different microRNAs, the circuit expresses the output molecule only under specific conditions, which are indicative of a particular cell type(s). For example, in a cell that expresses the first microRNA(s) but not the second microRNA(s), the RNA molecule encoding the protein that specifically binds to a RNA motif and inhibits protein production is not translated or is degraded, which then permits expression of the output molecule. If the second microRNA(s) is present, then the RNA molecule that includes the sequence encoding an output molecule is not translated or is degraded. In the absence of the first microRNA(s), the first RNA molecule expresses the protein that specifically binds to a RNA motif and inhibits protein production, which binds to and represses translation of or degrades the second RNA molecule that encodes the output molecule. Thus, only in cells in which the first microRNA(s) is present and the second microRNA(s) is absent is the output molecule produced. This allows for specific control over the expression of the output molecule.
For example, in some embodiments, expression is controlled by the presence and absence of certain microRNAs in a cancer cell. In one embodiment, a microRNA that is expressed in the cancer cell is miR-21, and microRNAs that are not expressed in the cancer cell are miR-141, miR-142 and/or miR-146.
In some embodiments, synthetic RNA circuits that are post-transcriptional cascades are provided. Such circuits can include a plurality of RNA molecules. A first RNA molecule includes at least one sequence recognized by a protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule. A second RNA molecule includes at least one sequence recognized by a second protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production. A third RNA molecule includes at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the second protein that specifically binds to a RNA motif and inhibits protein production. The synthetic RNA circuit also can include the siRNA molecule or microRNA molecule that binds to the third RNA molecule. The siRNA molecule can be a synthetic siRNA molecule. The microRNA molecule can be an endogenously expressed microRNA molecule.
Without the siRNA or microRNA, the second protein that specifically binds to a RNA motif and inhibits protein production is translated, and it represses translation of or degrades the second RNA molecule. This means that the first protein that specifically binds to a RNA motif and inhibits protein production, which is encoded on the second RNA molecule, is not expressed. As a result, the first RNA molecule can be translated, and this permits production of the output molecule. If the siRNA or microRNA is present, the second protein that specifically binds to a RNA motif and inhibits protein production is not translated, and it cannot repress translation of or degrade the second RNA molecule. This means that the first protein that specifically binds to a RNA motif and inhibits protein production, which is encoded on the second RNA molecule, is expressed. As a result, translation of the first RNA molecule is repressed (or the RNA is degraded), and the output molecule is not translated.
In some embodiments, the synthetic RNA circuits include a first RNA molecule that includes at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule; and a second RNA molecule that includes at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production. The synthetic RNA circuit of can also include the siRNA molecule or microRNA molecule that binds to the second RNA molecule. The siRNA molecule can be a synthetic siRNA molecule. The microRNA molecule can be an endogenously expressed microRNA molecule. In the presence of the siRNA or microRNA, the first protein that specifically binds to a RNA motif and inhibits protein production is not produced and the output molecule is produced, whereas in the absence of the siRNA or microRNA, the first protein that specifically binds to a RNA motif and inhibits protein production is produced and the output molecule is not produced.
In some embodiments, synthetic RNA circuits that are two-state switches are provided. Such circuits can include a plurality of RNA molecules. A first RNA molecule includes at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding a second protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a first siRNA molecule or microRNA molecule. A second RNA molecule includes at least one sequence recognized by the second protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a second siRNA molecule or microRNA molecule. The synthetic RNA circuit of can also include the siRNA molecule or microRNA molecule that binds to the second RNA molecule. The siRNA molecule can be a synthetic siRNA molecule. The microRNA molecule can be an endogenously expressed microRNA molecule.
In some embodiments, the first RNA molecule and/or the second RNA molecule further comprise a sequence encoding one or more output molecules that are not a protein that specifically binds to a RNA motif and inhibits protein production. The presence of the first siRNA molecule or microRNA molecule determines whether the first or second protein that specifically binds to a RNA motif and inhibits protein production is produced, and in some embodiments, whether one or more output molecules are produced.
In some embodiments, synthetic RNA circuits that are ON or OFF switches are provided. In some embodiments, a synthetic RNA circuit is provided including an RNA molecule that includes a sequence encoding a destabilization domain fused to an output protein. The destabilization domain facilitates degradation of the output protein in the absence of a small molecule that binds to the destabilization domain. In some embodiments, the destabilization domain is, or is derived from, the E. coli DHFR protein (DDd).
In some embodiments, a synthetic RNA circuit is provided including a plurality of RNA molecules. A first RNA molecule includes a sequence encoding a destabilization domain fused to a protein that specifically binds to a RNA motif and inhibits protein production. A second RNA molecule includes at least one sequence recognized by the protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule. The destabilization domain facilitates degradation of the protein that specifically binds to a RNA motif and inhibits protein production in the absence of a small molecule that binds to the destabilization domain. In some additional embodiments, the output molecule is a fusion of a TetR protein and a second protein; and the RNA molecule(s) further include an aptamer sequence and a second output molecule. The aptamer sequence is bound by the TetR protein in the absence of tetracycline. The aptamer sequence is positioned relative to the second output molecule so that it inhibits production of the second output molecule in the absence of tetracycline.
In some embodiments, a synthetic RNA circuit is provided that includes an RNA molecule comprising a sequence encoding a TetR protein and a sequence encoding an output protein, and an aptamer sequence that is bound by the TetR protein in the absence of tetracycline. The aptamer sequence is positioned relative to the sequence encoding the output protein so that it inhibits production of the output protein in the absence of tetracycline. In some embodiments, the aptamer is positioned in the 5′ untranslated region (UTR) of the sequence encoding an output protein. In other embodiments the TetR protein is a fusion protein.
The output molecule typically is a protein. However, the output molecule can be another type of molecule, such as a nucleic acid molecule, for example an RNA molecule that is an input for a strand displacement reaction. Protein output molecules include therapeutic proteins, cell death proteins, fluorescent proteins, antigen (and/or adjuvants), selection proteins, and immunomodulators.
Therapeutic proteins can be any protein that is used in therapy of disease. For example, a therapeutic protein can be a protein used for protein replacement therapy, such as for metabolic disorders; Myr-Akt for treating Duchenne muscular dystrophy; or follistatin for treating Becker muscular dystrophy, Duchenne muscular dystrophy, inclusion body myositis.
Selection proteins can be used for selection or purification of a cell in which the selection protein is expressed. For example, the selection protein can be a protein that confers drug resistance to a cell, or acts as a marker for the cell type for separation from other cells by separation techniques such as flow cytometry.
Fluorescent proteins include many different types of proteins known in the art, such as enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), enhanced blue fluorescent protein (EBFP), cyan fluorescent proteins (e.g., AmCyan1), other green fluorescent proteins (e.g., AcGFP1, and ZsGreen1), other yellow fluorescent proteins (e.g., ZsYellow1 and mBananna), orange fluorescent proteins (e.g., mOrange and mOrange2), red fluorescent proteins (e.g., DsRed, tdTomato, mStrawberry and mCherry), and far-red fluorescent proteins (e.g., mKate, HcRed1, mRaspberry and mPlum).
Antigens include proteins of infectious agents or cancer antigens, of which many are known in the art. Protein adjuvants also can be expressed, alone or in conjunction with antigen output proteins.
Immunomodulator proteins include cytokines, for example, IL-12, IL-15 or IL-21, or immunosuppressant proteins.
Cell death proteins include hBax.
In some embodiments, the synthetic RNA circuits described herein include RNA molecules that encode more than one output molecule.
Proteins that specifically bind to an RNA motif and inhibit protein production by a variety of mechanisms including repression of translation or degradation of RNA are included in many of the embodiments of the synthetic RNA circuits described herein. Such proteins may be referred to herein as a “protein that specifically binds to an RNA motif and inhibits protein production” or an “RNA binding protein” or the like. Such RNA binding proteins bind to a specific RNA sequence (also referred to as a “RNA motif” herein) and inhibit protein production by repressing translation of the RNA molecule to which they bind. Repression of translation can occur any of the several mechanisms known in the art for repression of translation. Alternatively, such RNA binding proteins bind to a specific RNA sequence (also referred to as a “RNA motif” herein) and inhibit protein production by degradation of RNA.
One example of a protein that specifically binds to an RNA motif and inhibits protein production is L7Ae. The L7Ae protein binds to one or more Box C/D, K-turn and/or K-loop motifs in an RNA molecule. In some embodiments more than one Box C/D, K-turn and/or K-loop motifs (such as two K-turn motifs) are included in an RNA molecule to confer better binding to the RNA molecule and repression of RNA translation. In some embodiments, the one or more Box C/D, K-turn and/or K-loop motifs are placed in the 5′ untranslated region (UTR) of the RNA molecule, i.e., upstream of a sequence encoding an output molecule. In addition, other proteins that bind specific RNA motifs and inhibit protein production can be used in the same manner as described herein for L7Ae.
Another example of a protein that specifically binds to an RNA motif and inhibits protein production is a fusion of MS2 protein and a protein degrades RNA. In some embodiments, MS2 protein can be fused to CNOT7 protein (to form MS2-CNOT7) or Dm-POP2 protein (to form MS2-Dm-POP2), each of which are deadenylases, but other proteins that degrade RNA also can be fused or linked to MS2. In addition, other proteins that bind specific RNA motifs but do not repress translation can be fused to a protein that degrades RNA, and used in the same manner as described herein for MS2-CNOT7.
MS2 protein binds to one or more MS2 coat protein binding sites. In some embodiments more than one MS2 coat protein binding sites (such as eight MS2 coat protein binding sites) are included in an RNA molecule to confer better binding to the RNA molecule and inhibition of protein production, e.g., by degradation of the RNA. In some embodiments, the one or more MS2 coat protein binding sites are placed in the 3′ untranslated region (UTR) of the RNA molecule, i.e., downstream of a sequence encoding an output molecule.
In some embodiments, the RNA molecule(s) of the synthetic RNA circuit includes modified RNA. Such modified RNA molecules can include, for example, 5-methylcytosine-triphosphate and/or pseudouridine-triphosphate. Other modifications of RNA molecules are known in the art, and may be useful, for example, to increase stability or resistance to RNAses.
In some embodiments, the RNA molecule(s) of the synthetic RNA circuit are encoded on one or more RNA replicons. RNA replicons are known in the art and include alphavirus derived replicons, Venezuelan equine encephalitis virus derived replicons or Sindbis derived virus replicons. In such embodiments, the RNA molecule(s) can be expressed from one or more subgenomic promoters of the one or more replicons. In some embodiments, the one or more subgenomic promoters are regulated by a small molecule, such as trimethoprim (TMP).
In some embodiments, the RNA molecule(s) of the synthetic RNA circuit are encoded on one or more plasmids.
Also provided are methods for treating disease using the synthetic RNA circuits described herein. In some embodiments, methods of treating cancer in a mammal are provided, in which a synthetic RNA circuit is administered to a mammal. In some embodiments, the synthetic RNA circuit produces an output protein that treats the cancer, including but not limited to a cell death protein such as hBax, or an immunomodulatory protein.
Also provided are methods for inducing an immune response in a mammal using the synthetic RNA circuits described herein. In some embodiments, the methods include administering to a mammal a synthetic RNA circuit, which produces an output protein that induces the immune response, or augments an immune response. Such methods may be used in vaccination of a mammal, or for other uses in which inducing an immune response is beneficial to the mammal. The output protein produced typically is one or more antigens, but may also include one or more adjuvants, and/or other immunomodulatory proteins. In addition, the methods include controlling the expression of the output protein(s) by administering molecules that control destabilization domains (e.g., trimethoprim) or that control binding of TetR protein to aptamers (e.g., tetracycline). This enables administering the synthetic RNA circuits described herein at one time and administering molecules that control expression of the output protein(s) at a different time, including at several times after the administration of the synthetic RNA circuits. Such administration of the synthetic RNA circuits described herein and the molecules that control expression of the output protein(s) can be used to produce expression of antigens and/or adjuvants at certain times relative to one another in order to produce an improved immune response in the mammal. The molecules that control expression of the output protein(s) can be administered by any suitable method, including by oral administration, intramuscular injection of lipid nanoparticles, or or by implantation of a polymeric implant for sustained release.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teachings that are referenced herein.
EXAMPLES Example 1In our initial circuits, we use two translational repressors, L7Ae (12) and a fusion protein MS2-CNOT7 (13). L7Ae is an archaeal protein that binds K-turn and K-loop motifs with high affinity. When the motif is placed in the 3′UTR of the target mRNA, L7Ae can strongly repress translation of the output gene by blocking ribosome scanning. As has been shown, using multiple repeats of the binding motif and placing the motif close to the transcription start result in enhanced repression (14). We used two repeats of the K-turn motif with an eighteen base pair spacer from the transcription start and such configuration resulted in a very strong repression even at low doses of L7Ae. MS2 is another RNA binding protein, a coat protein from bacteriophage MS2. CNOT7 is a human deadenylase that can efficiently repress translation of mRNA, if directed to its 3′UTR (13). In our system, the reporter mRNA contains eight repeats of the MS2 coat protein binding site in the 3′UTR and MS2 is fused with repression domain, CNOT7.
