MicroRNA-based logic gates and uses thereof
The present disclosure, at least in part, relates to a miRNA based logic gate that comprises an engineered RNA carrier that comprises an nuclear export signal, a target site for a first miRNA and a pre-miRNA sequence for a second miRNA. Also provided by the disclosure are recombinant viruses (e.g., recombinant adeno-associated viruses (rAAV)) for delivery of the miRNA based logic gates.
1. An miRNA based logic gate comprising an engineered RNA carrier that comprises
(i) a nuclear export sequence and a nuclear import sequence;
(ii) a target site for a first microRNA (miRNA); and
(iii) one or more pre-miRNA sequences of a second microRNA (miRNA),
wherein the nuclear export sequence is not a small nuclear RNA (snRNA) nuclear export sequence and the nuclear import sequence is not an snRNA nuclear import sequence.
2. The miRNA based logic gate of claim 1 , wherein the miRNA based logic gate is a NOT gate.
3. The miRNA based logic gate of claim 1 , wherein the miRNA based logic gate is a NOR gate.
4. The miRNA based logic gate of claim 3 , wherein the engineered RNA carrier further comprises a target site for a third miRNA.
5. An miRNA based logic gate, wherein the miRNA logic gate is a miRNA-based NAND gate, which comprises two miRNA based NOT gate of claim 2 .
6. An miRNA based logic gate, wherein the miRNA logic gate is a miRNA-based flip flop, which comprises two miRNA based NOR gates of claim 3 .
7. The miRNA based logic gate of claim 1 , wherein the engineered RNA carrier is an mRNA or a non-coding RNA.
8. The miRNA based logic gate of claim 7 , wherein the non-coding RNA is selected from a group consisting of small non-coding RNA and RNAs capable of being shuttled back and forth from the cytosol and the nucleus of the cell.
9. The miRNA based logic gate of claim 7 , wherein the mRNA is a viral nuclear RNA or a viral cytosolic RNA.
10. The miRNA based logic gate of claim 1 , wherein the engineered RNA carrier further comprises a sequence that binds to the survival of motor neuron complex (SMN complex).
11. The miRNA based logic gate of claim 1 , further comprising a 3′ cap.
12. An engineered nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding the miRNA based logic gate of claim 1 .
13. A recombinant virus comprising: a viral capsid containing a promoter operably linked to a nucleotide sequence encoding the miRNA based logic gate of claim 1 , optionally wherein the recombinant virus is a recombinant AAV (rAAV).
14. An isolated cell comprising the miRNA based logic gate of claim 1 , optionally wherein the isolated cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a plant cell, an insect cell, a mammalian cell or a human cell.
15. The isolated cell of claim 14 , wherein the isolated cell is a diseased cell or wherein the isolated cell is from a specific tissue.
16. The isolated cell of claim 14 , wherein the isolated cell expresses the first miRNA, and does express the second miRNA; or
wherein the isolated cell does not express the first microRNA, and expresses the second miRNA; or
wherein the isolated cell expresses both the first and the second miRNA, and the downregulation of the first miRNA leads to the upregulation of the second miRNA; or
wherein the isolated cell expresses both the first and the second miRNA, and the upregulation of the first miRNA leads to the downregulation of the second miRNA.
17. A pharmaceutical composition, comprising the miRNA based logic gate of claim 1 , optionally further comprising a pharmaceutically acceptable carrier.
18. A method for delivering a miRNA to a specific cell type in a subject, comprising administering to the subject an effective amount of the miRNA based logic gate of claim 1 .
19. A method for alleviating a disease in a subject in need thereof, comprising
administering to the subject an effective amount of the miRNA based logic gate of claim 1 , wherein the second miRNA further comprises a therapeutic RNA interference molecule or a nucleic acid enzyme.
20. The method of claim 19 , wherein the subject has or is at risk of having Alpha-1 antitrypsin deficiency, Hypercholesterolemia, Hepatitis B infection, Liver adenoma due to HIV infection, Hepatitis C virus infection, Ornithine transcarbamylase deficiency, Hepatocellular carcinoma, Amyotrophic lateral sclerosis, Spinocerebellar ataxia type 1, Huntington's disease, Parkinson disease, Spinal and Bulbar muscular atrophy, Pyruvate dehydrogenase deficiency, Hyperplasia, obesity, facioscapulohumeral muscular dystrophy (FSHD), Nerve Injury-induced Neuropathic Pain, Age-related macular degeneration, Retinitis pigmentosa, heart failure, cardiomyopathy, cold-induced cardiovascular dysfunction, Asthma, Duchenne muscular dystrophy, or prostate cancer.