Artificial expression constructs for selectively modulating gene expression in selected neuronal cell populations
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 GABAergic neurons generally; and/or GABAergic neuron cell types such as lysosomal associated membrane protein 5 (Lamp5) neurons; vasoactive intestinal polypeptide-expressing (Vip) neurons; somatostatin (Sst) neurons; and/or parvalbumin (Pvalb) neuron cell types. Certain artificial expression constructs additionally drive selective gene expression in Layer 4 and/or layer 5 intratelencephalic (IT) neurons, deep cerebellar nuclear neurons or cerebellar Purkinje cells.
1 . An artificial expression construct comprising (i) an enhancer consisting of the sequence of SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; (ii) a heterologous promoter; and (iii) a heterologous coding sequence, wherein the enhancer is operatively linked to the heterologous promoter and the heterologous coding sequence.
2 . An artificial expression construct comprising an enhancer having the sequence as set forth in SEQ ID NO: 1, a promoter, and a coding sequence.
3 . The artificial expression construct of claim 1 , wherein the coding sequence encodes an effector element or an expressible element.
4 . The artificial expression construct of claim 3 , wherein the effector element includes a reporter protein or a functional molecule.
5 . The artificial expression construct of claim 4 , wherein the reporter protein comprises a fluorescent protein.
6 . The artificial expression construct of claim 4 , wherein the functional molecule comprises a functional neurotransmitter, ion transporter, enzyme, transcription factor, receptor, membrane protein, cellular trafficking protein, signaling molecule, calcium reporter, channelrhodopsin, CRISPR/Cas molecule, editase, guide RNA molecule, homologous recombination donor cassette, or a designer receptor exclusively activated by designer drugs (DREADD).
7 . The artificial expression construct of claim 1 , wherein the artificial expression construct is encapsidated into a capsid that crosses the blood brain barrier.
8 . The artificial expression construct of claim 7 , wherein the capsid includes PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, adeno-associated virus serotype 9 (AAV9), or AAVrh.10.
9 . The artificial expression construct of claim 1 , wherein the artificial expression construct includes or encodes a skipping element.
10 . The artificial expression construct of claim 9 , wherein the skipping element includes a 2A peptide or an internal ribosome entry site (IRES).
11 . The artificial expression construct of claim 10 , wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.
12 . The artificial expression construct of claim 1 , wherein the artificial expression construct includes or encodes: SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, adeno-associated virus (AAV), self-complementary recombinant adeno-associated virus (scAAV), recombinant adeno-associated virus (rAAV), beta globin minimal promoter (minBglobin), cytomegalovirus (CMV) promoter, minimal cytomegalovirus (minCMV) promoter, minimal Rho (minRho) promoter, minimal Rho promoter with a SacI restriction site removed (minRho*), fluorescent protein, cyclization recombinase (Cre), improved Cre recombinase (iCre), DHFR-GFP-Cre (dgCre), codon-optimized recombinase flippase (FIpO), heat shock protein 68 (Hsp68) promoter, tetracycline transactivator 2 (tTA2), spermatid specific promoter 10 (SP10) insulator, woodchuck hepatitis virus post-transcriptional regulatory element 3 (WPRE3), and/or bovine growth hormone polyA sequence (BGHpA).
13 . The artificial expression construct of claim 1 , wherein the artificial expression construct includes or encodes from 5′ to 3′:
SEQ ID NO: 9, minimal Rho promoter (minRho), coding sequence, woodchuck hepatitis virus post-transcriptional regulatory element 3 (WPRE3), and bovine growth hormone polyadenylation (BGHpA);
SEQ ID NO: 1 or SEQ ID NO: 42, heat shock protein 68 minimal promoter (Hsp68), coding sequence, WPRE3, and BGHpA;
SEQ ID NO: 1 or SEQ ID NO: 42, beta-globin minimal promoter (minBglobin), coding sequence, WPRE3, and BGHpA;
SEQ ID NO: 2, Hsp68, coding sequence, WPRE3, and BGHpA;
SEQ ID NO: 3, minBglobin, coding sequence, WPRE3, and BGHpA;
SEQ ID NO: 4, minBglobin, coding sequence, WPRE3, and BGHpA;
SEQ ID NO: 9, minRho, coding sequence, and post-transcriptional regulatory elements;
SEQ ID NO: 1 or SEQ ID NO: 42, Hsp68, coding sequence, and post-transcriptional regulatory elements;
SEQ ID NO: 1 or SEQ ID NO: 42, minBglobin, coding sequence, and post-transcriptional regulatory elements;
SEQ ID NO: 2, Hsp68, coding sequence, and post-transcriptional regulatory elements;
SEQ ID NO: 3, minBglobin, coding sequence, and post-transcriptional regulatory elements; or
SEQ ID NO: 4, minBglobin, coding sequence, and post-transcriptional regulatory elements.
14 . The artificial expression construct of claim 1 , wherein the artificial expression construct is within a vector for delivery into a cell.
15 . The artificial expression construct of claim 14 , wherein the vector comprises 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 15 , wherein the cell is a GABAergic neuron.
19 . The artificial expression construct of claim 15 , wherein the cell is a lysosomal associated membrane protein (LAMP) neuron, a vasoactive intestinal polypeptide-expressing (VIP) neuron, a somatostatin (Sst) neuron, a parvalbumin (PVALB) neuron, a layer 4 (L4) intratelencephalic (IT) neuron, a layer 5 (L5) IT neuron, or a cerebellar Purkinje cell.
20 . An artificial expression construct comprising the sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.