Towards the goal of creating a platform for future applications through a plug-and-play post-transcriptional regulation framework we engineered a set of diverse regulatory circuits including a multi-input cell type classifier, a cascade and a two-state switch. Additional capabilities, or further tuning of the synthetic regulatory pathways can be achieved with the use of small molecule dependent aptamers or degradation domains.
Regulation with RNA-Binding Proteins (RBP)
To demonstrate that the RBP-based repressors can be utilized to create variable functional circuits we engineered a multi-input microRNA sensor, a cascade and a two-input switch. The microRNA sensing circuit is a post-transcriptional only version of our previously designed (15) HeLa cell classifier. The circuit recognizes microRNA profile that is specific for HeLa cells (high miR-21, low miR-141, miR-142(3p) and miR-146a,
Our next circuit (
We next tested a two-layer version of the cascade encoded on self-amplifying viral replicon for RNA-only delivery (
Our third circuit is a two-state switch where two repressors mutually regulate their expression (
Another form of regulation of RNA circuits, especially useful in a clinical setting would be with the use of a small molecule switch. Here, we have engineered an ON/OFF switch to regulate expression from self-replicating RNA using an FDA-approved small molecule and have achieved more than 20-fold induction. A potential application of this method may include the regulated delivery of antigens for safer programmable vaccines.
To build the ON/OFF switch, we used destabilization domains, which mark proteins for degradation. Upon addition of a small molecule ligand, the ligand binds the domain, and the protein is stabilized and no longer degraded. To test destabilization domains (DDs) as a control mechanism for the replicon, we fused the domain, FKBP12 (16), to the N-terminus of a yellow fluorescent protein, mVenus, electroporated into BHK-21 cells and induced with Shield (17) to test the ON switch. See
Next, we created the OFF switch by fusing a destabilization domain, ecDHFR (18), to the L7Ae repressor as shown in
Tunable expression can also be achieved with an RNA based circuit whose dynamics are governed by TetR/Dox, and by TetR-homolog/small-molecule. Tet Repressor protein (TetR)-binding RNA elements is placed in the 5′-untranslated region (5′-UTR) of mRNA, such that translation of a downstream antigen coding sequence is directly controlled by TetR and tetracycline analogs (20); see
Proteins vary in solubility, are difficult to purify and expensive to store. DNA vaccinations and therapy present potential risks such as integration into the host genome or induction of pathogenic anti-DNA antibodies.
Recently, RNA-based vaccines employing alphavirus replicons, which undergo sustained self-replication of RNA sequences encoding protein antigens within infected cells, have gained attention as a potential strategy for safe and effective vaccination. Such RNA-based vaccines are expected to be safer than DNA-based vectors (lacking the potential for integration into the host genome), and because their function requires delivery only to the cytosol (but not the nucleus) of target cells, synthetic materials may be capable of delivering RNA vaccines without the manufacturing and safety issues of viral vectors. Self-replicating RNA and modified RNA have gained much interest as potential therapeutic agents and in stem cell reprogramming.
No control mechanisms have been developed/used for self-replicating or modified RNA. We propose multiple ways of such control that would allow for e.g. tunable or delayed expression of a therapeutic agent and switching between two different agents.
Example 2. Mammalian Synthetic Circuits with RNA Binding Proteins Delivered by RNA Materials and Methods Cell CultureHEK293FT and HEK293 (293-H) cell lines were purchased from Invitrogen. HeLa (CCL.2) and MCF7 (HTB-22) cell lines were originally obtained from ATCC. The performance of DNA-encoded miRNA sensors in these cell lines had been characterized previously (15). HEK293FT were freshly purchased from the supplier. HeLa, MCF7 and BHK21, although not recently authenticated, were tested for mycoplasma. All cell lines used in this study were maintained in Dulbecco's modified Eagle medium (DMEM, Cellgro) supplemented with 10% FBS (Atlanta BIO), 1% penicillin/streptomycin/L-Glutamine (Sigma-Aldrich) and 1% non-essential amino acids (HyClone) at 37° C. and 5% CO2. In the case of MCF7 cells, DMEM without phenol red was used. BHK21 cells were maintained in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS.
DNA Preparation and TransfectionAll transfections were carried out in 24-well format. Parallel transfections in HEK293, HeLa and MCF7 cells (4-input sensor,
Modified RNA Preparation and mRNA Transfection
A template DNA for in vitro transcription was generated via PCR, using a forward primer containing T7 promoter and a reverse primer containing 120-nucleotide-long Poly(T) tract transcribed into a Poly(A) tail. PCR products amplified from plasmids were subjected to digestion by Dpn I restriction enzyme and purified. Reactions of in vitro transcription were performed using MegaScript T7 kit (Life Technologies) under a modified condition, in which GTP, CTP and UTP was replaced by GTP mixed with Anti Reverse Cap Analog (New England Biolabs) at the ratio of 1 to 4, 5-methylcytosine-triphosphate and pseudouridine-triphosphate (TriLink BioTechnologies), respectively. Transcripts were treated with Turbo DNase (Life Technologies) for 30 min at 37° C. and purified using RNeasy MiniElute Cleanup Kit (QIAGEN). Resulting mRNAs were incubated with Antarctic Phosphatase (New England Biolabs) for 30 min at 37° C. and purified again. Modified mRNAs were transfected into the cells using TransIT-mRNA transfection kit (Mirus Bio) according to manufacturer's protocol. StemFect (Stemgent) was used to perform co-transfections of modified mRNAs with siRNAs, according to manufacturer's instruction. The medium was exchanged 4 hours after the transfection, and transfected cells were subjected to the analysis after 24 hours. Transfection details for each experiment are shown in Table 1. Detailed configurations for modified mRNA and sequences of mRNA used in this study are shown in Tables 3 and 4.
All replicon experiments were performed in BHK21 cells (a kind gift from Dr. Odisse Azizgolshan(34)) using an alphaviral replicon derived from the genome of the Sindbis virus TE12 strain (35) containing a P726S mutation in nsP2 (36) as described previously (37) or an alphaviral replicon derived from the Venezuelan equine encephalitis (VEE) TC-83 strain containing a A3G mutation in the 5′UTR and a Q739L mutation in nsP2 (38) constructed in this study. Briefly, BHK21 cells cultured at 37 degrees C. and 5% CO2 in EMEM (ATCC) medium containing 10% FBS (PAA) were electroporated using the Neon® Transfection System (Life Technologies) per the manufacturer's instructions with ˜1-6 ug of replicon RNA per ˜100,000 cells and plated in 24 well plates (Corning). Transfection details for all experiments are provided in Table 1. Sindbis replicon RNA was produced by run-off in vitro transcription (IVT) of SacI-HF (NEB)-digested replicon plasmid DNA using the mMESSAGE mMACHINE® SP6 Kit (Life Technologies) and purified using the RNeasy® Mini Kit (Qiagen). VEE replicon RNA was produced by run-off in vitro transcription (IVT) of I-SceI (NEB)-digested replicon plasmid DNA using the MEGAscript® T7 Transcription Kit, followed by purification using the RNeasy® Mini Kit (Qiagen), denaturation of the RNA at 65 degrees C., enzymatic (cap1) capping of the RNA using the ScriptCap™ 2′-O-Methyltransferase Kit (Cellscript) and ScriptCap™ m7G Capping System (Cellscript), and a final purification using the RNeasy® Mini Kit (Qiagen) following the manufacturers' protocols. siRNAs (IDT) were co-electroporated (0-10 nM final concentration) along with replicon RNA. Cells were analyzed by flow cytometry 24 h post electroporation. Replicon encoding plasmids used as templates for IVT are listed in Table 5.
qRT-PCR
In the case of pDNA and modRNA, total RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Resulting cDNA was subjected to qPCR on StepOnePlus (Life Technologies) for modRNA using Power SYBR Green PCR Master Mix (Life Technologies). Same Master Mix and Mastercycler ep Realplex (Eppendorf) was used for pDNA experiments. For qRT-PCR of RNA replicons, total RNA was purified from BHK21 cells using the RNeasy® Mini Kit (Qiagen). RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) and qPCR was performed on a Mastercycler ep Realplex (Eppendorf) using the KAPA SYBR® FAST Universal 2× qPCR Master Mix (Kapa Biosystems) or the KAPA PROBE FAST Universal 2× qPCR Master Mix (Kapa Biosystems) following the manufacturer's recommended protocol. Primers unique to the genomic RNA regions were used to calculate the absolute copy number of genomic and antigenomic RNA using a standard curve of synthetic DNA. Subgenomic RNA copy numbers were calculated by subtracting the copy numbers of genomic and antigenomic RNA from the absolute copy numbers of all replicon RNA (i.e. genomic, antigenomic, and subgenomic RNA) using primers spanning the regions downstream of the SGP. Genomic and subgenomic RNA quantities were then normalized to 18S rRNA (internal control) levels quantified using QuantumRNA™ Universal 18S Internal Standard (Life Technologies) or Eukaryotic 18S rRNA Endogenous Control (FAM™/MGB probe, non-primer limited; Life Technologies).
Cells were analyzed with LSR Fortessa or FACSAria flow cytometer, equipped with 405, 488 and 561 nm lasers (BD Biosciences). We collected 30,000-100,000 events per sample and fluorescence data were acquired with the following cytometer settings: 488 nm laser and 530/30 nm bandpass filter for EYFP/EGFP, 561 nm laser and 610/20 nm filter for mKate, and 405 nm laser, 450/50 filter for EBFP. In detecting mKate by FACSAria, a 780/60 nm bandpass filter was used. Data analysis was performed with FACSDiva software (BD Biosciences) and FlowJo (flowjo.com). For all fluorescence assays, populations containing live, single cells were first determined based on forward and side scatter. Red fluorescent protein (mKate) was used in all pDNA experiments as a transfection marker. Reported fluorescence values of pDNA experiments present normalized mean output fluorescence (EYFP, EGFP or EBFP) for all mKate positive cells. Non-transfected cells were used to set the gate determining mKate positive cells. For replicon electroporations and modRNA transfections the efficiency of nucleic acid delivery usually exceeds 90% and therefore all live, single cells were taken into account for calculating mean output fluorescence.
In
Fluorescence microscopy images of live cells were taken in 24-well plates using Zeiss Axiovert 200 microscope and Plan-Neofluar 10×/0.30 Ph1 objective. The filters used were 390/22 (excitation) and 460/50 (emission) for EBFP2, 500/20 (excitation) and 535/50 (emission) for EYFP and 565/30 (excitation) and 620/60 (emission) for mKate. Data collection and processing were performed using AxioVision software (Zeiss).
Apoptosis and Cell Death AssaysSample cells including those in supernatant were collected 24 h post-transfection, washed with PBS and stained with Pacific Blue conjugated 1 μL of Annexin V (Life Technologies) or 0.5 μL of SYTOX AADvanced (Life Technologies) in 50 μL of binding buffer for 30 min at room temperature. The cells were analyzed by flow cytometry. Percentage of apoptosis induction was defined as the percentage of Annexin V positive cells. In the case of HEK/HeLa co-culture assay, HeLa cells were labeled with stable expression of EBFP2 fluorescent protein (excitation/emission maxima of 383 nm and 448 nm) and therefore SYTOX AADvanced (excitation/emission maxima of 546 nm and 647 nm) was used instead Pacific Blue Annexin V (excitation/emission maxima of 415 nm and 455 nm). HEK293 and HeLa-EBFP2 cells were mixed in 1:1 ratio, cultured together and the cell mixture was transfected with modRNA-encoded circuit or controls. Cells were stained with SYTOX AADvanced and analyzed by flow cytometry 24 h post-transfection. % of cell death was calculated as follows: (number of HEK (or HeLa) AAdvanced positive cells/total number of HEK (or HeLa) cells)*100%.
