SOD1 dual expression vectors and uses thereof
In some aspects, the disclosure relates to compositions and methods useful for inhibiting SOD1 expression in cells (e.g., cells of a subject). In some embodiments, the disclosure describes isolated nucleic acids engineered to express an inhibitory nucleic acid targeting endogenous SOD1 and an mRNA encoding a hardened SOD1 protein. In some embodiments, compositions and methods described by the disclosure are useful for treating Amyotrophic Lateral Sclerosis (ALS) in a subject.
1. An isolated nucleic acid comprising:
(a) a first region that encodes one or more first miRNAs comprising a nucleic acid having sufficient sequence complementary with an endogenous mRNA of a subject to hybridize with and inhibit expression of the endogenous mRNA, wherein the endogenous mRNA encodes a SOD1 protein; and
(b) a second region encoding an exogenous mRNA that encodes a wild-type SOD1 protein,
wherein the one or more first miRNAs do not comprise a nucleic acid having sufficient sequence complementary to hybridize with and inhibit expression of the exogenous mRNA,
wherein the wild-type SOD1 protein is encoded by a sequence comprising the sequence set forth in SEQ ID NO: 7.
2. The isolated nucleic acid claim 1 , wherein the one or more first miRNAs:
(a) targets an untranslated region of the nucleic acid encoding the endogenous mRNA;
(b) targets a coding sequence of the nucleic acid encoding the endogenous mRNA;
(c) hybridizes to a nucleic acid comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, or 21 consecutive nucleotides of a RNA encoded by a sequence as set forth in SEQ ID NO: 2; and/or
(d) is encoded by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, or 21 consecutive nucleotides of a sequence comprising the sequence as set forth in SEQ ID NO: 3 and/or 4.
3. The isolated nucleic acid of claim 2 , wherein the one or more first miRNAs further comprise flanking regions of miR-155 or miR-30.
4. The isolated nucleic acid of claim 1 , further comprising a first promoter operably linked to the first region.
5. The isolated nucleic acid of claim 1 , further comprising a second promoter, wherein the second promoter is operably linked to the second region.
6. The isolated nucleic acid of claim 1 , further comprising an enhancer sequence.
7. The isolated nucleic acid of claim 1 , wherein the first region is positioned within an untranslated region of the second region.
8. The isolated nucleic acid of claim 7 , wherein the first region is positioned within an intron of the second acid.
9. The isolated nucleic acid of claim 1 , wherein the first region is positioned 5′ with respect to the second region.
10. The isolated nucleic acid of claim 1 , further comprising at least one adeno-associated virus (AAV) inverted terminal repeat (ITR).
11. The isolated nucleic acid of claim 10 , comprising a full-length ITR and a mutant ITR, wherein the ITRs flank the first and second regions.
12. A recombinant adeno-associated virus (rAAV) comprising:
(i) the isolated nucleic acid of claim 1 ; and
(ii) an AAV capsid protein.
13. The rAAV of claim 12 , wherein the rAAV targets tissue of the central nervous system (CNS).
14. The rAAV of claim 12 , wherein the capsid protein is AAV9 capsid protein or AAVrh.10 capsid protein.
15. A composition comprising the isolated nucleic acid of claim 1 , and a pharmaceutically acceptable excipient.
16. The isolated nucleic acid of claim 1 , wherein the exogenous mRNA lacks a 5′ untranslated region (5′ UTR), lacks a 3′ untranslated region (3′ UTR), or lacks both a 5′ UTR and a 3′UTR.
17. The isolated nucleic acid of claim 1 , wherein the exogenous mRNA encoding the SOD1 protein has one or more silent base pair mutations relative to the endogenous mRNA.
18. The isolated nucleic acid of claim 17 , wherein the exogenous mRNA comprises a nucleic acid sequence that is at least 95% identical to the endogenous mRNA.