Therapeutic editing
Disclosed herein are compositions that comprise engineered polynucleotides, pharmaceutical compositions comprising the same, methods of making the same, and methods of treatment comprising the compositions that comprise the engineered polynucleotides.
1. A composition that comprises: an engineered guide RNA comprising a targeting sequence that has complementarity to a region of a target RNA sufficient for the targeting sequence to hybridize to the region of the target RNA, wherein the region of the target RNA comprises a sequence that encodes an amyloid precursor protein (APP) polypeptide; and
wherein the engineered guide RNA is configured to hybridize to the region of the target RNA and to facilitate editing of an adenosine in the target RNA by an RNA editing entity, thereby generating an edited target RNA,
wherein the adenosine is comprised in a codon which encodes an amino acid in proximity to a cleavage site of the APP polypeptide, and wherein the amino acid is at position 670 or 671, or both positions of the APP polypeptide comprising the polypeptide sequence of SEQ ID NO: 2,
wherein the cleavage site is selected from the group consisting of: an alpha-secretase cleavage site, a beta-secretase cleavage site, a beta′-secretase cleavage site, a gamma-secretase cleavage site, and any combination thereof,
wherein the target RNA encodes an unmodified APP polypeptide that comprises at least one amino acid residue difference as compared to a modified APP polypeptide generated from translation of the edited target RNA, and
wherein the modified APP polypeptide comprises a K670G substitution, a M671V substitution, or both substitution of the unmodified APP polypeptide comprising the polypeptide sequence of SEQ ID NO: 2 and wherein the modification of the APP polypeptide results in reduced levels of Abeta 40, Abeta 42, or both peptides produced by a cell as compared to the unmodified APP polypeptide.
2. The composition of claim 1 , wherein the engineered guide RNA, when hybridized with the target RNA, forms a structural feature.
3. The composition of claim 2 , wherein the structural feature comprises a bulge, an internal loop, a hairpin, a mismatch, a wobble base pair, or any combination thereof.
4. The composition of claim 3 , wherein the structural feature comprises the bulge.
5. The composition of claim 4 , wherein the bulge comprises an asymmetric bulge.
6. The composition of claim 4 , wherein the bulge comprises a symmetric bulge.
7. The composition of claim 4 , wherein the bulge comprises from about 1 to about 4 nucleotides of the engineered guide RNA and from about 0 to about 4 nucleotides of the target RNA.
8. The composition of claim 4 , wherein the bulge comprises from about 0 to about 4 nucleotides of the engineered guide RNA and from about 1 to about 4 nucleotides of the target RNA.
9. The composition of claim 4 , wherein the bulge comprises 3 nucleotides of the engineered guide RNA and 3 nucleotides of the target RNA.
10. The composition of claim 4 , wherein the bulge comprises 3 nucleotides of the engineered guide RNA and 2 nucleotides of the target RNA.
11. The composition of claim 4 , wherein the engineered guide RNA, when associated with the target RNA, forms a second structural feature.
12. The composition of claim 11 , wherein the second structural feature is a bulge that comprises 2 nucleotides of the engineered guide RNA and 2 nucleotides of the target RNA.
13. The composition of claim 11 , wherein the second structural feature is a G/G mismatch followed by an A/C mismatch.
14. The composition of claim 13 , wherein the base of the nucleotide of the target RNA is an adenosine of the A/C mismatch.
15. The composition of claim 11 , wherein the structural feature is a bulge that comprises 2 nucleotides of the engineered guide RNA and 2 nucleotides of the target RNA; and wherein the second structural feature is a bulge that comprises 3 nucleotides of the engineered guide RNA and 2 nucleotides of the target RNA.
16. The composition of claim 3 , wherein the structural feature comprises the internal loop.
17. The composition of claim 16 , wherein the internal loop comprises an asymmetric internal loop.
18. The composition of claim 16 , wherein the internal loop comprises a symmetric internal loop.
19. The composition of claim 16 , wherein the internal loop is formed by from about 5 to about 10 nucleotides of either the engineered guide RNA or the target RNA.
20. The composition of claim 3 , wherein the structural feature comprises the mismatch and wherein the mismatch comprises a base in the targeting sequence of the engineered guide RNA opposite to and unpaired with the base of the nucleotide of the target RNA.
21. The composition of claim 20 , wherein the mismatch comprises a base in the targeting sequence of the engineered guide RNA opposite to and unpaired with the base of the nucleotide of the target RNA.
22. The composition of claim 20 , wherein the mismatch comprises a guanine-guanine mismatch.
23. The composition of claim 20 , wherein the mismatch comprises an adenosine-cytosine mismatch, and wherein the adenosine is in the target RNA and the cytosine is in the targeting sequence of the engineered guide RNA.
24. The composition of claim 23 , wherein the adenosine in the adenosine-cytosine mismatch is the adenosine in the target RNA edited by the RNA editing entity.
25. The composition of claim 3 , wherein the structural feature comprises the wobble base pair.
26. The composition of claim 25 , wherein the wobble base pair comprises a guanine paired with a uracil.
27. The composition of claim 1 , wherein the RNA editing entity comprises an adenosine deaminase acting on RNA (ADAR) polypeptide or biologically active fragment thereof, wherein the ADAR polypeptide or the biologically active fragment thereof comprises ADAR1, ADAR2, or a biologically active fragment of any of these.
28. The composition of claim 1 , wherein the editing of the adenosine in the target RNA by the RNA editing entity is sufficient to reduce β-amyloid peptide, which comprises preventing or eliminating formation of the β-amyloid peptide.
29. The composition of claim 28 , wherein the editing is sufficient to eliminate the β-amyloid peptide formation.
30. The composition of claim 28 , wherein the editing is sufficient to increase an amount of secreted ectodomain APP alpha (sAPPa).
31. The composition of claim 1 , wherein the engineered guide RNA is configured to facilitate editing of two bases of the target RNA by the RNA editing entity, wherein the two bases are comprised in codons that encode amino acids at position 670 and position 671.
32. The composition of claim 1 , wherein the engineered guide RNA is encoded by a polynucleotide sequence comprised in a vector.
33. The composition of claim 32 , wherein the vector is an adeno associated viral (AAV) vector.
34. The composition of claim 33 , wherein the AAV vector is of a serotype selected from the group comprising: AAV2, AAVS, AAV6, AAV8, AAV9, a fusion product thereof, and any combination thereof.