Generation of HeLa-EBFP2 Cells for Co-Culture Cell Death AssayHeLa-EBFP2 cells were generated through lentiviral infection and antibiotic selection. First, HEK293FT packaging cells (Invitrogen) were used for virus production. 2×106 cells were seeded in a 60 mm dish (to ˜80% confluency), approximately 3 h later supplemented with 3 ml of fresh complete medium and co-transfected with the following plasmids:
-
- 0.5 μg pLV-hEF1a-EBFP2-P2A-Bla (hEF1a—human elongation factor 1alpha promoter, P2A—ribosomal skipping 2A sequence from porcine teschovirus-1(39), Bla—blasticidin resistance gene)
- 1.1 μg pCMV-dR8.2 dvpr helper plasmid (40) (Addgene plasmid 8455)
- 0.55 μg pCMV-VSV-G helper plasmid (40) (Addgene plasmid 8454)
Transfections were performed using attractene (Qiagen) and standard manufacturer's protocol. Transfection complexes were added dropwise to the adhered cells without additional media change. 2 days later, media from virus producing cells were collected into 3 ml syringe, and pressed through a low protein binding 0.45 μm sterile filter. 1 ml of the filtered virus containing media was mixed with 4×103 HeLa cells in 0.5 ml fresh culture media and placed in a 12-well dish. Cells were supplemented with fresh media the next day and 10 μg/ml blasticidin (Invivogen) was added to the media on days 3-8 post-infection. Selected cells were over 99% BFP positive throughout the course of experiments as determined by flow cytometry. We additionally performed a fluorescent assay using our classifier circuit (as described in
I. pDNA Model
Species:pC nuclear MS2-CNOT7 plasmid
pL nuclear L7Ae plasmid
mC MS2-CNOT7 mRNA
mL L7Ae mRNA
LmC L7Ae protein bound to cytoplasmic MS2-CNOT7 mRNA
CmL MS2-CNOT7 protein bound to cytoplasmic L7Ae mRNA
L2mC L7Ae protein doubly bound to cytoplasmic MS2-CNOT7 mRNA
C2 mL MS2-CNOT7 protein doubly bound to cytoplasmic L7Ae mRNA
C MS2-CNOT7 protein
L L7Ae protein
Transcription is assumed to be first-order upon cell division when the pDNA enters the cell nucleus.
pC→pC+mC kTS [1]
pL→pL+mL kTS [2]
Translation is assumed to be first-order. While MS2-CNOT7 binding does not have any steric effect on L7Ae translation, bound L7Ae greatly inhibits translation. When one L7Ae protein is bound to the RNA it inhibits translation by a factor, σ, and when two copies of L7Ae are RNA-bound translation is inhibited twice as much.
mC→mC+C kTL [3]
LmC→LmC+C kTL·σ [4]
L2mC→L2mC+C kTL·σ/2 [5]
mL→mL+L kTL [6]
CmL→CmL+L kTL [7]
C2mL→C2 mL+L kTL [8]
For simplicity, two binding sites were assumed for both MS2-CNOT7 and L7Ae RNA. A second-order association rate is used and first-order dissociation rate.
L+mC↔LmC2·kON,L,kOFF,L [9]
C+mL↔CmL2·kON,C,kOFF,C [10]
L+LmC↔L2mC kON,L,2·kOFF,L [11]
C+CmL↔C2mL kON,C,2·kOFF,C [12]
First-order degradation rates were assumed. When the deadenylase MS2-CNOT7 is bound to L7Ae RNA it increases the RNA's degradation rate by a factor, α. In addition to these reactions, all species (including plasmids) are diluted by cell division.
mC→0 degR [13]
mL→0 degR [14]
LmC→L degR [15]
CmL→C degR·α [16]
LmC→mC degP [17]
CmL→mL degP [18]
L2mC→2·L degR [19]
C2mL→2·C degR·2·α [20]
L2mC→LmC degP [21]
C2mL→CmL degP [22]
C→0 degP [23]
L→0 degP [24]
rC cytoplasmic MS2-CNOT7 replicon (genomic)
rL cytoplasmic L7Ae replicon (genomic)
rfC MS2-CNOT7 replicon in spherule (replication factory)
rfL L7Ae replicon in spherule
LrC L7Ae protein bound to cytoplasmic MS2-CNOT7 replicon
CrL MS2-CNOT7 protein bound to cytoplasmic L7Ae replicon
L2rC L7Ae protein doubly bound to cytoplasmic MS2-CNOT7 replicon
C2rL MS2-CNOT7 protein bound to cytoplasmic L7Ae replicon
mC MS2-CNOT7 mRNA (subgenomic)
mL L7Ae mRNA (subgenomic)
LmC L7Ae protein bound to cytoplasmic MS2-CNOT7 mRNA
CmL MS2-CNOT7 protein bound to cytoplasmic L7Ae mRNA
L2mC L7Ae protein doubly bound to cytoplasmic MS2-CNOT7 mRNA
C2 mL MS2-CNOT7 protein doubly bound to cytoplasmic L7Ae mRNA
C MS2-CNOT7 protein
L L7Ae protein
In this simplified model, the transport of replicons to the plasma membrane and the creation of spherules is assumed to be a first-order process. The transport of replicons into spherules depends on nonstructural proteins and other cellular factors and occurs independently for each replicon. In the replicon case, we also consider the inhibition of replicon transport through RBP binding, where β is a fraction (1=no inhibition, 0=complete inhibition).
rC→rC+rfC kTR [1]
rL→rL+rfL kTR [2]
LrC→LrC+rfC kTR·β [3]
CrL→CrL+rfL kTR·β [4]
L2rC→L2rC+rfC kTR·β [5]
C2rL→C2rL+rfL kTR·β [6]
Transcription is assumed to be first-order upon the formation of spherules (replication factories). Spherules can also transcribe more genomic RNA (Equations 9 and 10). This positive feedback is tuned by the fraction ε.
rfC→rfC+mC kTS [7]
rfL→rfL+mL kTS [8]
rfC→rfC+rC kTS·ε [9]
rfL→rfL+rL kTS·ε [10]
Translation is assumed to be first-order as in the pDNA case.
mC→mC+C kTL [11]
LmC→LmC+C kTL·σ [12]
L2mC→L2mC+C kTL·σ/2 [13]
mL→mL+L kTL [14]
CmL→CmL+L kTL [15]
C2mL→C2mL+L kTL [16]
Second-order association rates and first-order dissociation rates were used as above. In the replicon system we assume RBPs can also bind the genomic RNA with the same efficacy (Equations 17-20).
L+rC↔LrC2·kON,L,kOFF,L [17]
C+rL↔CrL2·kON,C,kOFF,C [18]
L+LrC↔L2rC kON,L,2·kOFF,L [19]
C+CrL↔C2rL kON,C,2·kOFF,C [20]
L+mC↔LmC2·kON,L,kOFF,L [21]
C+mL↔CmL2·kON,C,kOFF,C [22]
L+LmC↔L2mC kON,L,2·kOFF,L [23]
C+CmL↔C2mL kON,C,2·kOFF,C [24]
First-order degradation rates were assumed as above. We assume that the degradation factor for mRNAs bound by MS2-CNOT7 also applies to genomic replicon RNAs bound by MS2-CNOT7. Spherules are assumed to be stable for the 4 hours simulated here and are only diluted through cell division.
rC→0 degR [25]
rL→0 degR [26]
LrC→L degR [27]
CrL→C degR·α [28]
LrC→rC degP [29]
CrL→rL degP [30]
L2rC→2·L degR [31]
C2rL→2·C degR·2·α [32]
L2rC→LrC degP [33]
C2rL→CrL degP [34]
mC→0 degR [35]
mL→0 degR [36]
LmC→L degR [37]
CmL→C degR·α [38]
LmC→mC degP [39]
CmL→mL degP [40]
L2mC→2·L degR [41]
C2mL→2·C degR·2·α [42]
L2mC→LmC degP [43]
C2mL→CmL degP [44]
C→0 degP [45]
L→0 degP [46]
Gene delivery using messenger RNA (mRNA) rather than plasmid DNA (pDNA) may be safer owing to a reduced risk of genomic integration (2). Advances in chemical mRNA modification technology have made it possible to use stable in vitro synthesized mRNA with low immunogenicity for gene therapy (21). Self-replicating RNAs that couple RNA-only delivery with prolonged gene expression are of interest for biomedical applications including vaccination and stem cell reprogramming (21). Synthetic biology, however, has so far relied exclusively or partially on transcriptional regulation, which requires introduction of foreign DNA (9, 10). RNA-based regulatory parts, such as aptamers or riboswitches (22-24) cannot currently be interconnected to build complex RNA-encoded circuits. RNA strand displacement reactions, used to date only in bacteria (25, 26) could be combined into logic circuits (27). However, such multi-layered RNA circuits have not yet been successfully implemented. We propose that RNA-binding proteins (RBPs) (12) can function as both the input and the output of RNA regulatory devices and be wired to regulate production of each other towards the construction of complex circuits. The synthetic circuits containing RBPs reported to date have not shown that one RBP can regulate another and have depended on both translational and transcriptional regulation, requiring the use of pDNA for circuit delivery (24). Additionally, general mechanisms to regulate expression from synthetic mRNA or RNA replicons have not yet been implemented. In this article we report that RBP regulatory devices can be wired together and interconnected with cellular and synthetic signaling pathways to build complex circuits that can be delivered to mammalian cells as RNA. We characterize and optimize of a set of RBP devices and then use them to engineer diverse regulatory circuits including a multi-input cell type classifier, a cascade and a switch (
As a first step toward creating RNA-encoded circuits, we optimized and characterized a set of RNA repressor devices comprising RBPs and their binding motifs (
As a first step toward creating RNA-encoded circuits, we improved the L7Ae:K-turn system (12). L7Ae is an archaeal protein that binds a K-turn motif with high affinity. When the K-turn motif is placed in the 5′UTR of target mRNA, L7Ae represses translation of the output gene. We increased repression of this system by using two repeats of the K-turn motif with a short 5′UTR (
To show that these RBP-based repressors can be used as a platform for composite RNA-encoded circuits, we engineered a multi-input microRNA sensing circuit that is a simplified post-transcriptional only version of our previously reported HeLa cell classifier (15). The circuit recognizes whether the cell has a microRNA expression profile indicative of HeLa cells (high miR-21, low miR141, 142(3p) and 146a) and triggers a response only if the profile is matched (
We next connected RBP devices to produce a scalable RNA-only circuit design platform. To generate a one-way information transmitter, we designed a post-transcriptional cascade with three repression stages (
A two-stage version of the cascade was encoded on self-replicating RNA derived from Sindbis virus (30) (
Plasmid DNA (pDNA). Plasmids have been widely used for delivery and expression of foreign genes in mammalian cells. The ease and cost efficiency of sequence modification and pDNA handling make plasmids a popular modality for delivery in many types of experiments. pDNA constructs are also relatively stable and less prone to folding than RNA. While pDNA delivery leads mostly to transient expression, the DNA can still randomly integrate into the host genome, posing serious safety concerns. Additionally, the many steps required between transiently transfected pDNA cell entry and gene expression (nuclear transport of pDNA, transcription, mRNA transport to the cytoplasm and translation) as well as cell-to-cell variability in transfection amount make it a relatively noisy method, which may be not desirable for certain applications.
Modified mRNA (modRNA). Instead of being produced from delivered DNA, mRNAs synthesized in vitro have also been transferred directly into target cells. The use of mRNAs is gaining interest particularly in therapeutic applications due to its safety profile (53). The 5′ end of endogenous mRNAs in eukaryotic cells is modified with a 7-methylguanosine cap structure, and their 3′ ends are polyadenylated. These end structures play an essential role in post-transcriptional processes and facilitate protein production (54). Modification of pyrimidine residues is also known to enhance transgene expression from delivered mRNAs mostly because these modifications to the RNA molecules result in lower stimulation of the innate immune system of host cells (55). modRNAs used in this study contain antireverse cap analog and 120-nt poly(A) tail. In addition, all cytosine and uridine residues are replaced with 5-methylcytosine and pseudouridine.
Self-replicating RNA (replicon). RNA replicons used in this study were derived from the single-strand positive-sense RNA viruses, Sindbis (52) or Venezuelan equine encephalitis (constructed here) viruses of the Alphavirus genus, Togaviridae family (30). The entire lifecycle of a positive strand RNA virus (and thus also the alphavirus) occurs in the cytoplasm of the cell (30) (
Expression noise with pDNA, modRNA and replicon. Complex regulatory networks are subject to gene expression noise, resulting in cell populations exhibiting cell-to-cell variation in protein levels (56, 57). It has been shown that regulatory motifs, such as negative feedback loops, acting at transcriptional (58) or post-transcriptional level (59) may reduce noise in gene expression, thus conferring robustness to biological processes.
