Artificial expression constructs for selectively modulating gene expression in excitatory cortical neurons
Artificial expression constructs for selectively modulating gene expression in selected central nervous system cell types are described. The artificial expression constructs can be used to selectively express synthetic genes or modify gene expression in excitatory cortical neurons, such as primarily within cortical layers 2/3, 4, 5, and 6 and including those with extratelencephalic (ET) projections, intratelencephalic (IT) projections, and pyramidal tract (PT) projections, among others.
1 . An artificial expression construct comprising
(i) an enhancer consisting of
the sequence of SEQ ID NO: 29,
the sequence of SEQ ID NO: 177,
the sequence of SEQ ID NO: 178,
the sequence of SEQ ID NO: 188,
the sequence of SEQ ID NO: 25,
the sequence of SEQ ID NO: 26,
the sequence of SEQ ID NO: 27,
the sequence of SEQ ID NO: 28,
the sequence of SEQ ID NO: 31,
the sequence of SEQ ID NO: 32,
the sequence of SEQ ID NO: 33,
the sequence of SEQ ID NO: 34,
the sequence of SEQ ID NO: 35,
the sequence of SEQ ID NO: 36,
the sequence of SEQ ID NO: 37,
the sequence of SEQ ID NO: 38,
the sequence of SEQ ID NO: 39,
the sequence of SEQ ID NO: 41,
the sequence of SEQ ID NO: 42,
the sequence of SEQ ID NO: 43,
the sequence of SEQ ID NO: 44,
the sequence of SEQ ID NO: 45,
the sequence of SEQ ID NO: 46,
the sequence of SEQ ID NO: 47,
the sequence of SEQ ID NO: 48,
the sequence of SEQ ID NO: 50,
the sequence of SEQ ID NO: 51,
a sequence having at least 95% sequence identity to the sequence consisting of SEQ ID NO: 29,
a sequence having at least 95% sequence identity to the sequence consisting of SEQ ID NO: 177,
a sequence having at least 95% sequence identity to the sequence consisting of SEQ ID NO: 178,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 188,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 25,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 26,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 27,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 28,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 31,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 32,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 33,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 34,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 35,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 36,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 37,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 38,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 39,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 41,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 42,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 43,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 44,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 45,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 46,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 47,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 48,
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 50, or
a sequence having at least 95% sequence identity with the sequence consisting of SEQ ID NO: 51,
(ii) a promoter, and
(iii) a coding sequence.
2 . The artificial expression construct of claim 1 , wherein the artificial expression construct comprises
three copies of the enhancer consisting of SEQ ID NO: 29,
three copies of the enhancer consisting of SEQ ID NO: 177,
three copies of the enhancer consisting of SEQ ID NO: 178,
three copies of the enhancer having at least 95% sequence identity to the sequence consisting of SEQ ID NO: 29,
three copies of the enhancer having at least 95% sequence identity to the sequence consisting of SEQ ID NO: 177, or
three copies of the enhancer having at least 95% sequence identity to the sequence consisting of SEQ ID NO: 178.
3 . The artificial expression construct of claim 1 , wherein the enhancer comprises
the sequence of SEQ ID NO: 30,
the sequence of SEQ ID NO: 40,
the sequence of SEQ ID NO: 49,
a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 30,
a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40, or
a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 49.
4 . The artificial expression construct of claim 1 , wherein the artificial expression construct comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 29, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 188, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 51.
5 . The artificial expression construct of claim 1 , wherein the artificial expression construct comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the sequence of SEQ ID NO: 29, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 188, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 51.
6 . The artificial expression construct of claim 1 , wherein the coding sequence encodes an effector element or an expressible element.
7 . The artificial expression construct of claim 6 , wherein the effector element comprises a reporter protein or a functional molecule.
8 . The artificial expression construct of claim 7 , wherein the reporter protein comprises a fluorescent protein.
9 . The artificial expression construct of claim 7 , wherein the functional molecule comprises a functional ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular trafficking protein, signaling molecule, neurotransmitter, calcium reporter, channel rhodopsin, CRISPR/Cas molecule, editase, guide RNA molecule, homologous recombination donor cassette, or a designer receptor exclusively activated by designer drug (DREADD).
10 . The artificial expression construct of claim 1 , wherein the artificial expression construct is associated with a capsid that crosses the blood brain barrier.
11 . The artificial expression construct of claim 10 , wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, AAV9, or AAVrh.10.
12 . The artificial expression construct of claim 1 , wherein the expression construct comprises or encodes a skipping element.
13 . The artificial expression construct of claim 12 , wherein the skipping element comprises a 2A peptide and/or an internal ribosome entry site (IRES).
14 . The artificial expression construct of claim 13 , wherein the 2A peptide is selected from T2A, P2A, E2A, or F2A.
15 . The artificial expression construct of claim 1 , wherein the artificial expression construct is within a viral vector.
16 . The artificial expression construct of claim 15 , wherein the viral vector comprises a recombinant adeno-associated viral (AAV) vector.
17 . The artificial expression construct of claim 16 , wherein the AAV vector is replication-competent.
18 . The artificial expression construct of claim 1 , comprising a sequence having 95% sequence identity to the sequence of SEQ ID NO: 73, SEQ ID NO: 179, SEQ ID NO: 78, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 76, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 101, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 112.
19 . The artificial expression construct of claim 1 , comprising the sequence of SEQ ID NO: 73, SEQ ID NO: 179, SEQ ID NO: 78, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 76, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 101, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 112.