Recombinant adeno-associated virus vectors to target medullary thyroid carcinoma
Provided herein are nucleic acids, recombinant adeno-associated virus (rAAV) particles, and compositions, as well as methods of use thereof for transducing medullary thyroid carcinoma cells and in treatment of disease, such as medullary thyroid carcinoma. In some aspects, the nucleic acid comprises a truncated calcitonin promoter, which is optionally encapsidated within a rAAV particle. In other aspects, the rAAV particle is a rAAV particle having a mutation in a surface-exposed amino acid, such as tyrosine, threonine, or serine, that enhances transduction of the particle into medullary thyroid carcinoma cells.
1 . A method of delivering a nucleic acid to a medullary thyroid carcinoma cell, the method comprising:
administering to a medullary thyroid carcinoma cell a rAAV2 particle comprising:
(a) a modified capsid protein comprising a non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid in a wild-type AAV2 capsid protein; and
(b) a nucleic acid comprising an expression construct containing a promoter operably linked to a coding sequence of a gene of interest,
wherein the promoter is a truncated calcitonin promoter consisting of the nucleic acid sequence of SEQ ID NO: 1.
2 . The method of claim 1 , wherein the non-native amino acid substitution is selected from:
(a) a non-tyrosine amino acid at Y730,
(b) a non-serine amino acid at S662,
(c) a non-threonine amino acid at T491,
(d) a non-serine amino acid at S662 and a non-threonine amino acid at T491,
(e) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, and a non-tyrosine amino acid at Y730, or
(f) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, a non-tyrosine amino acid at Y730 and a non-threonine amino acid at T491;
wherein each substitution is relative to the wild-type AAV2 sequence given as SEQ ID NO: 2.
3 . The method of claim 2 , wherein the non-native amino acid substitution is selected from:
(a) Y730F,
(b) S662V,
(c) T491V,
(d) S662V and T491V,
(e) Y444F, Y500F, and Y730F, or
(f) Y444F, Y500F, Y730F and T491V.
4 . The method of claim 3 , wherein the non-native amino acid substitution is selected from (a) Y730F or (e) Y444F, Y500F, and Y730F.
5 . The method of claim 1 , wherein the gene of interest is CYP2B6.
6 . The method of claim 1 , wherein the expression construct further comprises a tissue-specific enhancer region of the calcitonin gene having the sequence of nucleotides 980 to 1200 of SEQ ID NO: 3, and wherein the enhancer region is positioned 5′ of the truncated calcitonin promoter.
7 . The method of claim 1 , wherein the expression construct is flanked on each side by an AAV2 inverted terminal repeat sequence (ITR).
8 . The method of claim 1 , wherein the medullary thyroid carcinoma cell is in a human subject.
9 . The method of claim 8 , wherein the human subject has metastatic medullary thyroid carcinoma.
10 . A method of treating medullary thyroid carcinoma in a subject comprising administering to the subject:
(i) an rAAV2 particle comprising
(a) a modified capsid protein comprising a non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid in a wild-type AAV2 capsid protein, and
(b) a nucleic acid comprising an expression construct containing a promoter operably linked to a coding sequence of a gene of interest, wherein the gene of interest is CYP2B6 wherein the promoter is a truncated calcitonin promoter consisting of the nucleic acid sequence of SEQ ID NO: 1; and
(ii) cyclophosphamide.