Since they avoid transcriptional bursting, which is often a major source of intrinsic noise (57), RNA encoded circuits might exhibit less variability in protein expression in comparison to their pDNA counterpart. For this, we analyzed the coefficient of variation (CV), that is the relative deviation of protein expression in each cell compared with the population average, which is used as a measure of noise (57, 59). We computed the CV for cells where constitutive expression of EGFP was delivered with pDNA, modRNA, or replicon. A smaller CV corresponds to a tight distribution centered around the mean, therefore a smaller cell-to-cell variability; a large CV corresponds to a wide distribution, indicating larger cell-to-cell variability (59). Indeed pDNA delivery shows higher CV than modRNA and replicon, suggesting that RNA based circuits might provide in this experimental setup more robust gene expression than DNA counterparts (
Finally, we created an RNA-based switch circuit in which two RBPs cross-repress each other to demonstrate two-way signal transmission and feedback regulation (
In the absence of siRNA FF4 or FF5, the replicon-based and plasmid-based switch systems exhibit different behaviors (
To investigate these observations, simple computational models of the pDNA and replicon systems were implemented and analyzed. Stochastic simulations using the Gillespie Algorithm were performed in MATLAB27 using HTCondor queued computer cluster at MIT Computer Science and Artificial Intelligence Laboratory. The reaction equations and rates are reported below, and model schematic diagrams are displayed in
First, to better understand the nature of the unimodal state achieved by the pDNA system, several of the parameter values were varied. The resulting behavioral trends are shown in
The next question to investigate was why the replicon system does not go through this high/high state. Based on the results from the pDNA analysis, we hypothesized that the replicon system either avoids the simultaneous burst of expression or it has a faster switching time due to the feedback mechanisms involved in the first few hours post-infection (ongoing negative strand synthesis). As depicted in
We performed global sensitivity analysis by randomly sampling 2000 parameter sets from the log-transformed realistic parameter space (Table 6).
There is, however, a strong relationship between mutual exclusivity and both R0 and kTR, the initial replicon copy number and the transport rate. Both relate to the independent and stochastic nature of spherule formation. Decreasing either R0 or kTR leads to an increase in MEx score. This occurs because stochasticity in the transport reaction increases, allowing an initial bias in replication. As expected, their effects are also correlated (
Overall, these results suggest that the individualized and stochastic nature of spherule formation and transport results in an initial bias in replication. The resulting bimodality can be realized in the first four hours postinfection. The effects are amplified by an increase in stochasticity through a decrease in replicon copy number, and by a fast replication rate (kTS). These differences in dynamics are likely to have important implications when using replicons in synthetic biology circuits, especially when the expression timing of various species is important to circuit functionality.
To our knowledge no previous study has shown that complex cellular logic can be encoded exclusively at the post-transcriptional level in mammalian cells, offering potentially significant benefits for in vivo applications. This is made possible through the use of RBPs, which can act as both the input and the output of a regulatory device, and are promising candidates for creating scalable and modular control and information processing circuits. Our engineered circuits are functional when encoded either on modified mRNA (transient response) or self-replicating RNA (prolonged circuit operation). The inherently transient nature of RNA makes it an appealing platform for applications where safety is a primary concern, as RNA circuits could be programmed to act for a defined period of time and do not leave a long-term genetic footprint. Additionally, the different expression dynamics, lifetime (
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The creation of a safe and cost-effective prophylactic/therapeutic vaccine which can induce potent broadly-neutralizing antibody (bNAb) and cytotoxic T lymphocyte (CTL) responses is urgently needed to end the global HIV/AIDS epidemic. Here we hypothesize that a programmable RNA replicon-based vaccination platform developed through a collaboration between the Weiss and Irvine groups may be used to effectively support this goal by precisely engineering and optimizing the kinetics of antigen/adjuvant expression.
Rationale and Preliminary DataIn vitro transcribed RNA as a vaccine platform is cheaper and easier to manufacture than recombinant proteins and safer than DNA to administer to patients due to the low risk of potentially harmful integration of the vector into the genome (Sahin et al. 2014). Furthermore, vaccination can be readily scaled-up to humans using synthetic lipid nanoparticle (LNP)-based delivery systems (Sahin et al. 2014). Previously, we demonstrated that the expression of a firefly luciferase (fluc) reporter gene from our Venezuelen Equine Encephalitis (VEE) Virus-based self-replicating RNA replicon vector can be prolonged by packaging it into a cationic LNP (
The quality and durability of immune responses elicited by vaccination can be dramatically impacted by the kinetics with which the immune system is exposed to antigen and adjuvant cues, yet vaccine kinetics are not typically engineered by immunization. For example, it had been previously shown that the augmentation of humoral responses against HIV-1 gp120 by expression of a cytokine (IL-2/Ig) requires the cytokine vector to be injected two to five days after injection of the antigen expressing vector and not before or coincident with the antigen vector (Barouch et al. 1998). Furthermore, we and others have shown that CTL and antibody responses can be drastically improved by exponential dosing and exposure of antigens to the immune system (Johansen et al. 2008 and unpublished results;
The expression of cytokines such as IL-2/Ig, IL-12/Ig, IL-15/Ig can be used to significantly enhance an immune response against an antigen, however, the timing of cytokine expression in relation to antigen expression must be carefully tuned. Here, we propose to program the optimal adjuvant expression kinetics (expression of adjuvant two to seven days after antigen expression) into our replicon vaccine using the small molecule-regulated OFF switch shown in
Our programmable RNA replicon platform presents a practical means to provide the ideal (exponential) exposure pattern of an antigen (
In order to test whether it is possible to program the sequential expression of rationally designed gp120 immunogens to guide the immune system to induce cross-reactive antibodies focused on the conserved CD4 binding site, a “stripped core” gp120 immunogen and a variant gp120 immunogen containing mutations outside of the CD4 binding site (Wang et al. 2015) are encoded on the small molecule-regulated replicon cascade shown in
Self-amplifying RNA replicons are an attractive alternative to traditional nucleic acid based expression platforms, providing relatively high, sustained expression compared to non-replicating RNA, without the risk of genomic integration associated with DNA-based therapies. When expressing multiple genes, encoding these genes on a single replicon is an attractive alternative to co-transfection. Here, we propose a comprehensive strategy for the assembly and characterization of multi-gene replicon. In order to control expression of multiple genes from a single Venezuelan Equine Encephalitis (VEE) replicon, we created a library of subgenomic promoters (SGPs) of varying strengths, both higher and lower than the wild type VEE SGP. We found that introducing additional 3′-UTR sequences between translational units also significantly increased expression. Finally, we adapted a Modular Cloning (MoClo) assembly strategy for VEE replicons, demonstrating controlled expression from one hundred and forty different two and three SGP variants expressing fluorescent proteins.
Technical DescriptionInterest has been growing in RNA replicons as an alternative to standard DNA-based gene delivery methods.1 Replicons are not only self-amplifying, but are also regarded as safer than competing gene delivery technologies, making replicons attractive for medical applications such as vaccine delivery, gene therapy, and cellular reprogramming.2-4 Because they are self-amplifying, replicons can generate higher expression of a gene from a relatively low initial dose, compared to non-replicating RNA. Moreover, with regard to safety, replicons remain in the cytoplasm of the cell, so the risk of undesired integration into the genome is minimal.5,6
We have previously demonstrated that expression of multiple genes from co-transfected replicons can be modeled and predicted with a high degree of precision.7 However, there are disadvantages associated with the use of multiple replicons for gene delivery. First, a cell must contain at least one copy of each replicon if more than one gene is required for a given therapy or any type of regulation. In addition, we have found that after three days, in those cells that are co-transfected with two replicons, there is a gradual decrease in the number of double positive cells, preventing sustained regulation using multiple replicons, as shown in
In order to have controlled expression of multiple genes from a single replicon, we needed to independently affect translation of each gene. At the RNA level, this was achieved creating a library of Venezuelan Equine Encephalitis (VEE)8 subgenomic promoters (SGPs) of varying strengths, both higher and lower than the wild type VEE SGP. We also experimented with other means, such as introducing additional 3′-UTR sequences between translational units, which had a significant effect on expression. To truly characterize multi-gene replicons and understand how these components affect expression, we adapted a Modular Cloning (MoClo) assembly strategy for VEE replicons and generated all combinations of two and three SGP constructs expressing fluorescent proteins, using low, midrange, and high strength SGPs with and without 3′UTRs.
Controlling Expression Using Subgenomic Promoters and Additional 3′UTRsA subgenomic promoter library was created for VEE replicon by truncating the full length SGP from either the plus or minus side. The SGP library was tested in a tandem format, depicted in
Another particularly important finding from this experiment was the effect of position on expression, i.e. expression from the second unit is 8 times stronger than expression from the first unit when using two full-length −241/+30 SGPs. As a first attempt to overcome this disparity, an additional 3′UTR was inserted in between the translational units because it is known to play a role in minus strand RNA synthesis.9 As shown in
Because the SGP library could be generated by mutating only the positive side of the SGP, we next set out to validate the results from the tandem library in a single SGP setting. We chose three SGPs with 5, 30, and 15 base pairs on the positive side, representing low, midrange, and high SGPs, respectively. However, our initial round of experiments did not show the same expression pattern as we observed in a tandem format. Specifically, SGP5, which was the weakest of the three in tandem, was now the strongest, as shown on the left side of
We have demonstrated that expression of multiple genes from a replicon can be modulated using SGPs, additional 3′UTRs, and position. However, to more comprehensively characterize expression of two or more genes launched from a single replicon, a more efficient, preferably scarless, assembly strategy was necessary. As shown in
The following is a sequence level description of the Replicon MoClo Assembly, beginning with the various Level 0 destination vectors. These Level 0 destination vectors were made for use with either of the following Type IIS enzymes: SapI or BbsI. SapI has a 7-base pair (bp) recognition site and a 3-bp overhang while BbsI has a 6-bp recognition site and a 4-bp overhang. Typically, we mutate BsaI and SapI sites within any new ORFs to make the Level 0→1 and Level 1→2 reactions more efficient, respectively, but this is not required if a final ligation step is added to the MoClo reaction. Our SGP library and the VEE 3′UTR do not contain recognition sites for either of these enzymes, so this problem most commonly arises with ORFs, although introduction of aptamer sequences or modified 3′UTRs should also be considered. The Level 0 destination vectors contain Ampicillin resistance, with the BsaI site in the AmpR gene mutated to facilitate a more efficient Level 0→1 reaction. In addition, we have mutated the BsaI site in the ccdB gene, allowing us to also create Level 0's via a digest/ligation reaction with BsaI, which is very efficient because the ccdB gene kills the cells that do not receive the insert.
Each Level 0 destination vector is shown below, along with an example of how to insert a given unit (SGP, ORF, or UTR).
Once a library of SGPs, ORFs, and UTRs is established, one can combine Level 0's to make Level 1's, which are individual translational units. However, as we have shown, position on the replicon has a significant effect on expression, so the Level 1 destination vectors must also contain information on the translational unit's position in the final construct. In addition, some units have 3′UTR sequences while others do not. Finally, we have previously established (data not shown) that a truncated E1 structural protein is essential for replication, so the final (3′-most) translational unit must end with an E1-3′UTR sequence. These constraints leave us with the following seven Level 1 destination vectors (Table 8,
Finally the Level 1's are combined to generate Level 2's using the following destination vector:
Notice that for a single translation unit, this strategy is cumbersome, requiring two rounds of reactions: first combining SGP, ORF, and E1-3′UTR into a Level 1 and then inserting this single translational unit into a Level 2. To speed up cloning for single gene replicon, we have also created Level 0S, as shown below. These Level 0S can be combined directly into a Level 2 to test the function of a specific ORF before more in depth characterization. After such characterization, the Level 0S can easily be transferred to Level 0 (using SapI) for use with the MoClo strategy above. Note that Level 0S have Kanamycin resistance similar to Level 1 vectors.
Characterization Strategy for Multi-Gene ExpressionUsing this MoClo-based assembly strategy, were able to construct over 250 different multi-unit replicons in under a month. Over 75% of the created constructs sequenced correctly from a single colony, with 100% correct after picking 3 colonies. One hundred and forty of these constructs, a fraction of which are shown in
We have demonstrated that we are able to modulate expression of multiple genes from a single replicon using position, a novel SGP library, and through incorporation of additional 3′UTR sequences. Coupled with our MoClo assembly strategy we are able to efficiently construct and characterize large libraries of construct. There has recently been a large amount of interest in self-replicating RNA, but such characterization has yet to occur for VEE or any other alphavirus replicon. Using this characterization, prediction and rational design of multi-gene replicons based upon the desired expression is provided.
REFERENCES FOR EXAMPLE 4
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- (5) Robertson, J. S. (1994) Safety considerations for nucleic acid vaccines. Vaccine 12, 1526-1528.
- (6) Klinman, D. M., Takeno, M., Ichino, M., Gu, M., Yamshchikov, G., Mor, G., and Conover, J. (1997) DNA vaccines: safety and efficacy issues. Springer Semin. Immunopathol. 19, 245-256.
- (7) Beal, J., Wagner, T. E., Kitada, T., Azizgolshani, O., Parker, J. M., Densmore, D., and Weiss, R. (2015) Model-Driven Engineering of Gene Expression from RNA Replicons. ACS Synthetic Biology 4, 48-56.
- (8) Kulasegaran-Shylini, R., Thiviyanathan, V., Gorenstein, D. G., and Frolov, I. (2009) The 5?UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins. Virology 387, 211-221.
- (9) Frolov, I., Hardy, R., and Rice, C. M. (2001) Cis-acting RNA elements at the 5′ end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis. RNA 7, 1638-1651.
Nucleic acids have shown promise as an alternative to protein therapeutics for many applications, including vaccination, cancer immunotherapy, genetic reprogramming, and protein-replacement therapies3-5. While tremendous strides have been made in protein engineering since the approval of recombinant human insulin, the cost of production, due to protein modification and purification, can discourage its use for some applications. Nucleic acid therapies avoid this cost by producing the desired protein within the target cells, allowing for correct folding and protein modifications, as well as longer exposure to the therapeutic protein6. In both cases, tissue-specific delivery and clearance rate are of great importance, leading to increased research in those areas. However, while targeted protein delivery is primarily extracellular via modified liposomes, nanoparticles or protein-protein interactions, nucleic acids have the ability to determine cell specificity inside the cell using genetic parts, such as tissue-specific promoters or microRNA (miRNA) target sites7-9. This intracellular control, which can be coupled with extracellular modes of targeted delivery, is one of the key benefits of nucleic acid therapies, but is still very much in its infancy in a clinical setting.
DNA, the primary delivery platform for nucleic acid therapies, is generally introduced as either a viral vector or plasmid DNA (pDNA). Non-replicating RNA has recently emerged as a potential therapeutic platform, in part, due to the development of novel modifications that decrease immunogenicity and increase RNA half-life6,14,22. Unmodified mRNA has been shown to express in vivo as long as a week, but results in a significant innate immune response23,24. By incorporating modified bases, such as pseudouridine and 5-methylcytidine, into the mRNA, expression has been observed up to 4 weeks with a diminished innate immune response25-29. Additional optimization of the 5′ cap, untranslated regions (UTRs), poly-A tail length, and open reading frame (ORF) have also been shown to affect mRNA stability and expression6. Unlike transcription of pDNA, translation of RNA occurs in the cytoplasm, making it possible in both dividing and non-dividing cells. However, because it cannot replicate, dilution becomes an issue in rapidly dividing cells. Additionally, modified RNA generally has lower expression levels than self-replicating RNA. Nonetheless, many of the genetic parts created for replicons can also be used with modified mRNA, and for some applications a much lower immune signature may be preferable.
Replicons are self-amplifying RNA, capable of producing high amounts of protein expression up to 7 weeks after administration in vivo, from relatively low initial doses compared to pDNA and non-replicating RNA30. Of the numerous replicon systems developed, two replicons derived from the alphavirus genus, Sindbis virus (SIN) and Venezuelan Equine Encephalitis virus (VEE) are used for the studies described herein. The invention is not limited to these examples. Replicons from both of these viruses are well-characterized and variants with reduced cytopathicity have been established35-37. Alphaviruses are a group of positive-strand RNA viruses with genomes between 11-12 kilobases. The genome is divided into two parts: the 5′ two-thirds encodes four non-structural proteins used in RNA replication and the 3′ one-third, or subgenomic RNA, encodes the structural proteins38. The genome is preceded by a 5′-7-methylguanosine cap and ends with a 3′-poly-A tail, mimicking cellular mRNA to facilitate translation of the non-structural proteins using host cell machinery.
As self-replicating RNA, replicons offer several advantages over other nucleic acid delivery systems. Because replication occurs outside of the nucleus and replicons do not reverse transcribe, there is minimal risk of integration, a major concern with viral particles. In addition, replicons have shown low vector immunity, expanding its applications to those requiring multiple doses. Replicons are also able to persist in both dividing and quiescent cells, presumably with lower dilution rates in rapidly dividing cells than non-replicating RNA. A high dose of a therapeutic protein can also be produced from as little as one replicon entering a target cell, minimizing the impact of delivery efficiency compared to pDNA and mRNA.
Self-amplifying nature of a replicon presents a major hurdle with respect to dosing. The majority of replicon-based technologies constitutively express a therapeutic protein without any regulation. It is demonstrated herein that protein production cannot be controlled by initial dose alone, as it can for pDNA and mRNA, but requires intracellular control of replicon expression. Control devices that not only govern output of the desired protein, but also determine tissue specificity using miRNA sensing, in a manner similar to tissue-specific promoters used in pDNA are described herein and provided as aspects of the invention. The external input for many of the genetic parts described herein are small molecules, as they are the simplest means to establish tunable and dose-dependent control after a replicon is inside a cell. However, other external inputs are also encompassed within the invention. Because it may not be optional for these drugs to be continuously administered to patients over long periods of time, we have focused the genetic circuits of the invention include ON/OFF switching in response to brief pulses of small molecule or other external inputs.
Many genetic parts for RNA have already been generated, including RNA binding proteins (RBPs), endoribonucleases, riboswitches, and RNA sensors. The examples described herein utilize two RBPs for the majority of the circuits, L7Ae and TetR. L7Ae is a ribosomal protein from Archaeoglobus fulgidus that has been shown to bind RNA motifs called kink-turns (K-turns) with high affinity, as well as K-loops to a lesser degree. The Tet repressor (TetR) protein derived from Escherichia coli is traditionally used for regulation of pDNA genetic circuits. However, using systematic evolution of ligands by exponential enrichment (SELEX), RNA aptamers were found to which TetR bound tightly. Placing either K-turns or TetR aptamers in the 5′UTR upstream of an ORF has been shown to repress expression of the output protein. In the case of TetR, this repression is relieved by the addition of a tetracycline derivative, such as doxycycline, showing small molecule regulation from RNA is possible. Another useful genetic part, Csy4, is a CRISPR-associated endoribonuclease found in Pseudomonas aeruginosa. The Csy4 protein recognizes a 28-nucleotide RNA repeat and cleaves between nucleotides 20 and 2146. Due to the inherent cytotoxicity of the replicon, a Csy4 site-specific “kill switch” is a very useful genetic part of the constructs described herein. Surprisingly, while L7Ae and TetR function in both replicon and modified RNA contexts, we have observed that Csy4 is unable to cleave modified RNA, presumably due to structural changes caused by the modified bases.
Single replicon circuits require multiple proteins to be expressed from a given RNA. Because these proteins must be independently regulated for predictable circuit design, and subgenomic promoter strength had been shown to be sequence dependent in Sindbis virus59 we generated a subgenomic promoter library for VEE by truncating the full-length SGP from either the plus or minus side (
Because a ten-fold range in expression was attainable by truncating only the plus side of the SGP, we were able to validate the results of our tandem experiment in a single SGP setting without risking mutations in nsP4. We chose three SGPs, representing low (SGP5), midrange (SGP30), and high (SGP15) expression in a tandem format, and placed them upstream of an mVenus reporter. These three SGPs exhibited the same pattern of expression strengths in a single SGP format, with a 22-fold range of expression (
During this experiment, we also found that cloning scars can have a profound impact on the range of expression of the SGP library. While cloning for the tandem SGP library left a minimal scar, initial cloning for the single SGP experiment was performed using standard Gateway® cloning (Life Technologies) techniques, resulting in a recombination scar that appeared to buffer expression and exhibited a low dynamic range. The maximal range of 22-fold was observed when the SGP was followed immediately by a Kozak sequence.
After establishing control of expression using an SGP library, additional 3′UTRs, and position on the replicon, a more in depth characterization of two and three SGP constructs was pursued. As we began testing the scalability of our approach in a three SGP format, it quickly became apparent that a large collection of SGP combinations, with and without additional 3′UTRs, would need to be tested to adequately characterize the system and understand positional effects. Due to the large number of combinatorial assemblies, as well as the need for scarless assembly, we adapted a Modular Cloning (MoClo)63 assembly strategy for replicons, which was used to generate all two and three SGP constructs discussed hereafter.
RNA Binding Proteins, Destabilization Domains, and EndoribonucleasesA subset of RNA binding proteins (RBPs) can serve as translational repressors, recognizing specific RNA structures and blocking ribosome initiation. Many RBPs have been characterized, but for the following replicon circuits, we have chosen to focus on two RBPs with varying repressive capabilities, L7Ae and TetR. The archaeal protein L7Ae binds the kink-turn (K-turn) motif, repressing expression very strongly. We have enhanced this repression further by including multiple K-turn repeats (e.g. 2xK-turn). As shown in
After characterizing these translational regulators, an input signal, either applied externally or in response to intracellular cues, was necessary to create a responsive replicon circuit. In has been demonstrated that destabilization domains (DDs) fused to proteins can promote reversible, dose-dependent small molecule regulation. These domains signal rapid degradation of the fusion protein unless the small molecule is present. We began by testing two orthogonal DDs engineered from E. coli dihydrofolate reductase (DDd) and human estrogen receptor ligand binding domain (DDe), which respond to trimethoprim (TMP) and 4-hydroxytamoxifin (4-OHT), respectively. The dose-response curves were produced by fusing each DD to a firefly luciferase (Fluc2) reporter and observing expression in C2C12 mouse myoblast cells (
After independently demonstrating the efficacy of both RBPs and DDs, we began to study DD-RBP fusions. It was observed in the previous experiment that DDs decrease protein expression, so focus was primarily on DD-L7Ae fusions, as weakening TetR would further decrease its fold repression. In these experiments, a 2xK-turn sequence was placed upstream of the reporter. If the small molecule was absent, DD-L7Ae would be degraded and the reporter would express. Alternatively, if the small molecule was present, DD-L7Ae would be stabilized and repress the output. Because L7Ae is such a strong repressor, initial experiments conducted in both BHK-21 and C2C12 cell lines used a relatively weak SGP driving DDd-L7Ae, and resulted in 18-fold and 22.5-fold repression, respectively, upon addition of TMP (
Another genetic part with potential for irreversible switching, Csy4 acts as a site-specific endoribonuclease. The 28-base pair Csy4 recognition site is relatively short, and a single recognition site inserted downstream of a reporter was able to decrease expression 23-fold. Because Csy4 can be used to cleave the poly-A tail off of a replicon, it has tremendous potential as a “kill-switch” and could be used to limit the immune response caused by the replicon over time. In order for this application to be feasible, DD-Csy4 fusions are designed to enable timed control of expression. Four constructs are co-transfected with a replicon containing mVenus and a Csy4 recognition site. Unlike TetR and L7Ae, Csy4 is irreversible, so a small amount of leaky expression would prevent proper circuit function. To prevent leaky expression, Csy4 expression is lowered by incorporating a second DDe or a PEST sequence, which decreases protein half-life64. These fusions are tested under a weak (SGP5) and wild type (SGP30) subgenomic promoter in both BHK-21 and C2C12 cell lines.
Characterize Replicon-Based Platforms for Expression of Multiple Genes Co-transfection of Multiple RepliconsBefore the SGP library was generated or destabilization domains were fused to RBPs, the most straightforward way to control the level of expression of a given protein was co-transfection with a second replicon species. As shown in
While co-transfection can be useful for the transfection of independent, constitutively expressed proteins, it presents some hurdles with regard to genetic circuits. As previously demonstrated, after three days the percentage of double positive BHK-21 cells transfected with VEE replicon gradually decreased, with one of the two replicon species gaining prominence. This behavior would pose problems for circuit design and functionality, as regulatory devices could be out-competed. Furthermore, with co-transfection, it can be difficult to ensure that each component of a genetic circuit or therapy is transfected into a given cell, which affects circuit performance or therapeutic efficacy. To avoid these drawbacks, we began to pursue single replicon platforms that could be used to express multiple genes.
Multi-SGP RepliconsAfter determining the elements governing expression from multi-SGP systems, namely position, SGP strength, and the presence of additional 3′UTR sequences, we planned to characterize constitutive expression from two and three SGP replicons using fluorescent reporters. It became clear that such characterization could not be completed without a high-throughput workflow, so a Modular Cloning (MoClo) assembly strategy was adapted for VEE replicons. As shown in
Using this MoClo-based cloning strategy, we were able to generate all combinations of two and three SGP constructs containing low (SGP5), midrange (SGP30), and high (SGP15) subgenomic promoter strengths, with and without additional 3′UTRs.
These results also indicate an additional parameter with a lesser impact on expression: SGP length. The results for mVenus expression from the first SGP behave as expected, with a systematic increase in expression from the weak SGP5 to the strong SGP15, and slightly higher expression of each after including another 3′UTR. While mKate expression shows this same general increase from SGP5 to SGP15 under the second SGP, notice that the first SGP in front of mVenus also affects mKate expression, but not in a strength-dependent manner. We expect that higher mVenus expression may take resources away, leading to slightly lower mKate expression. However, when holding the second SGP constant, mKate expression is inversely correlated to the length of the first SGP. Replicon position, additional 3′UTRs, and SGP choice are most important when determining expression level (in that order).
Constructs with three SGPs were created to validate the results observed with two SGPs (
Another platform that was explored along with co-transfection of replicons was expression from a defective interfering (DI) RNA using a helper replicon. A defective interfering viral genome is produced when large portions of the genome are deleted due to recombination, leaving the remaining fragment defective and incapable of replication on its own. Instead, the DI genome must be complemented by a “helper” virus in order to replicate, interfering with the helper's own replication through competitive inhibition.
A VEE DI RNA was adopted for this study60. As shown in
We have validated results reported by Kulasegaran-Shylini et al. that a G3→A mutation significantly increased DI RNA expression, even though this mutation increases the ratio of genomic to subgenomic RNA in a full-length replicon (
We next verified that the SGP library carried over into this new platform and that co-transfection of a helper with multiple DI species behaved in a predictable manner. As with co-transfected replicons, we observed constant total expression, with a linear response in expression based upon the initial ratio of the two DI species. Finally, we changed the ratio of helper to DI RNA, as multiple regimes of DI RNA interference have been reported against wild type viruses based on the amount of DI RNA present. Here, we see that while helper expression drops with decreasing initial dose, DI RNA expression does have a maximum in the tested system that is dependent on the helper-DI RNA ratio (
The helper-DI system may not experience the gradual decrease of double positive cells observed with co-transfection of multiple replicons. If the DI RNA begins to out-compete the helper, then the decrease in helper could lead to a decrease in non-structural proteins, and a subsequent decrease in DI RNA. If the helper begins to out-compete the DI RNA, then more non-structural proteins are produced, and more DI RNA is replicated. A helper-DI RNA time course is performed to determine if equilibrium exists in this system, preventing the domination of one species and averting one of the major obstacles of circuit function using co-transfection. In addition, because DI RNA replication is dependent on the presence of the helper, by encoding the circuit output on DI RNA and regulatory elements on a helper, it is possible to ensure that the output is always be regulated, even using co-transfection. There are three possible cases: (i) the DI RNA enters the cell alone, is not replicated, and the protein is not be expressed, (ii) the helper enters the cell alone, replicates, but does not contain the output protein, and (iii) both the helper and DI RNA are co-delivered, replicates, and permits desired circuit function. Using this format, a reversible and irreversible small molecule inducible OFF switch is created using DD-L7Ae and DD-Csy4, respectively.
To circumvent any co-delivery issues, we have proposed a novel self-cleaving helper-DI RNA platform (
To test the validity of this approach, helper-CRS-DI RNA lacking an IRES-Csy4 was co-transfected with a replicon expressing either active or dead Csy4 (
As shown, using dead Csy4 to prevent cleavage results in low helper and DI RNA expression. Expression still exists at low levels because the helper-CRS-DI RNA acts as a modified two SGP replicon. When active Csy4 is added, cleavage occurs, resulting in higher expression of mKate from the helper because it no longer experiences a positional effect. Here, we also observe the effect of the scar left by the full-length CRS compared to the minimal 3′ CRS. The scar left by the full-length CRS makes DI RNA replication very inefficient, leading to low mVenus expression from the DI RNA. On the other hand, using the minimal 3′ CRS results in substantial DI RNA expression. As a rapid test of the amount of Csy4 necessary, we also tested Csy4 expressed from a wild type VEE replicon. The wild type replicon produces higher levels of Csy4, enhancing cleavage and thus DI RNA expression, approaching levels comparable to the positive control of co-transfected helper-DI RNA.
As a next step, helper-CRS-DI RNA constructs containing Csy4 driven by an IRES from encephalomyocarditis virus (EMCV) is compared to co-transfection of a replicon expressing Csy4. These results indicate that optimization of the IRES sequence may produce higher expression of Csy4. Finally, we introduce ON/OFF switches that employ RNA degradation based regulation that would not be possible using a multi-SGP replicon.
Develop RNA-Only Circuits, with Emphasis on Small Molecule Inducible ON/OFF Switches
Inducible Single Replicon Switch with Cascade Topology
After characterizing our parts and expression platforms, we created a functional genetic circuit housed on a single replicon. While testing DDs fused to L7Ae, we effectively created an OFF switch, in which the addition of a small molecule stabilized L7Ae and repressed the output. Because we have not characterized any RBPs that act as translational activators, to create a single replicon ON switch required optimization of a three SGP system, containing a cascade of repressors (
In the circuit topology shown, if no TMP is present, DDd-L7Ae is destabilized, allowing TetR to repress mVenus-PEST. Alternatively, if TMP is present, DDd-L7Ae is stabilized, represses TetR, and mVenus-PEST is expressed. The PEST sequence shortens the half-life of mVenus. This rapid turnover would allow for more sensitive studies of circuit dynamics in the future. Doxycycline (Dox) was added in conjunction with TMP to further decrease TetR binding and increase expression of the ON state. The 96 variants shown were constructed using the replicon MoClo assembly system and tested in BHK-21 cells. Flow cytometry was performed 48 hours post-transfection and the optimal construct resulted in an OFF (−TMP/−Dox) to ON (+TMP/+Dox) fold-change of 10.75-fold (
Surprisingly, the eight constructs with the highest fold changes all had TetR expressed under the first SGP and DDd-L7Ae expressed under the second SGP (Orientation 2). This result was unexpected because it was thought that not enough TetR would be translated under the first SGP to provide sufficient repression. However, expression of TetR in either the first or second position appears to result in similar OFF states. Therefore, the high fold changes observed are a product of high ON states, caused by increased amounts of DDd-L7Ae translated from the second SGP. While this switch functions, the OFF state has leaky expression due to incomplete repression by TetR.
To further decrease the OFF state of this circuit, repression enhancers fused to TetR using fluorescence activated cell sorting (FACS) are screened in conjunction with next generation RNA sequencing (RNA-seq). A library of 513 Dox-inducible TetR ON switches, testing multiple SGPs and 57 different repression enhancers, are constructed in a one-pot batch reaction using replicon MoClo assembly (
Irreversible Switch using Csy4
While the aforementioned switches are reversible by the addition or removal of small molecule, we have also devised an irreversible switch using Csy4 (
In State 1, to produce low mVenus and high mKate, TMP is added to stabilize DDd-L7Ae. Expression of mVenus should already be very low, as it is in the first position of a three SGP replicon, but the stabilized DDd-L7Ae should reduce expression further. No 4-OHT is present, so DDe-Csy4 is degraded, but also is repressed by DDd-L7Ae to prevent leaky expression and premature cleavage. In State 2, TMP is removed and 4-OHT is added. This combination of small molecules eliminates DDd-L7Ae repression and induce DDe-Csy4 cleavage, resulting in a single replicon with high mVenus expression.
Helper-CRS-DI miRNA High Sensor
RNA degradation-based regulation has remained elusive in a single replicon format because any degradation affects the entire replicon, and thus the entire circuit. However, using Csy4 to intracellularly split a single RNA into independently replicating components allows us to overcome this barrier. A microRNA (miRNA) high sensor, termed as such because when the target miRNA is present, the output has high expression is created (
Unlike competing nucleic acid technologies, the replicon circuits of the invention utilize small molecule regulation rather than relying on integration or repeat administration of nucleic acids. Here, we propose a treatment for Duchenne muscular dystrophy (DMD) using a replicon switch to initially convert human dermal fibroblasts to a myogenic lineage to facilitate fusion, followed by expression of a therapeutic protein, follistatin.
DMD is a recessive X-linked disease characterized by continual degeneration and regeneration of muscle fibers. It is caused by a mutation in the dystrophin gene, which plays an important role in muscle stability by interacting with a dystrophin-glycoprotein complex at the muscle cell membrane. Over time the muscle tissue wastes away and is replaced by fibrotic and adipose tissue, leading to eventual paralysis and death. One in 3,500 males is born with DMD and those with the disease have a life expectancy of 25 years65.
Because DMD is recessive and female carriers of the DMD allele retain muscle stability66, initial therapies for DMD attempted to restore dystrophin to muscle tissue by implanting healthy donor myoblasts into dystrophic fibers. However, paternal biopsies used in clinical trials resulted in low engraftment efficiency and thus low dystrophin expression67. Additionally, using cells from a donor can lead to immune rejection of the implanted cells. Next, cell therapies were pursued to engineer a patient's own cells to express the therapeutic gene, follistatin. Unfortunately, a patient's preexisting myogenic cells would have already undergone many cycles of degeneration and regeneration, making them difficult to expand68. Dermal fibroblasts are one of the most abundant and easily accessible cell types. They are also capable of myogenic conversion and fusion into myotubes using transient expression of MyoD, a transcription factor involved in skeletal muscle differentiation69,70. Initially, it was believed that MyoD alone can facilitate fibroblasts' conversion into myotubes. However, recent studies suggest that while MyoD is essential to initiate differentiation, Myogenin (MyoG) is required later to retain this fate71.
A replicon circuit similar to that shown in
Sindbis replicon plasmids were linearized using SacI-HF (NEB) prior to run-off in vitro transcription (IVT) using the mMESSAGE mMachine® SP6 Kit (Life Technologies). For experiments conducted in BHK-21 cells, VEE replicon plasmids were linearized using I-SceI (NEB) prior to in vitro transcription using the mMESSAGE mMachine® T7 Kit (Life Technologies). Following IVT, the resulting RNA was purified using the RNeasy® Mini Kit (Qiagen) and the concentration was measured using the NanoDrop™ 2000. For experiments conducted in C2C12 myoblasts or myotubes, IVT was performed using the MEGAscript® T7 Transcription Kit (Life Technologies), followed by purification using the RNeasy® Mini Kit (Qiagen). The resulting RNA was denatured at 65° C. and enzymatic capping was performed using the ScriptCap 2′-O-methyltransferase Kit (Cellscript) and ScriptCap m7G Capping System (Cellscript). A final purification step using the RNeasy® Mini Kit (Qiagen) was performed prior to transfection.
TransfectionBHK-21 cells (a kind gift from Dr. James H. Strauss) were cultured in EMEM (ATCC) supplemented with 10% FBS (PAA) at 37° C. and 5% CO2. BHK-21 cells at approximately 70% confluence were electroporated using the Neon™ Transfection System (Life Technologies) following optimization, according to the manufacturers' instructions. In general, for a single well of a 24-well plate (Corning), approximately 100,000 cells were electroporated with 1,000 ng of RNA, unless otherwise stated.
C2C12 cells were cultured on gelatin coated plated in DMEM (ATCC) supplemented with 10% FBS (PAA) at 37° C. and 5% CO2. The Neon™ Transfection System (Life Technologies) was independently optimized for C2C12 cells, following the manufacturer's instructions. In general, for a single well of a 24-well plate (Corning), approximately 50,000 cells were electroporated with 100 ng of RNA, unless otherwise stated.
To differentiate C2C12 cells into myotubes, 150,000 cells were plated per well in a 24-well plate and allowed to grow for one day in DMEM supplemented with 10% FBS. Once the cell population was confluent, the media was changed to DMEM supplemented with 2% horse serum (Thermo SH30074). The media was replaced each day for 4-5 days. After this time, the media was changed back to DMEM supplemented with 10% FBS and transfections were performed with Lipofectamine™ MessengerMAX™ Reagent (Life Technologies) using 100 ng of RNA.
Data CollectionFor fluorescent reporters, cells for each time point were washed with 1×PBS, trypsinized, and resuspended in 1×PBS. Flow cytometry was performed using the BD LSRFortessa™ Flow Cytometer System (BD Biosciences), equipped with 405, 488, and 561 nm lasers. 20,000-40,000 events were collected per sample. FACSDiva software (BD Biosciences) was used for initial data collection and FlowJo was used for subsequent data analysis. For luciferase assays, 250 μL of Glo Lysis Buffer (Promega) was added to each well of a 24-well plate. 25 μL of lysate was mixed with 25 μL of Steady-Glo® reagent (Promega) in black 96-well clear bottom plates (Corning) and incubated at room temperature for 5 minutes. Luminescence was measured using a Tecan Safire2 plate reader.
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Comparison of Luciferase Expression Levels from Different RNA Platforms and Delivery Formats in Wild-Type and SCID Mice
To obtain a general understanding of the relative performances (translational capacity and duration) of different mRNA (RNA replicon and modified mRNA [modRNA]) platforms for intramuscular (i.m.) delivery into mice using various non-viral delivery methods (lipid nanoparticles (LNP) and electroporation (e.p.)) is performed.
To this end, Venezuelan equine encephalitis (VEE) replicon RNA and modRNA encoding firefly luciferase (Fluc) is produced by in vitro transcription (IVT) using bacteriophage T7 RNA polymerase. DNA templates for run-off IVT of the VEE replicons (wildtype (WT) and non-cytopathic nsP2Q739L replicon (NCP)) and modRNA (containing the 5′ and 3′ UTRs of the VEE subgenomic RNA (sgRNA)) are prepared by plasmid linearization followed by removal of the 3′ overhang by Klenow fragment. For modRNA IVT, N1-methylpseudouridine (m1Y) is incorporated into the RNA instead of uridine. Both mRNAs (replicon and modRNA) are capped co-transcriptionally using cap analogues (e.g. anti-reverse cap analogue (ARCA)) and subsequently treated with phosphatase to remove 5′ triphosphates from uncapped RNA. ModRNA is purified by high performance liquid chromatography (HPLC) and RNA replicon is purified by denaturing urea polyacrylamide gel electrophoresis combined with electroelution to remove contaminating dsRNA or RNA/DNA hybrids from the sample. Quality control (QC) of the RNAs is performed by denaturing gel electrophoresis or capillary electrophoresis using an Agilent Bioanalyzer to quantify the amount of full length RNA in the sample. Furthermore, dot blot is performed using a dsRNA specific antibody to quantify the levels of contaminating dsRNA in the sample, if any. The RNAs are subsequently transfected into mouse myotubes using Lipofectamine MessengerMAX (Life Technologies). Myotubes are differentiated from a mouse myoblast cell line (C2C12) using differentiation medium containing donor equine serum.
The RNAs that pass the QC test, are used for bilateral injection (6 ug) into the gastrocnemius muscles of WT (Balb/c) or severe combined immunodeficiency (SCID) mice. The levels of Fluc reporter proteins expressed from the various RNAs are monitored in vivo by bioluminescence imaging (BLI) over the course of 77 days. Administration occurs at day 0, (bilateral, i.m.) and assays of in vivo bioluminescence occurs at days 2, 4, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, and 77. After the last BLI measurement, the mice are sacrificed and quantitative reverse transcription PCR (qRT-PCR) analysis is performed on RNA extracted from the gastrocnemius muscle to detect the levels of replicon RNA in the tissue. I.m. delivery of RNA is accomplished by packaging the RNAs into LNPs or by naked injection followed by e.p. using a Harvard Apparatus BTX ECM830 electroporator (100V, 3 pulses, 60 ms duration/100 ms delay). Experimental groups are summarized in Table 9. LNP packaging of the RNAs is performed using the ethanol dilution method by complexing RNA with a cationic lipid and fusogenic lipids via electrostatic interactions and subsequently grafting with DSPE-PEG. QC of LNP-packaged RNA is performed by measuring the zeta-potential and size of the particles using dynamic light scattering (DLS) and by checking the RNA packaging efficiency using a RiboGreen® (Life Technologies) assay. The formulated RNAs are transfected in vitro into C2C12 myotubes to measure protein expression. The RNA and LNP QC procedures described are used to verify the quality of the IVT RNA and LNP-packaged RNA for all subsequent tasks.
The capabilities of the various RNA expression platforms (replicon and modRNA) to induce an immune response against the RSV F antigen by homologous prime/boost when delivered i.m. using LNPs or by e.p. as described above are compared.
To this end, two doses (1.5 and 6 ug) of WT or NCP VEE replicon or m1Y modRNA encoding the RSV F antigen are unilaterally injected and delivered into the gastrocnemius muscles of Balb/c mice by e.p. or using LNPs (prime; day 0). Three weeks after this prime injection, the mice receive a unilateral i.m. booster shot of the same amount/type of RNA using the same delivery method (boost; day 21). An aluminum-adjuvanted RSV protein prime/boost group following the same injection schedule as the RNA groups is included as a benchmark for the immune response against RSV F protein. Prime-only groups of the above are also included as a control. At Day 0, prime unilateral, i.m. is delivered, at day 21 a boost is administered, and on day 35, the mice are sacrificed, immune response is measured, and qRT-PCT is performed. See Table 10.
The immune responses against the RSV F antigen on day 35 (two weeks after the boost injection or five weeks after the prime injection for prime-only groups) for each experimental group is determined by measuring 1) the serum antibody (Ab) titers against RSV F, 2) serum virus-neutralizing Ab (VNA) titers against RSV, and 3) antigen specific activation and cytokine secretion (interferon (IFN)-γ) of spleen CD4+ and CD8+ T cells upon RSV F peptide stimulation (quantified by an Enzyme-Linked ImmunoSpot (ELISpot) assay.
Furthermore, the immunogenicity of the VEE replicase proteins are evaluated by measuring the serum Ab levels against the replicase proteins as well as the replicase specific immune response of splenocytes by IFN-γ ELISPOT. Systemic toxicity induced by the different RNA platforms and delivery methods is determined by measuring blood markers of liver toxicity (including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase) as well as pro-inflammatory cytokines (using cytometric bead array (CBA) assays).
Finally, after sacrificing the mice, qRT-PCR analysis is performed on RNA extracted from the gastrocnemius muscle to detect the levels of replicon RNA in the tissue.
- 1. Serum RSV F Ab titers (Crucell)
- 2. Serum RSV VNA titers (Crucell)
- 3. Immune response against RSV F by splenocyte (CD4+, CD8+ T cell) cytokine (IFN-γ) ELISpot (MIT)
- 4. Immune response against VEE replicase by serumAb titers and splenocyte (CD4+, CD8+ T cell) cytokine (IFN-γ) ELISpot (MIT)
- 5. Liver toxicity markers and pro-inflammatory cytokine measurements from the blood (MIT)
- 6. qRT-PCR of replicon RNA from muscle (MIT)
The magnitude and quality of the immune responses against RSV F following homologous (RNA-prime/RNA-boost or protein-prime/protein-boost) or heterologous (RNA-prime(/RNA-boost)/protein boost) prime/boosting of the antigen is compared.
Based on the results of the above, the optimal RNA platform, delivery method, and two RNA doses to express the RSV F antigen are determined. For the homologous RNA prime/boost, using this optimal setup, RNA are unilaterally injected into the gastrocnemius muscles of Balb/c mice (prime; day 0). Three and six weeks after this prime injection, the mice receive a unilateral i.m. booster shot (boost; days 21, 42). As a homologous protein prime/boost control, aluminum-adjuvanted RSV F protein prime-only or prime (day 0)/boost (day 21) injections is performed. These RNA or protein homologous prime/boost groups are compared with heterologous prime/boost injection groups in which an aluminum-adjuvanted RSV F protein booster injection is administered following a single RNA prime injection (day 0) or RNA prime (day 0)/boost (day 21) injections. Prime-only groups for replicon (1.5 ug) as well as aluminum-adjuvanted protein are also included as a control (experimental groups and injection schedules are summarized in
The immune responses against the RSV F antigen on days 14, and/or 35, and/or 56 depending on the experimental group (as described in
The magnitude and quality of an immune response against an antigen is established and may be improved by modulating the in vivo quantity of the antigen expressed from an RNA replicon.
To this end, we first establish whether it is possible to regulate the expression levels of a Fluc reporter protein in a manner that would be meaningful for the purpose of modulating the adaptive immune response. Regulation of target protein expression is done by adapting the L7Ae/K-turn translational repression system. The L7Ae repressor is fused to a destabilizing domain derived from the E. coli DHFR protein (DDd). When fused to a protein of interest, DDd targets the protein to the proteasome for degradation. However, targeting of the protein to the proteasome can be blocked by binding of the small molecule trimethoprim (TMP) to DDd. A set of configurations to identify an optimal TMP regulatable RNA replicon is screened (Circuit 1; “OFF switch”) with tandem subgenomic promoters (SGPs). The first SGP expresses a (2×)DDd-L7Ae fusion protein and the second SGP expresses a Fluc reporter whose translation can be controlled by binding of DDd-L7Ae to K-turn motifs as follows:
-
- SGP1(15) (2x)DDd-L7Ae SGP2(X) NxKt Fluc (IRES E3) (X=16, 30; N=2, 3, 4) (+TMP: DDd-L7Ae binding to motif→Fluc OFF; −TMP: DDd-L7Ae degradation and no binding to motif→Flue ON)
- Circuit 1
- SGP1(15) (2x)DDd-L7Ae SGP2(X) NxKt Fluc (IRES E3) (X=16, 30; N=2, 3, 4) (+TMP: DDd-L7Ae binding to motif→Fluc OFF; −TMP: DDd-L7Ae degradation and no binding to motif→Flue ON)
The ON/OFF ratio (circuit performance) of each replicon in the Circuit 1 library is first evaluated in C2C12 myotubes. The most promising member (high ON/OFF ratio and low OFF state expression) is subsequently tested in vivo. For this, two doses (1 and 6 ug) of the optimal Circuit 1 replicon is packaged with LNPs and bilaterally injected into the gastrocnemius muscles of Balb/c mice. TMP is added to the drinking water of the mice in the following periodic pattern: (1 week−TMP [Fluc ON], 2 weeks+TMP [Fluc OFF])×3 to see whether it would be possible to induce three pulses of Fluc expression in vivo in mice. “No TMP” and “constant TMP” groups as well as a constitutively repressed replicon group expressing L7Ae are used as controls (experimental groups and BLI schedules are summarized in
Based on the in vivo performance of the injected replicon circuit, up to two more attempts are made to reconfigure the replicon and improve the performance of the circuit (if necessary).
Once it has been established that it is possible to provide sequential pulses of the Fluc reporter using the DD-L7Ae TMP OFF switch in vivo, next, we regulate the expression of the RSV F antigen using a replicon with the optimal circuit topology identified above but encoding the antigen instead of Flue (Circuit 2). The optimal dose (1 or 6 ug depending on the results of the optimization experiment above) of Circuit 2 are packaged with LNPs and unilaterally injected into the gastrocnemius muscles of Balb/c mice (day 0). TMP is added to the drinking water of the mice in the following pattern: (1 week−TMP [RSV F ON], 2 weeks+TMP [RSV F OFF])×3 in order to modulate the expression of RSV F in vivo. “No TMP” and “constant TMP” groups as well as a constitutively repressed L7Ae replicon group are included as controls. The immune responses against the RSV F antigen on days 21, 42, and 63 are assessed by measuring the serum Ab titers, serum VNA titers, and antigen specific T cell activation levels. Experimental groups and assay schedules are summarized in
If the optimal TMP-based RSV F antigen OFF switch (Circuit 2) contains IRES E3, control groups using a replicon identical to Circuit 2 except in which the E3 protein is replaced with a “dummy” protein (e.g. mVenus) are included to make sure that the E3 innate immune inhibitor protein does not negatively affect the adaptive immune response elicited against the RSV F antigen.
Materials: In Vitro ReagentsDNA synthesis (IDT, GenScript), oligonucleotides (IDT), restriction enzymes (NEB), PCR reagents (Agilent), T4 DNA ligase (Promega), VEE replicon DNA template (manuscript in press), plasmid DNA purification columns (Qiagen), DNA sequencing services (Quintara), IVT kit (Life Technologies), modified NTPs (TriLink), ARCA (TriLink), phosphatase (epicentre), RNA purification columns (Qiagen), in vitro lipid transfection reagents (Life Technologies), dsRNA-specific monoclonal Ab J2 (English & Scientific Consulting), C2C12 myoblasts (kind gift from Dr. Barbara J. Wold, Caltech), cell culture media (Life Technologies, ATCC), fetal bovine serum (Thermo Fisher Scientific), donor equine serum (Thermo Fisher Scientific), phosphate buffered saline (Corning), trypsin (Corning), pipette tips, plastic ware other basic reagents and supplies (VWR, Fisher, Westnet).
In Vivo ReagentsAnesthesia machine fee (Koch Institute), IVIS machine fee (Koch Institute), flow cytometry facility fee (Koch Institute), CBA assay FACS panel (BD Biosciences), dialysis device (Life Technologies), liver and kidney toxicity enzyme detection kit (Millipore), ELISpot reagents/plates (Millipore), ELISpot Abs (MAbTech), RiboGreen® kit (Life Technologies), lipids (Avanti Lipids), ACK lysis buffer (Sigma), isoflurane (MIT DCM Pharmacy), Balb/c mice (The Jackson laboratory), NOD.SCID mice (The Jackson laboratory), mouse facility charges (Koch Institute), pipette tips, buffers, syringes, needles, other basic reagents and supplies (VWR, Fisher, Westnet).
EquipmentElutrap electroelution system (Whatmann), Qubit® 3.0 Fluorometer (Life Technologies), C18 HPLC column (Transgenomic), ÄKTA pure (GE Healthcare), Agilent 2100 Bioanalyzer, ELISpot reader (Zeiss)
Small Molecule-Inducible RNA Replicon Translational “ON Switch” Respiratory Syncytial Virus (RSV) VaccinationReplicons have recently received attention as vaccine delivery vectors. Replicons can produce large quantities of an antigen with sustained expression over many weeks. Additionally, replicons have inherent adjuvant-like properties, stemming from their viral origin. However, constitutive expression of an antigen is often not enough to mount a sustained immune response. Most vaccination strategies require prime-boosting, or delivery of two different doses of antigen usually separated by several weeks. Prime-boosting results in increased humoral and cell-mediated immunity compared to a single dose of an antigen. Because replicons have been shown to persist up to seven weeks in vivo, replicon-encoded circuits may be used to create a single injection prime-boost vaccination platform. Such a platform would be extremely beneficial in areas of the world where it is difficult to make repeat visits to a clinic. Instead of receiving a second injection, the antigen could be regulated by a small molecule, taken orally by the patient at the correct time.
As previously mentioned, the optimal prime-boost circuit would be an ON switch, requiring two doses of a small molecule to turn on antigen production during the prime and boost phases. However, as we have shown, replicon-based ON switches are more complex and require multiple regulatory elements. On the other hand, OFF switches require only one DD-fused repressor, as shown in
An RNA replicon-encoded small molecule regulatable “ON switch” which functions robustly when injected i.m. into mice is developed. An RNA replicon is created with tandem SGPs expressing a TetR fusion protein (TetR-RE; RE=repression enhancer, to be identified using the screen described below) from the first SGP and a Fluc reporter whose translation can be controlled by binding of TetR-RE to TetR aptamers (TetR-Apt) from the second SGP in the following manner:
-
- SGP1(15) TetR-RE SGP2(X) NxTetR-Apt Fluc (RE=member of Table RE; X=5, 15, 30; N=2, 3, 4) (+Doxycycline [Dox]: No TetR-RE binding to Apt→Fluc ON; −Dox: TetR-RE binding to Apt→Fluc OFF)
- Circuit 3
- SGP1(15) TetR-RE SGP2(X) NxTetR-Apt Fluc (RE=member of Table RE; X=5, 15, 30; N=2, 3, 4) (+Doxycycline [Dox]: No TetR-RE binding to Apt→Fluc ON; −Dox: TetR-RE binding to Apt→Fluc OFF)
RNA (1 or 6 ug) for the optimal Circuit 3 (optimized as described below) is injected bilaterally into the gastrocnemius muscles of Balb/c mice. The mice injected with Circuit 3 receive either Dox or do not receive Dox in the drinking water and Fluc expression is monitored by BLI for two weeks. As a negative control, RNA identical to Circuit 3 except with TetR-RE replaced by a mock repressor (mVenus-RE) that does not bind TetR-Apt is injected into a different group of mice. Experimental groups are summarized in
The optimal Circuit 3 to be tested in the in vivo experiment in
-
- SGP1(15) mVenus-2A-TetR-RE (configuration barcode) SGP2(X) NxTetR-Apt mKate (RE=member of Table RE; X=5, 15, 30; N=2, 3, 4) (+Doxycycline [Dox]: No TetR-RE binding to Apt→Fluc ON; −Dox: TetR-RE binding to Apt→Fluc OFF)
- Circuit 4
- SGP1(15) mVenus-2A-TetR-RE (configuration barcode) SGP2(X) NxTetR-Apt mKate (RE=member of Table RE; X=5, 15, 30; N=2, 3, 4) (+Doxycycline [Dox]: No TetR-RE binding to Apt→Fluc ON; −Dox: TetR-RE binding to Apt→Fluc OFF)
Candidate REs to be screened and their functions related to translational regulation are described in Table RE.
In order to enhance the throughput and reduce the cost of the screen, cloning, DNA preparation, and IVT is performed in batch (in one-pot reactions) for the entire library. The entire Circuit 4 library is then transfected into C2C12 myoblasts at a predetermined low transfection efficiency by e.p. to ensure that the majority of the transfected cells received one RNA circuit from the Circuit 4 library. The transfected cells are then divided into two: one group is cultured in media containing Dox and the other group without Dox. 24 h later, each group is separately processed by FACS. For either group, cells that are mVenus negative are not collected as those cells do not contain replicons from the Circuit 4 library. The mVenus positive cells are then be sorted into eight different bins by FACS based on their mKate expression levels using predetermined cell standards (e.g. negative cells, cells harboring SGP(5) mKate, SGP(15) mKate, SGP(30) mKate, etc.) as guides for partitioning of the experimental sample. The RNA from each bin (2 [+/−Dox]×8 [expression levels]=16 total bins) are then extracted and barcoded in batch (per bin). Subsequently, the barcoded samples are pooled and processed for RNA-Seq to read the configuration barcodes and determine the identities of TetR-RE, SGP2(X), NxTetR-Apt, and the mKate expression level bin that the replicon originated from (each mKate expression level bin is assigned an intensity score of 1-8). For each unique replicon, the geometric mean of the associated mKate intensity scores are calculated (separately for +Dox and −Dox conditions). The strategy of this screen is summarized in
Members of the library with the largest differences in the geometric means of the mKate scores under the two conditions (+/−Dox) are tested for follow-up transfection and evaluation in differentiated C2C12 myotubes. Promising TetR-RE and circuit configurations identified from the Circuit 4 library are used to construct Circuit 3 replicons for testing in vivo as described in
We discovered that enhancers of general translation such as protein kinase R (PKR) inhibitors can increase the dynamic range of small molecule-based regulation of replicon circuits in C2C12 myotubes. Therefore, to further improve the performance of the best performing member of the Circuit 4 library, a screen to identify general translation enhancers (GTEs) including but not limited to IFN response antagonist proteins that may further boost the performance of the optimal member of the Circuit 4 library when expressed from an internal ribosomal entry site (IRES) sequence is performed. Since it has been shown that certain IRES sequences may be more resistant to PKR-induced translational inhibition than others, we first identify the optimal IRES sequence to use for cap-independent expression from VEE replicons. To this end, we test the ability of known viral and synthetic IRES sequences (benchmarked against the EMCV IRES) to drive the expression of a Fluc reporter protein. Furthermore, we determine whether the magnitude of the intracellular antiviral innate immune response triggered by each IRES sequence is different by looking at the expression of IFN-β, PKR, and IL-6 by quantitative reverse-transcription PCR (qRT-PCR). Various IRES sequences (28 total) are tested in the following format initially in myotubes and then in vivo in mice for promising candidates (
-
- SGP1(15) TetR SGP2(30) 2xTetR-Apt mKate IRES Fluc (IRES=member of Table IRES)
- Circuit 5
- SGP1(15) TetR SGP2(30) 2xTetR-Apt mKate IRES Fluc (IRES=member of Table IRES)
IRES candidates to be screened and their origins are described in Table IRES.
Once an optimal IRES sequence is determined above (Circuit 5), that IRES is used to express candidate GTEs to enhance the performance of Circuit 4. To this end, a library of circuits (216 total) is constructed in the following format and screen by FACS/RNA-seq as described below:
-
- SGP1(15) mVenus SGP2(30) 2xTetR-Apt mKate IRES GTE (GTE=member of Table GTE)
- Circuit 6
- SGP1(15) mVenus SGP2(30) 2xTetR-Apt mKate IRES GTE (GTE=member of Table GTE)
Candidate GTEs to be used in this screen and their biological functions are described in Table GTE.
The workflow of this screen is similar to that of the screen for Circuit 4 in
Replicons of the Circuit 6 library containing the top GTE candidates (i.e. with the highest mKate scores) are evaluated further in C2C12 myotubes. The most promising GTEs are subsequently expressed from an IRES off of the best Circuit 4 replicon and tested for improved circuit performance in C2C12 myotubes. Secreted GTEs that are expected to have paracrine effects are not included in the FACS screen above but are individually cloned and tested directly in myotubes. Once improvement is confirmed, the specific circuit configuration is used to build a replicon in the Circuit 3 format for in vivo testing as described in
A library of replicons (216 total) is constructed in the following format:
-
- SGP1(15) mVenus-2A-TetR-RE SGP2(X) NxTetR-Apt mKate IRES GTE (X=5, 15, 30; N=2, 3, 4; GTE=member of Table GTE)
- Circuit 7
- SGP1(15) mVenus-2A-TetR-RE SGP2(X) NxTetR-Apt mKate IRES GTE (X=5, 15, 30; N=2, 3, 4; GTE=member of Table GTE)
The workflow of this screen is similar to that of the screen for Circuit 4 in
Members of the Circuit 7 library with the largest differences in the mKate scores under the two conditions (+/−Dox) are tested for follow-up transfection/evaluation in differentiated C2C12 myotubes. Promising circuit configurations identified from the Circuit 7 library are used to construct Circuit 3 replicons tested in vivo as described in
Circuit optimization screens are found in
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
Claims
1. A synthetic RNA circuit comprising
- a first RNA molecule comprising at least one sequence recognized by at least one first microRNA that is/are specifically expressed in a first cell type, and a sequence encoding a protein that specifically binds to a RNA motif and inhibits protein production; and
- a second RNA molecule comprising at least one sequence recognized by at least one second microRNA that is/are not expressed in the first cell type or is expressed at a low level relative to a second cell type, at least one RNA motif and a sequence encoding an output molecule.
2. The synthetic RNA circuit of claim 1, wherein the output molecule is a protein.
3.-21. (canceled)
22. The synthetic RNA circuit of claim 1, wherein in a cell that expresses the at least one first microRNA but does not express the at least one second microRNA, the at least one first microRNA represses translation of or degrades the sequence encoding the protein that specifically binds to a RNA motif and inhibits protein production, thereby allowing expression of the output molecule.
23.-39. (canceled)
40. A method of treating cancer in a mammal comprising administering to a mammal the synthetic RNA circuit of claim 1.
41. A method of inducing an immune response in a mammal comprising administering to a mammal the synthetic RNA circuit of claim 1.
42. (canceled)
43. A synthetic RNA circuit comprising:
- (1) a first RNA molecule comprising at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule; and/or
- a second RNA molecule comprising at least one sequence recognized by a second protein that specifically binds to a RNA motif and inhibits protein production or a second RNA molecule comprising at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production; and/or
- a third RNA molecule comprising at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the second protein that specifically binds to a RNA motif and inhibits protein production; or
- (2) an RNA molecule comprising a sequence encoding a destabilization domain fused to an output protein,
- wherein the destabilization domain facilitates degradation of the output protein in the absence of a small molecule that binds to the destabilization domain; or
- (3) an RNA molecule comprising a sequence encoding a TetR protein and a sequence encoding an output protein, and an aptamer sequence that is bound by the TetR protein in the absence of tetracycline;
- wherein the aptamer sequence is positioned relative to the sequence encoding the output protein so that it suppresses translation of the output protein in the absence of tetracycline.
44. The synthetic RNA circuit of claim 43, further comprising the siRNA molecule or microRNA molecule that binds to the third RNA molecule.
45. (canceled)
46. The synthetic RNA circuit of claim 43, wherein the output molecule is a protein.
47. The synthetic RNA circuit of claim 43, wherein the output molecule or output protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator
48.-79. (canceled)
80. A method of treating cancer in a mammal comprising administering to a mammal the synthetic RNA circuit of claim 43.
81. A method of inducing an immune response in a mammal comprising administering to a mammal the synthetic RNA circuit of claim 43.
82.-122. (canceled)
123. A synthetic RNA circuit comprising:
- (1) a first RNA molecule comprising at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding a second protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a first siRNA molecule or microRNA molecule; and
- a second RNA molecule comprising at least one sequence recognized by the second protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a second siRNA molecule or microRNA molecule, or
- (2) a first RNA molecule comprising a sequence encoding a destabilization domain fused to a protein that specifically binds to a RNA motif and inhibits protein production; and a second RNA molecule comprising at least one sequence recognized by the protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule;
- wherein the destabilization domain facilitates degradation of the protein that specifically binds to a RNA motif and inhibits protein production in the absence of a small molecule that binds to the destabilization domain.
124.-204. (canceled)
205. The synthetic RNA circuit of claim 123, wherein the output molecule is a protein.
206. The synthetic RNA circuit of claim 205, wherein the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator.
207.-240. (canceled)
241. A method of treating cancer in a mammal comprising administering to a mammal the synthetic RNA circuit of claim 123.
242. A method of inducing an immune response in a mammal comprising administering to a mammal the synthetic RNA circuit of claim 123.
243.-279. (canceled)
280. A synthetic RNA circuit comprising:
- (a) a first RNA molecule comprising a sequence encoding a destabilization domain fused to an RNA binding protein that is capable of inhibiting protein production; and
- (b) a second RNA molecule comprising at least one RNA motif that is capable of binding to the RNA binding protein, and a sequence encoding an output molecule; wherein the destabilization domain is capable of facilitating degradation of the RNA binding protein in the absence of a small molecule that binds to the destabilization domain.
281. The synthetic RNA circuit of claim 280, wherein the output molecule is a protein.
282. The synthetic RNA circuit of claim 281, wherein the protein comprises a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator.
283. The synthetic RNA circuit of claim 280, further comprising a subgenomic promoter.
Type: Application
Filed: May 3, 2022
Publication Date: Dec 15, 2022
Applicants: Massachusetts Institute of Technology (Cambridge, MA), Kyoto University (Kyoto)
Inventors: Ron Weiss (Newton, MA), Liliana Wroblewska (Wilmington, MA), Velia Siciliano (Naples), Tasuku Kitada (Ghent), Maria Hottelet Foley (Cambridge, MA), Katie Bodner (Stanford, CA), Hirohide Saito (Kyoto), Kei Endo (Chiba), Darrell J. Irvine (Arlington, MA), Tyler Wagner (Bel Air, MD), Jacob Becraft (Boston, MA)
Application Number: 17/735,627
